Conventional Phagocytosis Research Articles

Morphology, behavior, and phylogenomics of Oxytoxum lohmannii, Dinoflagellata.

Dinoflagellates are an abundant and diverse group of protists representing a wealth of unique biology and ecology. While many dinoflagellates are photosynthetic or mixotrophic, many taxa are heterotrophs, often with complex feeding strategies. Compared to their photosynthetic counterparts, heterotrophic dinoflagellates remain understudied, as they are difficult to culture. One exception, a long-cultured isolate originally classified as Amphidinium but recently reclassified as Oxytoxum, has been the subject of a number of feeding, growth, and chemosensory studies. This lineage was recently determined to be closely related to Prorocentrum using phylogenetics of ribosomal RNA gene sequences, but the exact nature of this relationship remains unresolved. Using transcriptomes sequenced from culture and three single cells from the environment, we produce a robust phylogeny of 242 genes, revealing Oxytoxum is likely sister to the Prorocentrum clade, rather than nested within it. Molecular investigations uncover evidence of a reduced, nonphotosynthetic plastid and proteorhodopsin, a photoactive proton pump acquired horizontally from bacteria. We describe the ultrastructure of O. lohmannii, including densely packed trichocysts, and a new type of mucocyst. We observe that O. lohmannii feeds preferentially on cryptophytes using myzocytosis, but can also feed on various phytoflagellates using conventional phagocytosis. O. lohmannii is amenable to culture, providing an opportunity to better study heterotrophic dinoflagellate biology and feeding ecology.

TLR4 signaling regulates PI3 kinase activation and macrophage exophagy (796.16)

We have characterized a novel process that macrophages use to degrade species such as aggregated lipoproteins (agLDL) that are too large to be internalized via conventional phagocytosis. Macrophages form an intimate interaction with agLDL through local actin polymerization and form an extracellular compartment to which hydrolytic enzymes are delivered through lysosome exocytosis. The process of extracellular macrophage degradation of large species prior to internalization has been termed “exophagy”. AgLDL degradation by macrophages leads to foam cell formation, a key feature of atherosclerotic plaque development. Therefore unraveling the signaling mechanisms involved in this process may allow therapeutic intervention.Upon engagement with agLDL, Toll‐like receptor 4 (TLR) interacts with its effector MyD88 and TLR4/MyD88 are critical for lysosome exocytosis and actin polymerization during exophagy. At contact sites with agLDL, PI(3,4,5)P3 is produced, suggestive of class I PI3 kinase activation. Inhibition of PI3 kinase impairs lysosome exocytosis and actin polymerization during exophagy. PI(3,4,5)P3 recruits and activates plekstrin homology (PH) domain containing proteins such as protein kinase B (Akt). AgLDL treatment stimulates PI3 kinase dependent Akt activation, and inhibition of Akt using Akti1/2 impairs lysosome exocytosis and actin polymerization. While PI(3,4,5)P3 levels at contact sites with agLDL peak rapidly (around 20‐30 min) and then decline, the sustained generation of PI(4,5)P2 may act as a lasting signal to promote actin polymerization and lysosome exocytosis at sites of exophagy.Exophagy may represent a general mechanism, which cells may use to degrade large extracellular species, and there exists a close relationship between this process and various human diseases. Therefore elucidation of signaling mediators regulating these processes would potentially be of widespread importance in a number of diseases other than atherosclerosis.Grant Funding Source: Supported by NIH

The innate and adaptive immune response induced by alveolar macrophages exposed to ambient particulate matter

Emerging epidemiological evidence suggests that exposure to particulate matter (PM) air pollution increases the risk of cardiovascular events but the exact mechanism by which PM has adverse effects is still unclear. Alveolar macrophages (AM) play a major role in clearing and processing inhaled PM. This comprehensive review of research findings on immunological interactions between AM and PM provides potential pathophysiological pathways that interconnect PM exposure with adverse cardiovascular effects. Coarse particles (10 μm or less, PM 10) induce innate immune responses via endotoxin-toll-like receptor (TLR) 4 pathway while fine (2.5 μm or less, PM 2.5) and ultrafine particles (0.1 μm or less, UFP) induce via reactive oxygen species generation by transition metals and/or polyaromatic hydrocarbons. The innate immune responses are characterized by activation of transcription factors [nuclear factor (NF)-κB and activator protein-1] and the downstream proinflammatory cytokine [interleukin (IL)-1β, IL-6, and tumor necrosis factor-α] production. In addition to the conventional opsonin-dependent phagocytosis by AM, PM can also be endocytosed by an opsonin-independent pathway via scavenger receptors. Activation of scavenger receptors negatively regulates the TLR4-NF-κB pathway. Internalized particles are subsequently subjected to adaptive immunity involving major histocompatibility complex class II (MHC II) expression, recruitment of costimulatory molecules, and the modulation of the T helper (Th) responses. AM show atypical antigen presenting cell maturation in which phagocytic activity decreases while both MHC II and costimulatory molecules remain unaltered. PM drives AM towards a Th1 profile but secondary responses in a Th1- or Th-2 up-regulated milieu drive the response in favor of a Th2 profile.

Virosomes can enter cells by non-phagocytic mechanisms

Phagocytosis of fine particles (1 μm) by macrophages is a ligand-receptor–mediated, actin-based process, whereas the entering of smaller particles (≤ 0.2 μm) in macrophages occurs also by other mechanisms. Virosomes with a diameter of 0.12–0.18 μm are widely used as carrier systems for drugs, vectors, and plasmids in cancer therapy or for vaccines. We investigated their interactions with airway cells, in particular penetration into monocyte-derived macrophages. The microscopic analysis of phagocytic cells incubated with virosomes and polystyrene particles showed that virosomes and particles penetrated cells even in the presence of cytochalasin D, a drug inhibiting actin-based phagocytosis. The charge of the virosomes and particles did not influence their penetration. Also, different inhibitors of endocytotic pathways did not prevent the particles and virosomes from penetrating into the cells. Additionally, to study the ability of virosomes to overcome the epithelial airway barrier, a triple cell co-culture model composed of epithelial cells, monocyte-derived macrophages and dendritic cells of the respiratory tract was used. We found virosomes and polystyrene particles in both populations of antigen-presenting cells, monocyte-derived macrophages, and dendritic cells, in the latter even if they were not directly exposed. In conclusion, virosomes are readily taken up by monocyte-derived macrophages, both by conventional phagocytosis and by actin-independent mechanisms. Further, they can penetrate the airway barrier and reach resident dendritic cells. Therefore, virosomes are promising vaccine candidates.

Cryptococcus neoformansEnters the Endolysosomal Pathway of Dendritic Cells and Is Killed by Lysosomal Components

Cryptococcus neoformans is an opportunistic fungal pathogen that primarily causes disease in immunocompromised individuals. Dendritic cells (DCs) can phagocytose C. neoformans, present cryptococcal antigen, and kill C. neoformans. However, early events following C. neoformans phagocytosis by DCs are not well defined. We hypothesized that C. neoformans traffics to the endosome and the lysosome following phagocytosis by DCs and is eventually killed in the lysosome. Murine bone marrow-derived DCs (BMDCs) or human monocyte-derived DCs (HDCs) were incubated with live, encapsulated C. neoformans yeast cells and opsonizing antibody. Following incubation, DCs were intracellularly stained with antibodies against EEA1 (endosome) and LAMP-1 (late endosome/lysosome). As assessed by confocal microscopy, C. neoformans trafficked to endosomal compartments of DCs within 10 min and to lysosomal compartments within 30 min postincubation. For HDCs, the studies were repeated using complement-sufficient autologous plasma for the opsonization of C. neoformans. These data showed results similar to those for antibody opsonization, with C. neoformans localized to endosomes within 20 min and to lysosomes within 60 min postincubation. Additionally, the results of live real-time imaging studies demonstrated that C. neoformans entered lysosomal compartments within 20 min following the initiation of phagocytosis. The results of scanning and transmission electron microscopy demonstrated conventional zipper phagocytosis of C. neoformans by DCs. Finally, lysosomal extracts were purified from BMDCs and incubated with C. neoformans to determine their potential to kill C. neoformans. The extracts killed C. neoformans in a dose-dependent manner. This study shows that C. neoformans enters into endosomal and lysosomal pathways following DC phagocytosis and can be killed by lysosomal components.

Francisella tularensisEnters Macrophages via a Novel Process Involving Pseudopod Loops

Intracellular bacterial pathogens employ a variety of strategies to invade their eukaryotic host cells. From an ultrastructural standpoint, the processes that bacteria employ to invade their host cells include conventional phagocytosis, coiling phagocytosis, and ruffling/triggered macropinocytosis. In this paper, we describe a novel process by which Francisella tularensis, the agent of tularemia, enters host macrophages. F. tularensis is a remarkably infectious facultative intracellular bacterial parasite--as few as 10 bacteria can cause life-threatening disease in humans. However, the ultrastructure of its uptake and the receptor mechanisms that mediate its uptake have not been reported previously. We have used fluorescence microscopy and electron microscopy to examine the adherence and uptake of a virulent recent clinical isolate of F. tularensis, subspecies tularensis, and the live vaccine strain (LVS), subspecies holarctica, by human macrophages. We show here that both strains of F. tularensis enter human macrophages by a novel process of engulfment within asymmetric, spacious pseudopod loops, a process that differs ultrastructurally from all previously described uptake mechanisms. We demonstrate also that adherence and uptake of F. tularensis by macrophages is strongly dependent upon complement receptors and upon serum with intact complement factor C3 and that uptake requires actin microfilaments. These findings have significant implications for understanding the intracellular biology and virulence of this extremely infectious pathogen.

Dendritic cells phagocytose and are activated by Treponema pallidum.

Cell-mediated immune processes play a prominent role in the clinical manifestations of syphilis, a sexually transmitted disease of humans caused by spirochetal bacterium Treponema pallidum. The immune cell type that initiates the early immune response to T. pallidum thus far has not been identified. However, dendritic cells (DCs) are the first immune-competent cells to encounter antigens within skin or mucous membranes, the principal sites of early syphilitic infection. In the present study, immature DC line XS52, derived from murine skin, was utilized to examine T. pallidum-DC interactions and subsequent DC activation (maturation). Electron microscopy revealed that T. pallidum was engulfed by DCs via both coiling and conventional phagocytosis and was delivered to membrane-bound vacuoles. The XS52 DC line expressed surface CD14 and mRNA for Toll-like receptors 2 and 4, molecules comprising important signaling components for immune cell activation by bacterial modulins. Both T. pallidum and a synthetic lipopeptide (corresponding to the 47-kDa major membrane lipoprotein) activated the XS52 DC line, as indicated by the secretion of interleukin-12 (IL-12), IL-1beta, tumor necrosis factor alpha, and IL-6 and elevated surface expression of CD54. The combined data support the contention that DCs stimulated by T. pallidum and/or its proinflammatory membrane lipoproteins are involved in driving the cellular immune processes that typify syphilis.

Human Dendritic Cells Very Efficiently Present a Heterologous Antigen Expressed on the Surface of Recombinant Gram-Positive Bacteria to CD4+ T Lymphocytes

Recombinant Streptococcus gordonii expressing on the surface the C-fragment of tetanus toxin was tested as an Ag delivery system for human monocyte-derived dendritic cells (DCs). DCs incubated with recombinant S. gordonii were much more efficient than DCs pulsed with soluble C-fragment of tetanus toxin at stimulating specific CD4+ T cells as determined by cell proliferation and IFN-gamma release. Compared with DCs treated with soluble Ag, DCs fed with recombinant bacteria required 102- to 103-fold less Ag and were at least 102 times more effective on a per-cell basis for activating specific T cells. S. gordonii was internalized in DCs by conventional phagocytosis, and cytochalasin D inhibited presentation of bacteria-associated Ag, but not of soluble Ag, suggesting that phagocytosis was required for proper delivery of recombinant Ag. Bacteria were also very potent inducers of DC maturation, although they enhanced the capacity of DCs to activate specific CD4+ T cells at concentrations that did not stimulate DC maturation. In particular, S. gordonii dose-dependently up-regulated expression of membrane molecules (MHC I and II, CD80, CD86, CD54, CD40, CD83) and reduced both phagocytic and endocytic activities. Furthermore, bacteria promoted in a dose-dependent manner DC release of cytokines (IL-6, TNF-alpha, IL-1beta, IL-12, TGF-beta, and IL-10) and of the chemokines IL-8, RANTES, IFN-gamma-inducible protein-10, and monokine induced by IFN-gamma. Thus, recombinant Gram-positive bacteria appear a powerful tool for vaccine design due to their extremely high capacity to deliver Ags into DCs, as well as induce DC maturation and secretion of T cell chemoattractans.

Coiling phagocytosis is the predominant mechanism for uptake of the colonic spirochetosis bacterium Serpulina pilosicoli by human monocytes.

Serpulina pilosicoli is a newly identified pathogenic spirochete that establishes persistent colonic infections in human beings and animals. Macrophages are one of the key defenses against invasion of mucosal surfaces by bacterial pathogens. Macrophages engulf many bacteria by conventional phagocytosis; however recent studies indicate coiling phagocytosis as a new and important mechanism for internalization of Legionella pneumophila and spirochetes of the genus Borrelia, Leptospira, and Treponema. In this study, THP-1 human monocytic cells were incubated with the human S. pilosicoli strain SP16 and the contribution of coiling and conventional phagocytosis to the total number of phagocytic events were determined by sequential ultrastructural examination between 5 and 45 minutes. The frequency of phagocytosis increased over time from 5.1% after 5 minutes up to 21.9% after 45 minutes with greater than 70% of the events involving coiling phagocytosis. The data indicate that coiling phagocytosis may be a universal mechanism for uptake of pathogenic spirochetes.

Coiling phagocytosis of trypanosomatids and fungal cells.

Coiling phagocytosis has previously been studied only with the bacteria Legionella pneumophila and Borrelia burgdorferi, and the results were inconsistent. To learn more about this unconventional phagocytic mechanism, the uptake of various eukaryotic microorganisms by human monocytes, murine macrophages, and murine dendritic cells was investigated in vitro by video and electron microscopy. Unconventional phagocytosis of Leishmania spp. promastigotes, Trypanosoma cruzi trypomastigotes, Candida albicans hyphae, and zymosan particles from Saccharomyces cerevisiae differed in (i) morphology (rotating unilateral pseudopods with the trypanosomatids, overlapping bilateral pseudopods with the fungi), (ii) frequency (high with Leishmania; occasional with the fungi; rare with T. cruzi), (iii) duration (rapid with zymosan; moderate with the trypanosomatids; slow with C. albicans), (iv) localization along the promastigotes (flagellum of Leishmania major and L. aethiopica; flagellum or posterior pole of L. donovani), and (v) dependence on complement (strong with L. major and L. donovani; moderate with the fungi; none with L. aethiopica). All of these various types of unconventional phagocytosis gave rise to similar pseudopod stacks which eventually transformed to a regular phagosome. Further video microscopic studies with L. major provided evidence for a cytosolic localization, synchronized replication, and exocytic release of the parasites, extending traditional concepts about leishmanial infection of host cells. It is concluded that coiling phagocytosis comprises phenotypically similar consequences of various disturbances in conventional phagocytosis rather than representing a single separate mechanism.

Characterization of mannose receptor-dependent phagocytosis mediated by Mycobacterium tuberculosis lipoarabinomannan.

The macrophage mannose receptor (MR) along with complement receptors mediates phagocytosis of the M. tuberculosis virulent strains Erdman and H37Rv. We have determined that the terminal mannosyl units of the M. tuberculosis surface lipoglycan, lipoarabinomannan (LAM), from the Erdman strain serve as ligands for the MR. The biology of the MR (receptor binding and trafficking) in response to phagocytic stimuli is not well characterized. This study analyzes the MR-dependent phagocytosis mediated by Erdman LAM presented on a 1-micron-diameter phagocytic particle. Erdman LAM microspheres exhibited a time- and dose-dependent rapid increase in attachment and internalization by human monocyte-derived macrophages (MDMs). In contrast, internalization of LAM microspheres by monocytes was minimal. Microsphere internalization by MDMs was visualized and quantitated by immunofluorescence and confocal and electron microscopy and resembled conventional phagocytosis. Phagocytosis of LAM microspheres by MDMs was energy, cytoskeleton, and calcium dependent and was mannan inhibitable. Trypsin treatment of MDMs at 37 degrees C, which depleted surface and recycling intracellular pools of the MR, reduced the subsequent attachment of LAM microspheres. Trypsin treatment at 4 degrees C allowed for subsequent recovery of LAM microsphere phagocytosis at 37 degrees C by recycled MRs. Pretreatment of MDMs with cycloheximide influenced LAM microsphere phagocytosis to only a small extent, indicating that MR-dependent phagocytosis of the microspheres was occurring primarily by preformed recycled receptors. This study characterizes the requirements for macrophage phagocytosis of a LAM-coated particle mediated by the MR. This model will be useful in further characterization of the intracellular pathway taken by phagocytic particles coated with different LAM types in macrophages following ingestion.

Coiling phagocytosis discriminates between different spirochetes and is enhanced by phorbol myristate acetate and granulocyte-macrophage colony-stimulating factor.

The mechanisms involved in coiling phagocytosis are not yet known, and it is not even clear whether this phenomenon is either an incidental event or a specific response. Therefore, the phagocytic uptake of Borrelia burgdorferi and other spirochetes by human monocytes in vitro was used to investigate the involvement of both sides--microbes and phagocytes--in coiling phagocytosis. As seen with electron microscopy, morphologically similar Borrelia, Leptospira and Treponema strains induced markedly different frequencies of coiling phagocytosis. The monocytes used coiling phagocytosis for both live (motile) and killed (nonmotile) B. burgdorferi, but pseudopod coils were observed neither with fragmented B. burgdorferi nor with cell-free supernatant from B. burgdorferi cultures. Investigation of the relationship of coiling phagocytosis with other pseudopod-based cellular mechanisms revealed that the use of bioreagents that inhibit conventional phagocytosis also inhibited coiling phagocytis but did not affect membrane ruffling. Bioreagents that increase membrane ruffling did not affect phagocytosis of B. burgdorferi, except for granulocyte-macrophage colony-stimulating factor and phorbol myristate acetate, which increased coiling phagocytosis selectively. These results demonstrate that coiling phagocytosis is not induced by microbial motility, viability, or a certain morphology and that it is not a random event. Rather, it is a selective uptake mechanism actively driven by the phagocytes. However, whether coiling phagocytosis represents an independent alternative to conventional phagocytosis or, alternatively, a fault in conventional phagocytosis remains to be determined.

Patients with chronic pancreatitis have an impaired oxidative burst ability of blood monocytes

The destruction by phagocytic cells of ingested microorganisms is best ensured by an intact respiratory burst with production of superoxide anion and other metabolites. The aim of the present study was to investigate the status of the reticulo-endothelial system, as assessed by superoxide anion generation of blood monocytes, in 18 patients with chronic pancreatitis (CP) as compared to 15 with chronic renal failure (CRF), 14 with diabetes mellitus (DM) and to 20 healthy volunteers. Macrophage suspensions (1 × 10 6) were prepared from blood samples withrawn after an overnight fast were tested for conventional phagocytosis function (carbon partical ingestion: percent and intensity) and for superoxide anion generation (chemiluminescent activation: cpm of light integrated intensity). Conventional phagocytosis function tests did not show any differemce among the groups examined. However, the peak value of generated susperoxide anion (× 10 4 cpm) was significantly lower in CP patients as compared to either healthy controls (14.9 ± 4.3 vs. 28.3 ± 4.7, P < 0.001) and CRF (24.8 ± 3.9, P < 0.01) and DN patients (22.8 ±_4.9, P < 0.05). Further, the time elapsed to generate such a peak was significantly longer in CP patients (374 ± 106 s) than in controls (208 ± 35 s, P < 0.01). This study suggests that patients with CP present a defective monocytes burst ability which is unrelated to flare-up of the disease and to the underlying malnutrition. Further, this phenomenon appears to occure to a far greater extent as compared to other long-standing chronic illnesses.

Salmonella stimulate macrophage macropinocytosis and persist within spacious phagosomes.

Light microscopic studies of phagocytosis showed that Salmonella typhimurium entered mouse macrophages enclosed in spacious phagosomes (SP). Viewed by time-lapse video microscopy, bone marrow-derived macrophages exposed to S. typhimurium displayed generalized plasma membrane ruffling and macropinocytosis. Phagosomes containing Salmonella were morphologically indistinguishable from macropinosomes. SP formation was observed after several methods of bacterial opsonization, although bacteria opsonized with specific IgG appeared initially in small phagosomes that later enlarged. In contrast to macropinosomes induced by growth factors, which shrink completely within 15 min, SP persisted in the cytoplasm, enlarging often by fusion with macropinosomes or other SP. A Salmonella strain containing a constitutive mutation in the phoP virulence regulatory locus (PhoPc) induced significantly fewer SP. Similar to Yersinia enterocolitica, PhoPc bacteria entered macrophages in close-fitting phagosomes, consistent with that expected for conventional receptor-mediated phagocytosis. These results suggest that formation of SP contributes to Salmonella survival and virulence.

Hypoexpression of major histocompatibility complex molecules on Legionella pneumophila phagosomes and phagolysosomes

Legionella pneumophila is a facultative intracellular pathogen that parasitizes host mononuclear phagocytes. Cell-mediated immunity is pivotal to host defense against L. pneumophila, and the infected host cell may play a central role in processing and presenting parasite antigens to lymphocytes mediating cell-mediated immune response. However, in the case of L. pneumophila and intracellular parasites in general, little is known about the intracellular trafficking of parasite antigens, the influence of parasite infection on major histocompatibility complex (MHC) expression, or the relationship of MHC molecules to sites of parasite replication. To learn more about this, we have used flow cytometry to study the expression of HLA-DR by monocytes infected with L. pneumophila and cryosection immunogold electron microscopy to study the distribution of MHC class I and II molecules on L. pneumophila phagosomes. Flow cytometry analysis demonstrated that L. pneumophila infection has little effect on the overall expression of HLA-DR by monocytes. Cryosection immunogold studies revealed abundant staining for MHC class I and II molecules on the plasma membrane of infected monocytes but little or no staining on the membranes of mature L. pneumophila phagosomes. Cryosection immunogold studies of an avirulent mutant of L. pneumophila that, unlike the wild type, does not inhibit phagosome-lysosome fusion and subsequently survives but does not multiply in a phagolysosome yielded similar results. We have previously found that MHC class I and II molecules are excluded from nascent phagosomes during coiling and conventional phagocytosis. The present work demonstrates that MHC molecules do not accumulate appreciably in the L. pneumophila phagosome as it matures and at a point in the life cycle of the organism in which it is replicating and producing immunoprotective T-cell antigens. This suggests that L. pneumophila does not reside in a typical endosomal compartment in the host cell and that L. pneumophila antigens may encounter MHC molecules at extraphagosomal sites within the host cell.

Coiling phagocytosis is the preferential phagocytic mechanism for Borrelia burgdorferi.

The uptake mechanism for the spirochete Borrelia burgdorferi, the causative agent of Lyme disease, was investigated by electron microscopy for human and murine phagocytes. Spirochetes of both a low- and a high-passage strain were preferentially internalized by coiling rather than by conventional phagocytosis. The spirochetes engulfed by coiling phagocytosis were found to disintegrate in an organelle exclusion zone without evident participation of lysosomes. Preincubation of B. burgdorferi with monoclonal antibody to the spirochetal OspA enhanced phagocytosis in general but did not consistently influence the uptake mechanism. Quantitative and kinetic differences concerning the phagocytic rate and mechanism were evident between cells from different lineages, different human individuals, and mice and humans. In general, when few phagocytes participated in spirochete uptake, the active cells displayed a high ratio of coiling versus conventional phagocytosis. These results suggest that coiling phagocytosis of B. burgdorferi plays a critical role in the control of spirochetal infection. More detailed studies on the molecular basis of this phagocytic mechanism may lead to new insights into the pathogenesis of Lyme borreliosis, a disease which is frequently characterized by the host's inability to eliminate the pathogenic spirochete.

S-layer-mediated association of Aeromonas salmonicida with murine macrophages.

The interaction of Aeromonas salmonicida with the murine macrophage (M phi) cell line P388D1 was used as a convenient model to study the involvement of the bacterial crystalline surface array (or A-layer) in the association with M phi s. A-layer-positive (A+) cells readily associated with M phi s in phosphate-buffered saline, whereas A- mutants were unable to do so, even when the bacterium-M phi interaction was forced by centrifugation. M phi s selectively interacted with A+ cells when challenged with mixtures of A+ and excess A- cells. Electron microscopy indicated that in phosphate-buffered saline only A+ bacteria were readily internalized, although by a nonconventional mechanism, suggesting that efficient phagocytosis in the absence of opsonins was A-layer mediated. Latex beads coated with a partially assembled A-layer were more efficiently taken up than uncoated or A-protein-coated beads, indicating that an organized A-layer was essential for M phi uptake. The reduced ability of M phi s plated on a substratum coated with the A-layer to bind A+ bacteria also suggested that association was both A-layer and receptor mediated. In the presence of tissue culture medium, competent M phi s interacted efficiently with A- bacteria and internalized them through conventional phagocytosis. A+ cells were markedly cytotoxic to M phi s, whereas the A-protein or A-layer was not. A- cells were cytotoxic to a lesser extent, suggesting that cytotoxicity was targeted.

Membrane sorting during phagocytosis: selective exclusion of major histocompatibility complex molecules but not complement receptor CR3 during conventional and coiling phagocytosis.

We have used immunocytochemical techniques and enzyme cytochemistry to examine the distribution of plasma membrane proteins during coiling phagocytosis of Legionella pneumophila and conventional phagocytosis of Escherichia coli. Whereas class I and class II major histocompatibility complex (MHC) molecules are relatively excluded from nascent phagosomes during conventional and coiling phagocytosis, the CR3 complement receptor persists in nascent phagosomes. The staining pattern for alkaline phosphatase activity resembles that of MHC molecules, with a marked exclusion of phosphatase activity from L. pneumophila coils and nascent phagosomes. The staining pattern for 5'-nucleotidase activity, on the other hand, resembles that of CR3 with intense staining in the inner layers of L. pneumophila coils. These results demonstrate that the cell has the ability to exclude selectively certain membrane proteins from the nascent phagosome during phagocytosis, thereby producing a phagosomal membrane markedly different from the plasma membrane from which it is derived.

Phagocytosis of leprosy bacilli is mediated by complement receptors CR1 and CR3 on human monocytes and complement component C3 in serum.

Mycobacterium leprae, an obligate intracellular pathogen, invades and multiplies within host mononuclear phagocytes. To understand M. leprae invasion better, we have investigated the role of phagocyte receptors and bacterium-bound ligands in phagocytosis of M. leprae by human monocytes. Complement receptors CR1 and CR3 mediate adherence and phagocytosis of M. leprae in nonimmune serum. Two MAbs used in combination against CR3 inhibit adherence by up to 90 +/- 3%. Two MAbs used in combination against CR1 and CR3 inhibit adherence by up to 70 +/- 1%. Single MAbs against CR1 or CR3 consistently inhibit adherence by 38-55%. In contrast, MAbs against other monocyte surface molecules, alone or in combination, do not significantly influence adherence. As studied by electron microscopy, 100% of monocyte-associated M. leprae are ingested in the presence of nonimmune serum and MAbs against CR3 markedly inhibit ingestion. Complement receptors CR1 and CR3 also mediate the low level of adherence observed in the absence of serum. Serum complement component C3 serves as a ligand on the bacterial surface in monocyte phagocytosis of M. leprae. Adherence of M. leprae to monocytes is enhanced by preopsonization (3.1 +/- 1.1-fold increase) and is markedly reduced in less than 0.5% fresh serum (66 +/- 7% reduction) or heat-inactivated serum (68 +/- 3% reduction). Adherence is also markedly reduced in C3- or factor B-depleted serum; repletion with purified C3 or factor B increases adherence 4.3 +/- 0.8- and 2.6 +/- 0.2-fold, respectively. C3 is fixed to M. leprae by the alternative pathway of complement activation, as determined by a whole bacterial cell ELISA. By electron microscopy, monocytes ingest M. leprae by conventional phagocytosis. This study demonstrates that (a) human monocyte complement receptors CR1 and CR3 mediate phagocytosis of M. leprae; (b) complement component C3 on the bacterial surface serves as a ligand for complement receptors; (c) complement component C3 binds to M. leprae by the alternative pathway of complement activation; and (d) monocytes phagocytize M. leprae by conventional phagocytosis.

Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3.

We have examined the receptor-ligand interactions and the method of phagocytosis of virulent Mycobacterium tuberculosis by human monocytes. mAb against complement receptors (CR) inhibit adherence and phagocytosis of M. tuberculosis in fresh nonimmune serum. A mAb against the type 1 CR (CR1) inhibits adherence of M. tuberculosis by 40 +/- 5%, and three different mAb against the type 3 CR (CR3) each inhibit adherence by 39 +/- 5% to 47 +/- 4%. A mAb against CR1 used in combination with one of the three mAb against CR3 inhibits adherence by up to 64 +/- 7%. Most strikingly, two mAb used in combination against CR3 inhibit adherence by up to 81 +/- 2%. mAb against other monocyte surface Ag do not significantly influence adherence. In like fashion, mAb against CR but not other monocyte surface Ag inhibit adherence of preopsonized M. tuberculosis in the presence of heat-inactivated serum. By electron microscopy, monocytes ingest all M. tuberculosis that adhere in the presence of nonimmune serum; mAb against CR3 markedly inhibit ingestion. In contrast to CR, the FcR and the beta-glucan-inhibitable receptor for zymosan play little or no role in mediating M. tuberculosis adherence or ingestion. Adherence of M. tuberculosis is serum-dependent, requiring greater than or equal to 2.5% serum for optimal adherence. Heat inactivation of serum markedly reduces adherence of M. tuberculosis (75.5 +/- 7%) and preopsonization of bacteria enhances adherence by 2.9 +/- 0.4-fold. Adherence is also markedly reduced in C3- or factor B-depleted serum; repletion with C3 or factor B increases adherence by 2.1 +/- 0.4-fold and 1.86 +/- 0.05-fold, respectively. Fab anti-C3 IgG markedly inhibits monocyte adherence of preopsonized M. tuberculosis (71 +/- 1%). C component C3 is fixed to M. tuberculosis by the alternative C pathway as determined by a whole bacterial cell ELISA. Human monocytes ingest M. tuberculosis by conventional phagocytosis as viewed by electron microscopy. This study demonstrates that human monocyte CR1 and CR3 mediate phagocytosis of M. tuberculosis and C component C3 in serum is acting as the major bacterium-bound ligand.