Experimental salmonellosis. VII. In vitro transfer of cellular immunity by ribosomal fraction of mouse mononuclear phagocytes

Sato, Ichiei (Gunma University, Maebashi, Japan), and Susumu Mitsuhashi. Experimental salmonellosis. VII. In vitro transfer of cellular immunity by ribosomal fraction of mouse mononuclear phagocytes. J. Bacteriol. 90:1194-1199. 1965.-The mononuclear phagocytes (termed monocytes) of mice hyperimmunized with live vaccine of Salmonella enteritidis inhibited the intracellular growth of virulent strain 116-54 of S. enteritidis. Also, the monocytes withstood the degeneration of cells caused by the phagocytosis of bacteria in the absence of immune serum in the tissue culture medium, termed cellular immunity. When the nonimmune monocytes were incubated with the ribosomal fraction of immune monocytes, obtained from the abdominal cavity of mice hyperimmunized with live vaccine of S. enteritidis, they acquired cellular immunity, but the monocytes did not acquire immunity when ribosomal fractions from normal mouse monocytes or from the monocytes of mice immunized with killed vaccine of S. enteritidis were used. The transfer agent present in the ribosomal fraction of immune monocytes was inactivated by treatment with ribonuclease but not with deoxyribonuclease or with trypsin.     

Experimental Salmonellosis XI. Induction of Cellular Immunity and Formation of Antibody by Transfer Agent of Mouse Mononuclear Phagocytes

When mice were injected intraperitoneally with a ribonucleic acid (RNA) preparation extracted from the peritoneal mononuclear phagocytes (termed monocytes) of immunized mice, these macrophages developed cellular immunity and cellular antibodies. The peritoneal monocytes were obtained from normal mice and maintained in tissue culture bottles in a homogeneous cell population. When they were treated in vitro with an immune RNA preparation, they acquired cellular immunity, and cellular antibodies were detectable in such monocytes. These results suggest that the mononuclear phagocytic cell line constitutes a cell line responsible for antibody formation.     

Experimental Salmonellosis X. Cellular Immunity and Its Antibody in Mouse Mononuclear Phagocytes

A cell-associated antibody was detected in the peritoneal mononuclear phagocytes (referred to as monocytes) of mice hyperimmunized with live vaccine of Salmonella enteritidis, by use of immune transfer and immune adherence hemagglutination techniques. The cellular antibody inhibited the growth of a virulent strain of S. enteritidis with the aid of complement and lysozyme on nutrient agar plates. This type of bactericidal antibody could not be detected in the monocytes of mice immunized with killed vaccine of S. enteritidis. The antibody extracted from the peritoneal monocytes of mice hyperimmunized with live vaccine was identified as a macroglobulin by ultracentrifugal analysis.     

Temporin A and related frog antimicrobial peptides use formyl peptide receptor-like 1 as a receptor to chemoattract phagocytes

Many mammalian antimicrobial peptides (AMPs) have multiple effects on antimicrobial immunity. We found that temporin A (TA), a representative frog-derived AMP, induced the migration of human monocytes, neutrophils, and macrophages with a bell-shaped response curve in a pertussis toxin-sensitive manner, activated p44/42 MAPK, and stimulated Ca(2+) flux in monocytes, suggesting that TA is capable of chemoattracting phagocytic leukocytes by the use of a G(ialpha) protein-coupled receptor. TA-induced Ca(2+) flux in monocytes was cross-desensitized by an agonistic ligand MMK-1 specific for formyl peptide receptor-like 1 (FPRL1) and vice versa, suggesting that TA uses FPRL1 as a receptor. This conclusion was confirmed by data showing that TA selectively stimulated chemotaxis of HEK 293 cells transfected with human FPRL1 or its mouse ortholog, murine formyl peptide receptor 2. In addition, TA elicited the infiltration of neutrophils and monocytes into the injection site of mice, indicating that TA is also functionally chemotactic in vivo. Examination of two additional temporins revealed that Rana-6 was also able to attract human phagocytes using FPRL1, but temporin 1P selectively induced the migration of neutrophils using a distinct receptor. Comparison of the chemotactic and antimicrobial activities of several synthetic analogues suggested that these activities are likely to rely on different structural characteristics. Overall, the results demonstrate that certain frog-derived temporins have the capacity to chemoattract phagocytes by the use of human FPRL1 (or its orthologs in other species), providing the first evidence suggesting the potential participation of certain amphibian antimicrobial peptides in host antimicrobial immunity.     

Lysophospholipid regulation of mononuclear phagocytes

Blood monocytes and tissue macrophages derived from monocyte differentiation in tissues are central elements of innate immunity in host defense against numerous pathogens and other challenges. These mononuclear phagocytes also participate in wound healing and normal tissue remodeling in development and growth. Pathological perversion of their physiological roles leads to participation of mononuclear phagocytes in fibrosing diseases including granulomatous disorders, chronic inflammation typical of arthritis, and atherosclerosis. Lysophospholipids, including lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), are platelet-derived lipid growth factors considered to participate in leukocyte differentiation and activation. This section summarizes our recent observations of the effects of lysophospholipids on mononuclear phagocytes.     

Effects of human peripheral blood monocytes,monocyte-derived macrophages,and spleen mononuclear phagocytes on Toxoplasma gondii

In vitro effects of human peripheral blood monocytes, peripheral blood monocyte-derived macrophages, and spleen mononuclear phagocytes on Toxoplasma gondii were studied. In almost all instances, over 80% of human monocytes and monocyte-derived macrophages infected with Toxoplasma in vitro destroyed the organism. Degeneration of intracellular Toxoplasma was not due to decreased viability of organisms in the challenge inoculum. Human monocytes did not elaborate into the culture medium substances which altered the capacity of Toxoplasma to survive and replicate within mouse macrophages. The early reduction in intracellular Toxoplasma was not affected by inhibitors of various intracellular processes or by diseases associated with altered cellular immunity (sarcoidosis, infectious mononucleosis, or lymphoma.) The Toxoplasma that remained after 6 hr within human monocytes and macrophages multiplied. This multiplication was observed both microscopically and in a radioassay which detects uptake of [³H]uracil or [³H]deoxyuridine into nucleic acids of intracellular Toxoplasma. Intracellular Toxoplasma in monocytes cultured with poly(I:C) or in monocyte-derived macrophages cultured with lymphokines showed decreased uptake of radiolabeled precursors into nucleic acids of intracellular Toxoplasma. Treatment of monocytes with endotoxin did not alter nucleic acid synthesis of surviving intracellular Toxoplasma. These results suggest that human mononuclear phagocytes in peripheral blood and in tissue (spleen) have the capacity to eliminate a large percentage of the Toxoplasma that they ingest or that invade them. The inhibition of nucleic acid synthesis of remaining Toxoplasma by exposure of monocyte-derived macrophages to lymphokines suggests that lymphocyte products may be important for elimination of the Toxoplasma that remain and multiply within a small proportion of mononuclear phagocytes.     

Neutral proteinases of human mononuclear phagocytes. Cellular differentiation markedly alters cell phenotype for serine proteinases, metalloproteinases, and tissue inhibitor of metalloproteinases

Mononuclear phagocytes have the capacity to directly participate in extracellular matrix turnover via secretion of neutral proteinases. We have studied the effects of in vivo and in vitro differentiation upon cellular content or secretion of a spectrum of neutral proteinases, along with a counter-regulatory metalloproteinase inhibitor (TIMP). We found 1) matrix-degradative serine proteinases (leukocyte elastase and cathepsin G) were lost during cellular maturation and/or differentiation; 2) the 92-kDa type IV/type V collagenase and TIMP were secreted earliest in mononuclear phagocyte differentiation, whereas stromelysin secretion was observed only by LPS-stimulated alveolar macrophages; 3) exposure of alveolar macrophages, but not monocytes, to phorbol esters and LPS resulted in markedly augmented secretion of all studied metalloproteinases and TIMP; 4) monocyte-derived macrophages partially (but not completely) mimicked the metalloproteinase secretory phenotype of alveolar macrophages; and 5) the secretory phenotype of alveolar macrophages for interstitial collagenase (but not TIMP) was largely lost during in vitro culture. These results underscore the complexity of the process of differentiation in human mononuclear phagocytes, and provide insights into the variable capacity of mononuclear phagocytes to degrade extracellular matrix components. Moreover, we anticipate that human mononuclear phagocytes at various stages of differentiation will provide a useful model system for study of the variable regulation of secretion of human matrix-degrading metalloproteinases.     

Effect of endotoxin on the production of colony-stimulating factor by human monocytes and macrophages

Colony-stimulating factor (CSF) is necessary for the clonal growth of human bone marrow in vitro. Human blood monocytes and macrophages produce CSF. Endotoxin was found to increase the level of CSF generated by macrophages, but had no stimulatory effect on monocytes. Several other substances known to influence the pinocytic or phagocytic activity of mononuclear phagocytes failed to enhance cellular CSF generation.     

Macrophages in resistance to candidiasis.

Candida albicans, an increasingly common opportunistic pathogenic fungus, frequently causes disease in immunodeficient but not immunocompetent hosts. Clarifying the role of the phagocytic cells that participate in resistance to candidiasis not only is basic to understanding how the host copes with this dimorphic pathogen but also will expedite the development of innovative prophylactic and therapeutic approaches for treating the multiple clinical presentations that candidiasis encompasses. In this review, we present evidence that a diverse population of mononuclear phagocytes, in different states of activation and differentiation and from a variety of host species, can phagocytize C. albicans blastoconidia via an array of opsonic and nonopsonic mechanisms and can kill C. albicans blastoconidia and hyphae by means of oxygen-dependent and -independent mechanisms. Reactive nitrogen intermediates should now be added to the well-established candidacidal reactive oxygen intermediates of macrophages. Furthermore, what were thought to be two independent pathways, i.e., nitric oxide and superoxide anion, have now been shown to combine to form a potent macrophage candidacidal molecule, peroxynitrite. In contrast to monocytes and neutrophils, which are important in resistance to early stages of C. albicans infections, more differentiated macrophages activated by cytokines such as gamma interferon participate in the acquired resistance of hosts with C. albicans-specific, cell-mediated immunity. Evidence presented in this review demonstrates that mononuclear phagocytes, in some instances in the absence of other professional phagocytes such as neutrophils, play an import role in resistance to systemic and mucosal candidiasis.     

Uptake of lactoferrin by mononuclear phagocytes inhibits their ability to form hydroxyl radical and protects them from membrane autoperoxidation.

Human mononuclear phagocytes do not contain the iron-binding protein lactoferrin that we have previously demonstrated inhibits the potential for human neutrophils to generate hydroxyl radical in the presence of an exogenous iron catalyst of the Haber-Weiss reaction. Previous work by other investigators has suggested that mononuclear phagocytes (monocytes and monocyte-derived macrophages (MDM] have the capacity to bind exogenous lactoferrin via lactoferrin-specific membrane surface receptors. Accordingly, we examined the possibility that uptake of iron-free (apo) lactoferrin by human mononuclear phagocytes could play a role in limiting the potential for generation of hydroxyl radical during the monocyte/MDM respiratory burst. When monocytes or MDM were incubated in the presence of apo-lactoferrin, cell-associated lactoferrin increased in proportion to the concentration of lactoferrin provided. Similar results were obtained with iron-loaded (diferric) milk lactoferrin. Consistent with the in vivo importance of these findings, we found that lactoferrin was intimately associated with human alveolar macrophages obtained by bronchoalveolar lavage. The fucose polymer fucoidan inhibited lactoferrin uptake whereas exogenous transferrin or MDM exposure to IFN-gamma was without effect. Scatchard binding analysis confirmed the presence of a lactoferrin-specific receptor with a calculated kDa of 3.56 x 10(-6) M and 3.4 x 10(7) binding sites per cell. Subcellular fractionation studies indicated that twofold more of the lactoferrin which became cell-associated over the 1-h incubation time could be found in the cytoplasmic fraction compared to the plasma membrane-containing fraction, consistent with previous evidence by others for internalization of lactoferrin by mononuclear phagocytes. When lactoferrin-loaded monocytes/MDM were incubated in lactoferrin-free media, evidence for release of lactoferrin was obtained by SDS-PAGE and immunoblot analysis, suggesting the presence of a recyclable pool of cell-associated lactoferrin. To assess the impact of lactoferrin loading on monocyte/MDM hydroxyl radical formation, lactoferrin-loaded phagocytes were stimulated with PMA in the presence of catalytic iron. Hydroxyl radical generation by lactoferrin-loaded cells was decreased to about 50% of control cells. Similarly, monocytes that had been lactoferrin-loaded demonstrated a 28% decrease in autooxidation of their membrane when stimulated in the presence of catalytic iron. These data suggest that lactoferrin binding may play an important role in maintaining optimal mononuclear phagocyte function and protecting adjacent tissue from untoward phagocyte-associated hydroxyl radical generation.     

The neurotoxic prion peptide fragment PrP(106-126) is a chemotactic agonist for the G protein-coupled receptor formyl peptide receptor-like 1

Prion diseases are transmissible and fatal neurodegenerative disorders which involve infiltration and activation of mononuclear phagocytes at the brain lesions. A 20-aa acid fragment of the human cellular prion protein, PrP(106-126), was reported to mimic the biological activity of the pathologic isoform of prion and activates mononuclear phagocytes. The cell surface receptor(s) mediating the activity of PrP(106-126) is unknown. In this study, we show that PrP(106-126) is chemotactic for human monocytes through the use of a G protein-coupled receptor formyl peptide receptor-like 1 (FPRL1), which has been reported to interact with a diverse array of exogenous or endogenous ligands. Upon stimulation by PrP(106-126), FPRL1 underwent a rapid internalization and, furthermore, PrP(106-126) enhanced monocyte production of proinflammatory cytokines, which was inhibited by pertussis toxin. Thus, FPRL1 may act as a "pattern recognition" receptor that interacts with multiple pathologic agents and may be involved in the proinflammatory process of prion diseases.     

Spontaneous aggregation as a mechanism for human monocyte purification

A previously unreported property of human mononuclear phagocytes is the ability of these cells to spontaneously aggregate. Fresh mononuclear cells obtained after plateletpheresis were noted to spontaneously form large cellular aggregates. Dual parameter immunofluorescence analysis demonstrated that the aggregating cells were positive for the monocyte marker CD11 (complement receptor, type 3) but were negative for the lymphocyte marker CD3 (T3 antigen). In addition, less than 5% of the nonaggregating cells were CD11+, suggesting that almost all CD11+ cells aggregated. Cellular aggregates were independent of cell concentration and formed more efficiently at 4 degrees C than at either 22 or 37 degrees C. Based on these observations, a purification procedure utilizing Ficoll-Hypaque separation, spontaneous aggregation at 4 degrees C, and transient plastic adherence resulted in a sevenfold enrichment of the CD11+ peripheral blood monocytes. Purified monocytes were contaminated with less than 2% CD3 cells. The size, growth, and adherence characteristics as well as cytologic stains indicated that the monocytes were not significantly altered by the purification procedure. Thus, spontaneous aggregation is an efficient and convenient method for the isolation of large numbers of purified monocytes.     

Changes in cellular dry mass during macrophage development.

Measurements of dry mass were made on developing human macrophages in suspension culture using a scanning and integrating microinterferometer. The mean dry mass (+/- SEM) of day 0 monocytes of 19 healthy individuals was 53.6 +/- 2.4 pg. There was a significant increase in cellular dry mass over the period of culture, mean values at 2, 4 and 6 days being 61.1, 79.8 and 110.4 pg, respectively. A comparison of the results with those obtained previously by ultrastructural morphometry inferred that there is a disproportionate increase in the water content of macrophages during development. The technique may have potential for investigating changes in mononuclear phagocytes in clinical states.     

EXPRESSION OF MONOCYTE CHEMOTACTIC PROTEIN-3 IN HUMAN MONOCYTES EXPOSED TO THE MYCOBACTERIAL CELL WALL COMPONENT LIPOARABINOMANNAN

Monocyte chemotactic protein-3 (MCP-3) is a C–C chemokine which interacts with the CCR1, CCR2 (MCP-1) and CCR3 receptors and has a distinct spectrum of action. The present study was designed to assess whether mycobacterial components were able to induce expression and production of MCP-3 in human monocytes. Mycobacterial lipoarabinomannan (LAM) induced expression of MCP-3 mRNA in human peripheral blood mononuclear cells. The non-mannose-capped version of lipoarabinomannan (AraLAM) was considerably more potent than the mannose-capped version ManLAM or the simpler version phosphatidylinositol mannoside (PLM). Among mononuclear cells, monocytes were responsible for LAM-induced MCP-3 mRNA expression. Whole mycobacteria (Mycobacterium bovisBCG) strongly induced MCP-3 expression. Pretreatment with actinomycin D abolished LAM-induced MCP-3 expression, whereas cycloheximide only partially reduced the expression. LAM-induced MCP-3 expression was associated with the production of immunoreactive PTX3. Interleukin 10 (IL-10) and IL-13 inhibited the induction of MCP-3 by LAM. Thus mycobacterial cell wall components induced expression of MCP-3 in human monocytes. MCP-3, a chemokine active on mononuclear phagocytes, NK cells, T cells and dendritic cells, may be relevant to the induction and expression of immunity against mycobacteria.     

Bacterial lipopolysaccharide, phorbol myristate acetate, and muramyl dipeptide stimulate the expression of a human monocyte surface antigen, Mo3e

Exposure of mononuclear phagocytes to bacterial lipopolysaccharide (LPS), phorbol myristate acetate (PMA), or muramyl dipeptide (MDP) is known to stimulate a variety of cellular activities that include increases in phagocytosis, oxidative metabolism, synthesis and secretion of monokines, and cytotoxicity of microbes and tumor cells. We now report that culture of human peripheral blood monocytes in medium containing LPS, phorbol compounds, or MDP also results in the acquired expression of a plasma membrane antigen. Mo3e, as identified by a murine monoclonal antibody. Mo3e is barely detectable (by immunofluorescence flow cytometry) on freshly isolated monocytes, but is expressed in high antigen density after exposure of cells to E. coli, Salmonella minnesota, or Serratia marcescens LPS (at concentrations exceeding 0.1 ng/ml), PMA (and other biologically active phorbol compounds) (0.5 to 1 X 10(-8) M), or MDP (0.01 to 1 X 10(-6) M). Mo3e expression stimulated by LPS is prevented by pretreatment of LPS with polymyxin B, suggesting that the lipid A portion of LPS is responsible for Mo3e induction (polymyxin B has no effect on Mo3e expression stimulated by PMA or MDP). Culture of monocytes in medium containing protein synthesis inhibitors (or at 4 degrees C) blocks the acquisition of Mo3e. Recombinant IFN-gamma, which is also known to "activate" mononuclear phagocytes, does not stimulate Mo3e expression, although both LPS and IFN induce enhanced expression of monocyte Ia antigen. Analogous to their stimulatory effect on monocytes, LPS and PMA induce Mo3e expression by the human monocytic cell line, U-937. On the basis of these observations, Mo3e may represent an immunologic marker for monocyte activation stimulated in vitro by LPS, PMA (and related compounds), and MDP.     

Respiratory syncytial virus infection of human mononuclear phagocytes stimulates synthesis of platelet-activating factor

Production of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine (PAF), a potent mediator of inflammation, by mononuclear phagocytes varies with their stage of cellular differentiation and the nature of the eliciting stimulus. The human monocytic cell line U937 can be induced to differentiate to a macrophage-like cell following phorbol myristate acetate exposure, and after differentiation, these cells efficiently support replication of respiratory syncytial virus (RSV). U937 cells induced to differentiate with phorbol myristate acetate demonstrated a time-dependent decrease in PAF synthesis. RSV infection of these differentiated U937 cells caused a sustained stimulation of PAF synthesis that paralleled viral replication and was dependent on infectious virus. Virus increased the activity of lyso-PAF:acetyl-CoA acetyl-transferase (PAF acetyltransferase) in cell lysates, thus enhancing the anabolic pathway of PAF synthesis without altering the activity of PAF acetylhydrolase, which regulates PAF catabolism. RSV infection of human monocytes also caused a marked increase in [3H] monocytes also caused to uninfected monocytes. Thus, virus infection serves as a novel stimulus to induce PAF synthesis in human mononuclear phagocytes and suggests that increased PAF production may have a critical role in the inflammatory response to RSV.