Nitrotyrosine formation after activation of murine macrophages with mycobacteria and mycobacterial lipoarabinomannan.

Murine peritoneal macrophages, elicited with thioglycollate, were stimulated in vitro with lipopolysaccharide (LPS). The production of nitrite, superoxide anion (SOA), and the accumulation of nitrotyrosine in the cells increased after treatment, and all were inhibitable by the NO synthase inhibitor NG-monomethyl-L-arginine monoacetate (L-NMMA). This effect suggests a direct correlation between the accumulation of those metabolites and NO synthase activity. Lipoarabinomannan (LAM) purified from Mycobacterium tuberculosis was added to peritoneal macrophages in the presence of interferon-gamma (IFN-gamma); the cells produced nitrite and SOA, both inhibitable by L-NMMA. There was, as well, accumulation of nitrotyrosine in the macrophage proteins. Strikingly, the amount of nitrotyrosine measured after LAM plus IFN-gamma, or LAM plus the low molecular weight adjuvant glutamylmuramyl dipeptide (GMDP), increased significantly in the presence of L-NMMA. These results suggest that murine macrophages, upon LAM stimulation, might generate reactive nitrogen metabolites by a route other than NO synthase. Nitrotyrosine accumulation after infection of macrophages in vitro, with either live bacille Calmette-Guérin (BCG) or live M. tuberculosis, in the presence or absence of IFN-gamma, showed no correlation with nitrite production, suggesting a low superoxide production.     

Bifidobacteria DNA induces murine macrophages activation in vitro

Previous studies have shown that oligodeoxynucleotides containing unmethylated CpG motifs were used as adjuvants for immunoregulation and immune response. This study was to explore the activation effects of Bifidobacteria DNA containing unmethylated CpG motifs （CpG DNA） on murine macrophage J774A.1 cells. The genomic DNA of Bifidobacteria was extracted and purified, and the methylation degree of CpG motifs was tested. The phagocytic ability of the macrophages was detected by flow cytometry. The cytokines （IL-1β, IL-6, IL-12p40 and TNF-α） levels in the culture supernatants of Bifidobacteria DNA treated J774A.1 cells were assayed by ELISA. The content of nitric oxide （NO） was detected by Griess reagent. After treated with Bifidobacteria DNA for 24h, Nile Red stain increased in J774A.1 macrophage, which suggested that the lipid metabolism increased in the macrophages. The phagocytic ability and levels of NO and cytokines of IL-1β, IL-6, IL-12p40 and TNF-α were significantly higher than PBS group and CT DNA group. The results indicated that Bifidobacteria DNA could activate murine macrophages J774A.1, which could provide scientific basis for the research and application of microorganism DNA preparation. Cellular ＆ Molecular Immunology. 2005;2（6）：473-478.     

Fungicidal activation of murine macrophages by recombinant gamma interferon.

Alveolar macrophages from BALB/c mice readily phagocytized endospores (2 to 5 micron) and arthroconidia of Coccidioides immitis in vitro. Within 24 to 30 h at 37 degrees C, the phagocytized endospores started developing into spherules, and the arthroconidia formed germ tubes and hyphae. However, these processes did not occur if the macrophages were incubated with murine recombinant gamma interferon (rIFN-gamma) during infection with C. immitis. Treatment with rIFN-gamma activated the fungicidal capabilities of the alveolar macrophages, as evidenced by the 50% reduction in the CFU which could be recovered from macrophages infected in the presence of gamma interferon compared with alveolar macrophages infected without gamma interferon (P less than 0.05). Similar results were seen with peritoneal macrophages incubated with rIFN-gamma and infected with C. immitis. As little as 10 U of rIFN-gamma per ml reduced by half the number of C. immitis CFU which could be recovered from the phagocytes 8 h after infection with arthroconidia, although interferon alone did not affect the viability of the fungi.     

Membrane damage and interleukin-1 production in murine macrophages exposed to listeriolysin O.

To obtain some insight into the interaction between listeriolysin O (LLO) and the macrophage membrane, we examined the effect of purified Listeria monocytogenes hemolysin on the viability and functions of mouse peritoneal exudate macrophages. The study showed that purified LLO impaired a variety of functions of the macrophages. First, it suppressed the luminol-dependent chemiluminescence response of macrophages. Second, it suppressed the phagocytic ingestion of opsonized sheep erythrocytes and latex beads. Third, exposure of macrophages to LLO resulted in an increase in dead cells, as determined by the trypan blue dye exclusion method. An interesting observation of this study is that the LLO-induced production of interleukin-1 from macrophages could not be blocked by preincubation with cholesterol, while the membrane-damaging ability could be blocked by cholesterol. The dissociation of the blocking effects of cholesterol suggests that the interleukin-1-inducing ability of LLO may be distinct from its membrane-damaging ability.     

Morphological and functional changes during cytomegalovirus replication in murine macrophages

Structural and functional changes were studied in murine peritoneal macrophages infected with murine cytomegalovirus by using centrifugal enhancement to achieve a high-level (greater than 90%) pulsed infection. During 3 d of culture the infected cells became enlarged and rounded with smooth surface contours. Transmission electron microscopy demonstrated various stages of viral maturation in the nucleus and cytoplasm. Intracellular organization was generally retained, apart from the development of large, irregular, intracytoplasmic vacuoles, in which enveloped virions and cell debris accumulated. The infected macrophages lost most surface markers tested (F4/80, Mac-1, FcR, and the receptor for gluteraldehyde-fixed sheep red blood cells), but H-2 expression was increased. Moreover, ingestion of colloidal gold or horseradish peroxidase was depressed, and the levels of acid phosphatase activity, lymphocytostatic activity, and interleukin 1 production were also decreased. The latter may explain the observed loss of accessory cell function.     

Cytokine activation of murine macrophages for in vitro killing of Entamoeba histolytica trophozoites.

Macrophage-mediated effector mechanisms against the protozoan parasite Entamoeba histolytica were studied. Unstimulated macrophages were inefficient at killing E. histolytica trophozoites in vitro and were killed by the trophozoites. Conversely, immature cells of the mononuclear phagocyte lineage (promonocytes) were shown to display a strong spontaneous amebicidal activity. The acquisition of macrophage amebicidal activity following cytokine treatment was investigated. Gamma interferon, tumor necrosis factor alpha, and macrophage colony-stimulating factor 1, or combinations thereof, were shown to endow murine bone marrow-derived macrophages with significant amebicidal activity. Low doses of gamma interferon and tumor necrosis factor alpha and of gamma interferon and colony-stimulating factor 1 were shown to act synergistically in this phenomenon. This enhancement of amebicidal activity was shown to operate on bone marrow-derived macrophages, elicited peritoneal macrophages, and, to a much lesser extent, spleen macrophages. Although acquisition of amebicidal activity was associated with a strong respiratory burst, the addition of oxygen-free radical scavengers showed that the killing activity was approximately 45% H2O2 dependent. In addition, amebicidal activity by macrophages was shown to be contact dependent and was inhibited by 61% with the protease inhibitor tosyl lysyl chloromethyl ketone. Our results indicate that immunologic production of gamma interferon, tumor necrosis factor alpha, and colony-stimulating factor 1 could be important in the activation of macrophages for host defense against amebiasis and that promonocytes are strong effector cells against virulent amebae.     

I-A restricted activation by T cell lines of anti-tuberculosis activity in murine macrophages.

Tuberculosis and leprosy remain two of the world's most significant diseases. Immunity involves the activation of macrophages by lymphokines but the details are unknown because there has been no objective assay for the relevant effector function using human pathogens. We previously reported the use of tritiated-uracil uptake by surviving mycobacteria as a measure of the anti-mycobacterial effect of human monocytes. We describe here the use of a modification of this assay to measure control of the proliferation of Mycobacterium tuberculosis in murine peritoneal macrophages. A bacteriostatic effect can be induced in macrophages infected with M. tuberculosis, by adding small numbers of Ly 1 +2- T cells from in vitro lines derived from immunized mice. The phenomenon is dependent on compatibility at the I-A locus of the major histocompatibility complex (MHC) and mediated by soluble factors. Such T cells also recognise and activate macrophages infected with other mycobacterial pathogens. Thus, T cells recognising shared mycobacterial antigens are active. The findings have implications for MHC linked susceptibility to mycobacterioses and the hypothesized ability of cross-reactive environmental mycobacteria to abrogate or pre-empt the protective efficacy of subsequent BCG vaccination.     

Human neutrophil-derived CAP37 inhibits lipopolysaccharide-induced activation in murine peritoneal macrophages

Human cationic antimicrobial protein, CAP37, is released from neutrophil granules during infection. CAP37 attracts monocytes, binds Gram-negative endotoxin (lipopolysaccharide, LPS), is bactericidal for a range of Gram-negative bacteria, and reduces mortality in murine polymicrobial sepsis. Here, we report that recombinant CAP37 specifically targets murine peritoneal macrophages. Under steady-state conditions, the bulk of cell-associated CAP37 was localized at the plasma membrane. However, a fraction of CAP37 gained access to the endocytic system, but did not accumulate in recycling endosomes or in the trans-Golgi network (TGN). Instead, CAP37 was internalized by fluid phase endocytosis and accumulated in a prelysosomal compartment. Macrophages that were preexposed to CAP37 exhibited diminished LPS responsiveness, as determined by analysis of c-Jun phosphorylation. Further examination showed that pretreatment with CAP37 reduced the ability of macrophages to bind LPS. Taken together, these observations demonstrate that prolonged exposure to CAP37 desensitizes macrophages to LPS and suggest that this protein plays a novel anti-inflammatory role in polymicrobial sepsis.     

Scanning electron microscopy of murine macrophages. Surface characteristics during maturation, activation, and phagocytosis.

The present report describes the surface architecture of critical point dried mouse peritoneal macrophages, after attachment and spreading on glass, and during maturation and active phagocytosis of rabbit erythrocytes and latex spheres. This study also compares the appearance of unstimulated cells with that of thioglycollate and endotoxin-stimulated cells. Activated cells, particularly thioglycollate-stimulated macrophages, showed more rapid and extensive spreading, a larger surface area, and more prominent ruffled membranes and filopodia. Many fine cytoplasmic pits were also evident and these may represent the sites of formation of numerous pinocytotic vesicles. The sequence of events during the various stages of phagocytosis was well visualized with the scanning electron microscope. Multiple large, round, and hemispherical craters were observed in these cells, and particles were engulfed through these structures and subsequently ingested. The findings are discussed in the light of current knowledge of the macrophage plasma membrane.     

The immunomodulatory glycan LNFPIII initiates alternative activation of murine macrophages in vivo

The early pathogen–macrophage interactions that help drive macrophage maturation towards classically or alternatively activated are largely unknown. To examine this question we utilized the immunomodulatory glycan Lacto-N-fucopentaose III (LNFPIII), which contains the Lewis X (LeX) trisaccharide, to activate murine peritoneal macrophages in vivo. Because LNFPIII is known to induce anti-inflammatory responses, we asked if LNFPIII stimulation of macrophages in vivo initiates alternative activation events such as upregulation of Arginase 1, Ym1, FIZZ-1, MGL-1 or macrophage mannose receptor (MMR). Examination of peritoneal exudate cells from mice 20 hr post-LNFPIII injection demonstrated increased Arginase 1 activity, at the mRNA and protein levels, coincident with undetectable inducible nitric oxide synthase expression or nitric oxide production. In addition to Arginase 1, Ym1 expression was also significantly upregulated at 20 and 48 hr after LNFPIII exposure in vivo. However, the expression of FIZZ-1, MGL-1, and MMR was not changed in these macrophages. In an attempt to determine activation requirements for functional activity, we adoptively transferred antigen-pulsed, in vivo LNFPIII activated macrophages into naïve recipients and found that they were capable of triggering recipient T cells to secrete elevated levels of interleukin (IL)-10 and IL-13 compared to mice receiving control macrophages. Together, these data demonstrate that upregulation of expression of Arginase 1 and Ym1 occur very early in activation of macrophages, and can be independent of other alternatively activated (AA) macrophage markers. Importantly, these early events appear to be IL-4/IL-13-independent in our model. In the future we hope to determine if upregulation of these initial AA maturational events is sufficient for these macrophages to exert immunoregulatory activity in vivo.     

In vitro activation of murine bone marrow-derived macrophages with cisplatin and mitomycin-C

Peritoneal macrophages from BALB/c mice after treatment for 24 h in vitro with cisplatin, lipopolysaccharide (LPS) or mitomycin-C were rendered significantly cytotoxic against L-929 tumor target cells. In a similar experiment none of these agents could induce tumoricidal activity of fresh non-adherent bone marrow cells (NABMC). NABMC when incubated in medium alone or in medium containing L-929 culture medium (L-929 CM), a form of macrophage colony stimulating factor (M-CSF), for three days matured to macrophages which were positive for non-specific esterase staining. These bone marrow-derived macrophages cultured with medium alone did not respond to cisplatin. LPS or mitomycin-C for induction of tumoricidal activity whereas bone marrow derived macrophages with that were incubated with L-929 CM showed also significantly enhanced cytotoxicity after treatment with cisplatin, LPS and mitomycin-C. Culturing of NABMC with L-929 CM significantly enhanced cell survival as compared to the cells incubated in medium alone. These results suggest that bone marrow cells not only mature in L-929 CM but also are primed by L-929 CM for induction of tumoricidal activity.     

Fate of vaccines of Propionibacterium acnes after phagocytosis by murine macrophages.

Stationary-phase (48 h) cells of Propionibacterium acnes VPI 0009, a potent stimulator of the reticuloendothelial system, persist unchanged within phagocytes for at least 24 h after ingestion. In contrast, exponential-phase (12 h) cells of the same strain (which do not induce splenomegaly) are extensively degraded within 5 h of phagocytosis. Suspensions of P. granulosum VPI 6500, which fails to induce splenomegaly in mice, also show considerable degradation after phagocytosis. Stationary-phase cells of strain VPI 0009 treated with sodium metaperiodate or with trichloroacetic acid, although without ability to induce splenomegaly, resist destruction almost as well as untreated vaccines. However, bacteria inactivated by acetic anhydride show about 50% breakdown in 24 h.     

The production of fibrinolysis inhibitors as a parameter of the activation state in murine macrophages.

Macrophages from peritoneal exudates which had been induced by various irritants were cultured, and the supernatants were tested for inhibitors of fibrinolysis using the plasmin-dependent lysis of ¹²⁵I-labeled fibrinogen as assay. Washout macrophages and casein or proteose peptone-elicited macrophages were found to release fibrinolysis inhibitors in contrast to lipopolysaccharide or thioglycollate-induced macrophages. The molecular weight of inhibitors was determined at 60 000, 45 000 and 15 000 by Sephadex chromatography. Whereas the inhibitors at 15 000 and 45 000 could be detected in all experiments, the inhibitor at mol. wt. 60 000 was not present in all preparations. The isoelectric point of inhibitor I (mol. wt. 15 000) and inhibitor II (mol. wt. 45 000) was determined at 4.15. The proteolytic and esterolytic activity of trypsin and chymotrypsin were both inhibited by each of the two inhibitors. On the other hand, only the proteolytic activity of plasmin could be inhibited. Evidence for the active synthesis of inhibitors by macrophages came from several experiments. Macrophages in serum-free cultures continued to release inhibitors for at least 48 h; normal mouse serum did not contain inhibitors of the same molecular size, and [³H]leucine was incorporated into the inhibitors which were specifically detected by the absorption of plasmin inhibitor complexes to lysyl-Sepharose. Since inhibitors and plasminogen activator could not be detected in the same macrophage culture supernatants, it appears that the production of inhibitors and plasminogen activator by the same macrophage population is mutually exclusive.     

Antimicrobial proteins of murine macrophages.

Three murine microbicidal proteins (MUMPs) were purified from cells of the murine macrophage cell line RAW264.7 that had been activated by gamma interferon. Similar proteins were also present in nonactivated RAW264.7 cells, in cells of the murine macrophage cell line J774A.1, and in resident and activated murine peritoneal macrophages. MUMP-1, MUMP-2, and MUMP-3 killed Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Mycobacterium fortuitum, and Cryptococcus neoformans in vitro. MUMP-1 resembled an H1 histone but was unusual because its N-terminal residue (serine) was not N acetylated. Although MUMP-2 was N terminally blocked, its high lysine/arginine ratio and its reactivity with an antibody to H1 histones suggested that it also belonged to the H1 histone family. MUMP-3 was identical to histone H2B in 30 of 30 amino-terminal residues. Although the antimicrobial properties of histones have been recognized for decades, this is the first evidence that such proteins may endow the lysosomal apparatus of macrophages with nonoxidative antimicrobial potential. Other MUMPs, including some with a more restricted antimicrobial spectrum and one that appeared to be induced in RAW264.7 cells after gamma interferon stimulation, were noted but remain to be characterized.     

Cisplatin-induced activation of murine bone marrow-derived macrophages require protein tyrosine phosphorylation

The aim of the present study is to evaluate the involvement of tyrosine phosphorylation in the signal transduction mechanism of cisplatin-induced macrophage activation in vitro. Stimulation of bone marrow-derived macrophages (BMDM) with cisplatin (CP) resulted in a time- and dose-dependent phosphorylation of several proteins having estimated molecular weights of approximately 18, 20, 21, 30, 33, 35, 39, 41, 44, 58 and 123 kD, detected by immunoblot using anti-phosphotyrosine antibody. CP-induced tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor genistein. Using this inhibitor, we were able to correlate tyrosine phosphorylation with several functional effects of CP on murine bone marrow-derived macrophages (BMDM) . Treatment of macrophages with genistein before incubation with CP completely inhibited the CP-induced tumoricidal activation of macrophages as well as production of TNF and NO, whereas pre-treatment of macrophages with phosphatase inhibitor sodium vanadate upregulated macrophage activation in addition to enhanced protein tyrosine phosphorylation. Taken together, these data suggest that tyrosine phosphorylation play a critical regulatory role in the activation of macrophages with CP.     

Role of calcium and calmodulin in the activation of murine peritoneal macrophages with cisplatin

When fura-2/AM loaded peritoneal macrophages were treated with cisplatin, it resulted in a rapid increase in the intracellular free calcium. Calcium modulating agents, EGTA, nifedipine, and TMB-8, and the calmodulin antagonist W-7 inhibited cisplatin-induced tumoricidal activity of murine peritoneal macrophages. Supernatants collected from macrophages treated with cisplatin and EGTA, nifedipine, TMB-8 or W-7 demonstrated decreased tumor necrosis factor (TNF) and interleukin-1 (IL-1) activity in comparison to supernatants collected from macrophages treated with cisplatin alone. Similarly, TNF and IL-1 activity were significantly inhibited in paraformaldehyde (PFA)-fixed macrophages and freeze-thaw lysates of macrophages treated with cisplatin and different calcium and calmodulin-modulating agents. These results suggest that calcium and calmodulin are important in the cisplatin-mediated activation of murine peritoneal macrophages to tumoricidal state.