Fibronectin Promotes Binding But Not Ingestion of Agarose Beads by Mouse Macrophages and Human Monocytes

We have examined to what extent human fibronectin associated with agarose beads with a 5- to 10-μm diameter mediates binding and uptake of the heads by mouse macrophages and human monocytes. Native agarose beads preincubated with ¹²⁵I-fibronectin were neither associated with nor taken up by mouse macrophages after 30 min of incubation under serum-free conditions. When fibronectin was cross-linked to cyanogen bromide-activated agarose heads or incubated with gelatinized heads, this resulted in a significant increase in particle binding by macrophages and monocytes as compared with gelatinized beads, whereas the fraction of cells with ingested particles remained unaltered. Native agarose heads activated by cyanogen bromide and treated with ethanolamine were to a greater extent associated with and taken up by phagocytes than fibronectin- or gelatin-coated heads. Our results indicate thai fibronectin acts as an adhesive glycoprotein and not as an opsonin. Since agarose beads are activators of the alternative pathway of complement, and fibronectin is reported to bind to factor C3, we speculate that cell-derived C3b is bound to the beads and fibroneetin-coaled beads arc ingested by the phagocytes via complement C3b receptors on the cells.     

Complement (C3)-Receptor-Mediated Phagocytosis of Agarose Beads by Mouse Macrophages

The phagocytosis by macrophages of C3bi-coated agarose heads reached a plateau after 15 min, compared with 30 min for C3b-coated beads. By using ¹²⁵I-Iabelled C3bi or C3b coupled to the agarose beads, we found that 70% and 95% of total radioactivity were removed from the heads after 12 h and 36 h of intracellular digestion, respectively. Intracellular degradation of C3bi linked to agarose beads was also demonstrated by testing binding of monoclonal antibodies against human C3c, C3g and C3d to beads extracted from the cells after phagocytosis. Such extracted beads also showed reduced attachment to new macrophages compared with non-ingested beads. Treatment of the cells with leupeptin, an inhibitor of the lysosomal enzyme cathepsin B, or with dextran sulphate to inhibit phagosome-lysosome fusion greatly reduced the release of labelled protein from the agarose during the first 12 h. These findings show that C3bi and C3b on agarose is destroyed intracellularly by lysosomal enzymes.     

Complement (C3)-Receptor-Mediated Phagocytosis of Agarose Beads by Mouse Macrophages

Phagocytosis of agarose beads by macrophages cultured under serum-free conditions was studied. 48 h was needed before a plateau in the uptake was reached. The ingested agarose beads were coated extracellularly with macrophage-derived protein before attachment and ingestion of the beads. Intracellularly, the agarose-linked protein was removed from the agarose. If the ingested agarose beads were extracted from the macrophages within 24 h after the plateau in the uptake was reached, a fraction of the beads could attach to new macrophages, demonstrating modification of the agarose beads by opsonin(s). Because of binding of anti-human C3c antibodies to beads extracted from the macrophages after 24 h of phagocytosis and the trypsin sensitivity of the protein on the agarose, we conclude that the main opsonin on the agarose beads is C3bi. Requirements for the stimulatory effect of agarose on macrophages are summarized.     

Complement (C3) Receptor-Mediated Attachment of Agarose Beads to Mouse Peritoneal Macrophages and Human Monocytes

We have determined the receptors on human monocytes and mouse peritoneal macrophages producing agarose binding. By using isolated human complement factors C3, B and D, agarose beads were coated with C3b. In some experiments C3b was converted to C3bi by using human serum diluted 1:20. Agarose beads coated with C3b or C3bi bound strongly to monocytes. Only agarose beads coated with C3bi were attached to mouse macrophages. Trypsinization of agarose beads coated with C3bi abolished the attachment of the beads to macrophages and monocytes, probably because of conversion of C3bi to C3d. Endocytosis by macrophages of agarose preincubated in human serum or in C5-deficient AKR mouse serum reached the same levels, indicating that the amount of C5 present in serum during preincubation is not important for the degree of endocytosis. It is concluded that internalization of agarose by macrophages is mediated via the C3bi receptor.     

Endocytosis of Agarose in Mouse Peritoneal Macrophages in Vitro

We have studied the endocytosis of tritium-labelled and of non-radioactive agarose in mouse macrophages in vilro. The endocytosis was greatest and most rapid in syngeneic mouse serum and in human serum, reaching a plateau after 12 h of incubation. Ten per cent serum was the minimum concentration giving optimal ingestion. The endocytosis appeared to be regulated by mechanisms involving complement factors C3 and B. Different pretreatments of sera, inactivating or depleting C3 and B, resulted in 70–80% reduction of endocytosis, Preincubation of agarose in untreated serum increased the endocytosis of agarose in heat-inactivated serum three-fold, indicating that the essential factors were bound to agarose. Antibodies against C3 and B reduced endocytosis moderately hut significantly.     

Evidence that Agarose Must Be Internalized to Stimulate Mouse Macrophages in Vitro

Agarose stimulation of macrophages in vitro was studied. Under conditions where agarose was ingested, stimulation was detected during 24-48 h of incubation at a time when the agarose increasingly was concentrated in the perinuclear region. Removal of extracellular agarose after 24 h when endocytosis had reached a plateau did not reduce the stimulatory effect. Preincubation for 4 days with dextran sulphate in concentrations reported to inhibit phagosome-lysosome fusion potentiated strongly the stimulatory effect. In all situations in which agarose was not internalized--in teflon tubes where the cells remain in suspension, on glass cover slips with inhibitors (2-deoxy-D-glucose, cytochalasin B), or with large, noningestible Sepharose beads--no stimulation was recorded. The possibility is discussed that stimulation of macrophages by agarose may be related to complement activation in phagosomes.     

In Vivo Activation of Mouse Macrophages with β-l,3-D-Glucan-Derivatized Plastic Beads

Macrophages obtained from animals treated with beta-1,3-D-glucan-derivatized plastic beads were greatly stimulated, as judged by morphology, esterase release, and cytostatic effect on L-929 tumour cells in vitro. The pretreatment of mice with such beads conferred an apparent absolute local resistance to an otherwise lethal pneumococcal infection but had no effect on the growth of intraperitoneal AA ascites sarcoma. Moreover, peritoneal cells from animals pretreated with glucan beads did not protect the animals in a Winn assay.     

Serum and Plasma Fibronectin Binds to Complement Reacted Immune Complexes Primarily via C1q

The binding of fibronectin to human Clq, C3b, and complement-reacted immune complexes (IC) was investigated by enzyme-linked immunosorbent assays. Microplates were coated with BSA followed by incubation with rabbit-anti-BSA IgG or F(ab')2 fragments of rabbit anti-BSA. Incubation of the solid phase with serum at 37 degrees C caused attachment of Clq and C3b. Addition of EDTA to the serum inhibited the binding of C3b, but not Clq, whereas substitution of the anti-BSA IgG on the solid phase with the F(ab')2 fragments abrogated the Clq, but not the C3b binding. Fibronectin binding was observed after incubation of the solid-phase IC with serum or plasma at conditions where Clq was also bound, whereas only minor amounts of fibronectin bound to the solid phase IC via C3b. Purified fibronectin showed a dose-dependent binding to solid-phase IC pretreated with Clq or fibronectin-depleted serum, confirming that the binding of fibronectin to IC largely occurred via Clq. Significantly smaller amounts of fibronectin were bound to solid-phase IC incubated with plasma instead of serum, despite the higher fibronectin concentration in plasma. This difference was found not to be due to a fibrinogen-fibronectin interaction in plasma. Binding of fibronectin to preformed fluid phase IC incubated with serum was demonstrated by SDS-PAGE analysis of PEG-precipitated IC.     

Phagocytosis of Agarose Beads by Receptors for C3b (CR1) and iC3b (CR3) on Human Alveolar Macrophages Cultured on Fibronectin in Vitro

We studied the phagocytosis of agarose beads by human alveolar macrophages in terms of the morphology, the receptors involved, and the cellular substrates (plastic or fibronectin) used. Beads coated with C3b (58%) and iC3b (42%) by treatment with serum, were ingested during 45 min by CR1 and CR3 on the macrophages. This ingestion was inhibited 80-90% by the presence of polyclonal F(ab')2 anti-C3 fragments. Since the phagocytosis of both C3b- and iC3b-coated beads was about threefold stronger than for C3b-coated beads (trypsinized serum-treated beads), the results indicate that the CR3 is more phagocytic than the CR1. The phagocytosis of initially complement uncoated beads, which are slowly opsonized with macrophage-produced C3b and iC3b in vitro, was also strongly inhibited (70-80%) by the presence of anti-human C3 F(ab')2 fragments. There was an increased phagocytosis (10-17%) of complement precoated beads by macrophages cultured on the fibronectin substrate versus the plastic substrate. The morphology and rapid phagocytosis of the complement precoated beads was demonstrated by SEM. The general impression was that membranous protrusions stretched towards the beads, which became increasingly enclosed by plasma membrane.     

The Macrophage Migration Inhibitory Factor Homolog of Entamoeba histolytica Binds to and Immunomodulates Host Macrophages

The host inflammatory response contributes to the tissue damage that occurs during amebic colitis, with tumor necrosis factor alpha (TNF-α) being a key mediator of the gut inflammation observed. Mammalian macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays an important role in the exacerbation of a wide range of inflammatory diseases, including colitis. We identified a MIF gene homolog in the Entamoeba histolytica genome, raising the question of whether E. histolytica MIF (EhMIF) has proinflammatory activity similar to that of mammalian MIF. In this report, we describe the first functional characterization of EhMIF. Antibodies were prepared against recombinantly expressed EhMIF and used to demonstrate that EhMIF is expressed as a 12-kDa protein localized to the cytoplasm of trophozoites. In a manner similar to that of mammalian MIF, EhMIF interacted with the MIF receptor CD74 and bound to macrophages. EhMIF induced interleukin-6 (IL-6) production. In addition, EhMIF enhanced TNF-α secretion by amplifying TNF-α production by lipopolysaccharide (LPS)-stimulated macrophages and by inhibiting the glucocorticoid-mediated suppression of TNF-α secretion. EhMIF was expressed during human infection, as evidenced by the presence of anti-EhMIF antibodies in the sera of children living in an area where E. histolytica infection is endemic. Anti-EhMIF antibodies did not cross-react with human MIF. The ability of EhMIF to modulate host macrophage function may promote an exaggerated proinflammatory immune response and contribute to the tissue damage seen in amebic colitis.     

Phagocytosis of Agarose Beads by Receptors for C3b (CR1) and iC3b (CR3) on Alveolar Macrophages from Patients with Sarcoidosis

Alveolar macrophages (AM) from sarcoidosis patients exhibit no detectable defect in their potential to synthesize the functional alternative and terminal pathway of complement. They also synthesize more C9 than AM from healthy controls. Various authors [4, 6] have suggested that sarcoid AM have decreased phagocytic ability. In the present work we studied whether there was any difference in C3 receptor-mediated phagocytosis of serum-treated and native agarose beads by AM recovered from patients with active sarcoidosis compared with controls, AM from seven patients with active sarcoidosis and seven healthy controls were cultured under serum-free conditions for 2, 12, 24. and 48 h. We found a significantly increased CR1 and CR3 receptor-mediated phagocytosis of native agarose heads by AM from the seven patients. CR1 and CR3 were also detected on AM directly recovered from bronchoalveolar lavage fluid using fluorescein-conjugated monoclonal anti-receptor antibodies. The percentage of AM expressing CR appeared to be increased in sarcoidosis. The reason for the enhanced phagocytosis of agarose beads by the sarcoid AM is probably the result of both increased synthesis and receptors of complement. Altered complement production and complement receptors may be important for the pathogenesis of this granulomatous disorder.     

Interactions of Thermoactinomyces vulgaris and Mouse Alveolar Macrophages

Interactions of mouse alveolar macrophages from three different inbred strains of mice and Thermoactinomyces vulgaris, a microbe associated with Farmer's lung disease, were studied. Alveolar macrophages were found to abolish the mitogenic activity of T. vulgaris. A prostaglandin synthesis inhibitor indomethacin, could not restore the activity. Alveolar macrophage supernatants generated by T. vulgaris treatment exerted strong suppression in secondary concanavalin A-induced lymphocyte transformation. Indomethacin partly relieved the suppression but a histamine 2 receptor blocker, cimetidine, had no effect. Interleukin 1 activity was practically undetectable by the thymocyte co-stimulation assay unless indomethacin was used. When indomethacin was used, interleukin 1 activity could be detected in all strains of mice tested. Major differences in the abolition of the mitogenic effect, in the suppressive effect, or in the release of interleukin 1 were not detected between inbred strains of mice tested. The results indicate that alveolar macrophages exert suppressive actions in vitro after T. vulgaris treatment but in vivo activities remain to be elucidated.     

Inhibition of Migration of Mouse Macrophages by Tuberculin-Sensitive Mouse Lymphocytes and by Mouse Migration Inhibitory Factor

The guinea pig migration inhibition technique, an accepted in vitro correlate of delayed hypersensitivity, has been adapted to a murine system. Peritoneal exudate cells from CF-1 mice vaccinated with viable cells of the H37Ra strain of Mycobacterium tuberculosis were inhibited in vitro by purified protein derivative (PPD) or whole H37Ra microorganisms. Peritoneal exudate cells from the inbred C57Bl/6 mice immunized with H37Ra cells also were inhibited in vitro by PPD or whole H37Ra microorganisms. Migration inhibitory factor (MIF) was produced by splenic lymphocytes from the H37Ra-immunized C57Bl/6 mice when incubated with either antigen. Intravenous injection of PPD or viable H37Ra organisms into H37Ra mice resulted in MIF production in vitro by splenic lymphocytes without further antigenic stimulation. Peritoneal exudate cells from nonimmunized C57Bl/6 mice and supernatant fluids from cultures of lymphocytes from nonimmunized C57Bl/6 mice were not inhibited in the presence of antigen. The production of MIF by splenic lymphocytes from immunized C57Bl/6 mice depended upon the conditions under which the lymphocytes were cultured, the time of exposure to antigen (3 days), the use of a higher concentration of PPD for stimulation of lymphocytes than that required for guinea pig cells, and also the use of cells from a highly inbred mouse strain.     

Mouse Hepatitis Virus 3 Binding to Macrophages Correlates with Resistance to Experimental Infection

Mouse hepatitis virus 3 (MHV3) infection of A/J and BALB/ c mice has been used as a model of resistance/susceptibility. A/J mice recover from a mild disease after 4–6 days of infection and the BALB/ c mice develop an acute hepatitis and die after 3–4 days of infection. In view of studying the MHV3 binding to cells or cell extracts, we performed an enzyme-linked immunosorbent assay-like virus-binding assay, preparing microplates with L929 cells, A/J or BALB/ c mouse macrophages and also with proteins extracted from these cells. Higher MHV3 bindings were observed to proteins of BALB/ c macrophages than to the A/J ones. The interferon-γ (IFN-γ) activation led to a reduction of MHV3 binding only to proteins of resistant A/J mouse macrophages. Our experiments contribute to the hypothesis that IFN-γ activation of macrophages plays an important role against MHV3 infection by downregulating the expression of viral receptors.     

Studies on Gonococcus Infection IX. In Vitro Decreased Association of Pilated Gonococci with Mouse Peritoneal Macrophages

Pili, in addition to enhancing attachment of gonococci to tissue culture cells, appear to reduce association (attachment/ingestion) of Neisseria gonorrhoeae with mouse peritoneal macrophages in vitro.     

Stimulation of Macrophages Increases, While Suppression of These Cells Inhibits Metastatic Dissemination of Two Transplantable Mouse Tumors in the Liver and Lungs

Stimulation of mouse tissue macrophages with carboxymethylated β-(1→3)-D-glycan 1 day before intravenous injection of tumor cells increased the number and weight of implants (experimental metastases) of mouse hepatocarcinoma and adenocarcinoma in the liver and lungs, respectively. Suppression of liver macrophages with gadolinium chloride or sequestration of cells during intraperitoneal administration of macrophage attractants inhibited metastatic dissemination of hepatocarcinoma and adenocarcinoma in the liver and lungs, respectively. In the latter case animal lifespan increased. Our results indicate that at certain stages of metastatic dissemination, activation of mononuclear phagocytes can stimulate the formation and growth of metastases. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 10, pp. 450–452, October, 2005     

Mouse Macrophages Are Permissive to Motile Legionella Species That Fail To Trigger Pyroptosis

Legionella pneumophila, a motile opportunistic pathogen of humans, is restricted from replicating in the lungs of C57BL/6 mice. Resistance of mouse macrophages to L. pneumophila depends on recognition of cytosolic flagellin. Once detected by the NOD-like receptors Naip5 and Ipaf (Nlrc4), flagellin triggers pyroptosis, a proinflammatory cell death. In contrast, motile strains of L. parisiensis and L. tucsonensis replicate profusely within C57BL/6 macrophages, similar to flagellin-deficient L. pneumophila. To gain insight into how motile species escape innate defense mechanisms of mice, we compared their impacts on macrophages. L. parisiensis and L. tucsonensis do not induce proinflammatory cell death, as measured by lactate dehydrogenase (LDH) release and interleukin-1β (IL-1β) secretion. However, flagellin isolated from L. parisiensis and L. tucsonensis triggers cell death and IL-1β secretion when transfected into the cytosol of macrophages. Neither strain displays three characteristics of the canonical L. pneumophila Dot/Icm type IV secretion system: sodium sensitivity, LAMP-1 evasion, and pore formation. Therefore, we postulate that when L. parisiensis and L. tucsonensis invade a mouse macrophage, flagellin is confined to the phagosome, protecting the bacteria from recognition by the cytosolic surveillance system and allowing Legionella to replicate. Despite their superior capacity to multiply in mouse macrophages, L. parisiensis and L. tucsonensis have been associated with only two cases of disease, both in renal transplant patients. These results point to the complexity of disease, a product of the pathogenic potential of the microbe, as defined in the laboratory, and the capacity of the host to mount a measured defense.Legionella pneumophila is a Gram-negative bacterium that opportunistically infects alveolar macrophages of the mammalian lung. Although 50 species of Legionella, comprising more than 70 serogroups, have been identified, only a subset have been associated with disease (39). The most prevalent cause of disease are serogroup 1 strains of Legionella pneumophila, which account for >75% of cases around the world. Non-L. pneumophila species contribute 5 to 10% of the disease burden, primarily in immunocompromised hosts. For example, the non-L. pneumophila species L. parisiensis and L. tucsonensis were each isolated from two renal transplant patients receiving immunosuppressive therapy (38, 69). Nevertheless, L. parisiensis strains can replicate in the human monocytic U937 cell line, in primary macrophages from C57BL/6 mice or guinea pigs, and in Acanthamoeba castellanii (3, 31, 33, 52). Whether these species use similar strategies to those of L. pneumophila to establish a replication niche or to evade the innate immune system is not known, since neither L. parisiensis nor L. tucsonensis has been studied in detail.Macrophages are key phagocytic defenders of the innate immune system that scout tissues for foreign materials, including pathogens. An important component of their surveillance are pattern recognition receptors that reside on the macrophage surface or within its cytoplasm. The Toll-like receptors (TLRs), present on the surfaces of many cell types, recognize microbe-associated molecular patterns (MAMPs), such as lipopolysaccharide, peptidoglycan, lipoproteins, microbial nucleic acids, and flagellin (2, 36). Much like the TLRs in function, the nucleotide-binding oligomerization domain (NOD-like) receptors (NLRs) monitor the cytoplasm (2, 78). Detection of microbial products by these receptors initiates a signaling cascade that culminates with the secretion of proinflammatory mediators that recruit other lymphocytes to respond to an infection (78).NLRs participate as components of a protein complex known as the inflammasome. Several different inflammasomes have been characterized based on the MAMPs that initiate their formation and activation. For example, Nalp1, Nalp3, and the more extensively studied NOD proteins each interact with muramyl dipeptides of peptidoglycan (2, 78). NLR family apoptosis inhibitory protein 5 (Naip5; also called Birc1e), which restricts replication of L. pneumophila in mouse macrophages (21, 23, 80), is composed of three domains: (i) an amino-terminal baculoviral inhibitor of apoptosis repeats, (ii) a central NOD domain, and (iii) carboxy-terminal leucine-rich repeats (LRRs) (32). Studies of other NLR proteins indicate that the LRR region is critical for recognition of microbial products, which then triggers oligomerization of the NLR with other inflammasome components through the NOD domain. Subsequently, activation of downstream signaling is carried out by the amino-terminal domains, such as the caspase recruitment domains (32). Unlike the NODs, the Nalps and Naip5 control posttranslational processing and secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18 (43). Once formed, the inflammasome recruits and activates caspase-1, which in turn processes pro-IL-1β and pro-IL-18 into their mature forms before their release into the extracellular space. Recruitment and activation of caspase-1 are mediated directly or through adapter proteins, such as ASC and Ipaf (1, 85). This proinflammatory reaction is a key element of pyroptosis, the “fiery” cell death seen in response to infection by the pathogens Salmonella enterica, Shigella flexneri, and L. pneumophila (11).L. pneumophila serogroup 1 has become a model intracellular organism to study both bacterial replication and host detection of pathogens (64). The pathogen requires a type IV secretion system to establish a replication vacuole in human and mouse macrophages, a key determinant of infection (29, 42, 74). However, mice can restrict L. pneumophila infection when the NLR proteins Naip5 and Ipaf detect flagellin (14, 20, 33, 49, 57, 81, 85). While C57BL/6 mice do not support L. pneumophila replication beyond the first 24 h (83), this bacterium replicates profusely in macrophages derived from the A/J mouse strain (82). The A/J Naip5 allele is hypofunctional, based on experiments using transgenic complementation, RNA knockdown, and Naip5^−/− mutant mice (21, 37, 80). A/J and Naip5 null mutant mice are defective for inflammasome activation during L. pneumophila infection (37, 49, 57). Likewise, L. pneumophila mutants that lack either flagellin (flaA) or type IV secretion (encoded by multiple dot/icm genes) do not stimulate pyroptosis. Whether components of the inflammasome directly bind flagellin is not yet known, but flaA mutant L. pneumophila subverts Naip5-mediated defenses and replicates in C57BL/6 macrophages (49, 57) and human macrophages (73).Unlike L. pneumophila serogroup 1, many other species of Legionella do replicate in C57BL/6 macrophages. These include strains of L. micdadei, L. bozemanii, L. dumoffii, L. feeleii, L. longbeachae, L. birminghamensis, L. maceachernii, L. sainthelensi, and L. parisiensis, as well as three non-serogroup 1 strains of L. pneumophila (7, 33, 48). With the exception of L. longbeachae (6, 7), the expression of flagellin and type IV secretion by these strains has not been reported. Therefore, we exploited two non-L. pneumophila strains to test the model that mouse macrophages rely on cytosolic flagellin to restrict growth of intracellular Legionella. We show that two motile strains of L. parisiensis and L. tucsonensis evade replication restriction by C5BL/6 murine macrophages, despite the host cells'' ability to detect their divergent bacterial flagellin when it contaminates the cytosol. Mouse macrophage restriction of replication correlates with three activities of the canonical L. pneumophila Dot/Icm type IV secretion system, namely, sodium sensitivity, late endosome and lysosome evasion, and phagosome perforation.     

Production of Prostaglandin E2 and Interleukin 1 by Mouse Peritoneal Macrophages Stimulated with β-l, 3-D-Glucan Derivatized Plastic Beads

Fluorescein-labelled plastic microbeads, with or without covalently attached beta-1,3-D-glucan, were injected into the peritoneal cavity of mice. Peritoneal cells were subsequently analysed by flow cytometry according to fluorescence and light scatter and separated into fluorescence positive and negative cells. We report that cells from animals treated with glucan-plastic beads produced large amounts of prostaglandin E2 (PGE2) whether the cells actually contained beads or not. On the other hand, cells from animals treated with glucan-plastic beads produced less thymocyte-stimulatory activity--presumably corresponding to interleukin 1 (IL-1)--than cells from control animals treated with commercial latex beads. However, when indomethacin was added, either in vivo or in vitro, cells from animals treated with glucan-plastic beads produced more thymocyte-stimulatory activity than controls. We interpret this to mean that glucan-plastic beads stimulate both IL-1 and PGE2, but that under circumstances where the cellular cyclo-oxygenase is not inhibited, the PGE2 will block IL-1 production.     

Human and Mouse Macrophages Collaborate with Neutrophils To Kill Larval Strongyloides stercoralis

Macrophages are multifunctional cells that are active in T_H1- and T_H2-mediated responses. In this study, we demonstrate that human and mouse macrophages collaborate with neutrophils and complement to kill the parasite Strongyloides stercoralis in vitro. Infection of mice with worms resulted in the induction of alternatively activated macrophages (AAMϕ) within the peritoneal cavity. These cells killed the worms in vivo and collaborated with neutrophils and complement during the in vitro killing process. AAMϕ generated in vitro killed larvae more rapidly than naive macrophages, which killed larvae after a longer time period. In contrast, classically activated macrophages were unable to kill larvae either in vitro or in vivo. This study adds macrophages to the armamentarium of immune components that function in elimination of parasitic helminths and demonstrate a novel function by which AAMϕ control large extracellular parasites.     

Interaction of Virulent and Avirulent Listeria monocytogenes with Cultured Mouse Peritoneal Macrophages

The interaction between smooth and rough Listeria monocytogenes and mouse peritoneal macrophages in culture was investigated. Initially, antibiotics were deleted from the culture medium, and no attempt other than the removal of unphagocytized bacteria by extensive washings was made to control extracellular growth. Under these conditions the monolayers were rapidly destroyed within an 8-h period, and this was associated with increases in the intracellular population of both strains. Extracellular viability counts revealed that washings failed to reduce the bacteria in the medium to less than 10% of the original inoculum. Continuous phagocytosis of Listeria which grew logarithmically in the maintenance media appears to account for the observed changes in the number of intracellular bacteria. The data also indicate that it is primarily the free bacteria in the culture medium which are responsible for the cytotoxic effects. In other experiments streptomycin-penicillin solutions were added to the maintenance media after an initial period of phagocytosis. In the presence of antibiotics, the total number of macrophages per field remained relatively constant, and no morphological alterations in the leukocyte cultures were observable. Extensive intracellular multiplication of either strain was not evident in fixed and stained cover slips. Viable intracellular counts reveal that after 24 h there is almost total killing of the rough variant, whereas the smooth strain tended towards complete survival.