Activation of murine macrophage-like cells by granulocytin

Granulocytin, a C-type lectin from Sarcophaga peregrina (flesh fly), stimulated glucose consumption and cytokine production by the mouse macrophage-like cell line J774.1. When J774.1 cells were pretreated with tunicamycin, their granulocytin-dependent TNF-alpha production was greatly reduced. These results suggest that the stimulus of granulocytin is transmitted to J774.1 cells via the carbohydrate chain of granulocytin receptors located on their surface.     

Metabolism of exogenous arachidonic acid by murine macrophage-like tumor cell lines

Murine macrophage-like cell lines, J774.2, P388D1, RAW264.7 and PU-5-1R, were incubated with exogenous arachidonic acid (AA). The major metabolites were identified by comigration with known standards in TLC and HPLC and by characteristic behavior following reduction. During a 30 min incubation J774.2 cells metabolized exogenous ¹⁴C-AA (10 μM) to PGE₂ (14.8%), 12-hydroxy-5,8,10-heptadecatrienoic acid (HTT)_ (13.0%), thromboxane B₂ (TXB₂) (7.4%), PGD₂ (4.4%) and PGF_2α (3.0%). The remainder was incorporated into phospholipids (39.0%), triglycerides (6.1%), and as yet unidentified metabolites (8.2%). No PGF_1α was found. Metabolism of exogenous AA was rapid, being >90% completed at 3.5 min. Metabolism of exogenous AA is not increased by the simultaneous addition of macrophage stimuli including the cation ionophore A-23187, particulate phagocytic stimuli and endotoxin. The synthesis of cyclooxygenase products was inhibited by low doses of indomethacin (ID₅₀=0.6 μM) while the synthesis of TXB₂ and HHT was selectively inhibited by benzylimidazole (ID₅₀=9.5 μM). Identification of a probable lipoxygenase product is being pursued. The synthesis of this product is not inhibited by indomethacin and migrates with an R_f value close to 5,12-diHETE in TLC. P388D1 and RAW264.7 cells metabolize exogenous AA to the same products as J774.2, in different proportions, while PU-5-1R does not produce cylooxygenase metabolites to any appreciable extent.     

Spontaneous lysosomal enzyme secretion by a murine macrophage-like cell line.

Lysosomal enzyme secretion by the murine macrophage-like cell line, P388D1, was compared with that of normal peritoneal macrophages. Unlike macrophages, lysosomal hydrolase secretion by P388D1 cells occurred spontaneously in vitro and was not further stimulated by the presentation of inflammatory agents such as zymosan and asbestos.     

Modulation of Fc-mediated phagocytosis by cyclic AMP and insulin in a macrophage-like cell line.

Studies on genetically selected mutants in phagocytosis of E[IgG] indicated that the defect in some mutants could be corrected by addition of 8 Br-cAMP, and suggested that cyclic AMP may be involved in the mechanism of phagocytosis through the Fc receptor. In order to elucidate the role of cyclic AMP in phagocytosis in the parental, nonmutant macrophage-like cell line, J774.2, it was necessary to employ restrictive conditions which rendered phagocytosis suboptimal. When 4774.2 cells were cultured in nontissue culture Petri dishes phagocytosis was markedly reduced. Addition of 8 Br-cAMP or inducers of intracellular cyclic AMP such as isoproterenol restored the phagocytic ability of these cells. Similarly, treatment of the parental J774.2 cells with insulin reduced the level of phagocytosis, and once again this suppression could be corrected by addition of 8 Br-cAMP. In no case did AMP mimic the effects of 8 Br-cAMP. The effect of cyclic AMP action in this system was not instantaneous, but rather reached optimal levels at 5 to 10 hr, suggesting that cyclic AMP is not the immediate signal for phagocytosis. The genetic analysis of macrophage variants may provide a useful model for studies on the mechanisms of phagocytosis, and also the effects of insulin and cyclic AMP on an easily measurable biologic function in a specialized cell type.     

Inactivation of DsbA alters the behaviour of Shigella flexneri towards murine and human-derived macrophage-like cells

The mutants of Shigella flexneri, Sh4 (dsbA::kan) and Sh42 (dsbA33G), behave differently towards murine and human-derived macrophage-like cells in vitro. Sh4 was trapped in the phagocytic vacuoles of the murine J774 cells as evidenced by its colony forming units plus and minus chloroquine exposure in a gentamicin protection assay, and by light and transmission electron microscopy (TEM). Sh42, similar to the wild-type M90TS, was able to escape from the vacuoles and kill host cells presumably by inducing apoptosis. In U937 cells, unlike M90TS that was free in the cytosol, both Sh4 and Sh42 grew poorly. TEM revealed that Sh4 and Sh42 were trapped within the U937 phagocytic vacuoles. Furthermore, the two mutants induced different patterns of interleukin-1beta and tumour necrosis factor-alpha expression, which might explain why they possess different immunogenic properties in vivo.     

Metabolism of modified LDL and foam cell formation in murine macrophage-like RAW 264 cells.

The uptake of modified low density lipoprotein (LDL) by arterial macrophages is a key event in the atherogenesis. We studied 1) the uptake and degradation of modified LDL, 2) LDL recognition by specific receptors, and 3) the foam cell formation with murine macrophage-like RAW 264 cells in vitro. The cells took up and degraded effectively 125I-labeled acetylated LDL (Ac-LDL) and aggregated LDL (Aggr-LDL). Also oxidized LDL (Ox-LDL) was taken up but it was degraded poorly. The degradation of 125I-Ac-LDL was efficiently competed by both unlabeled Ac-LDL and Ox-LDL, whereas the degradation of 125I-Ox-LDL was partially competed by unlabeled Ox-LDL and Aggr-LDL but not at all by unlabeled Ac-LDL. The incubation with increasing concentrations of Ac-LDL, Aggr-LDL or Ox-LDL resulted in marked foam cell formation in the RAW 264 cells. Ox-LDL was cytotoxic at 500 to 1000 microg/ml concentrations. The results show that RAW 264 cells have at least two classes of receptors for modified lipoproteins: one that recognizes both Ox-LDL and Ac-LDL, and is similar to the scavenger receptors, and another that recognizes Ox-LDL but not Ac-LDL. RAW 264 cells are a convenient model cell line for examining the metabolism of modified lipoproteins, not only that of Ac-LDL but also that of Ox-LDL and Aggr-LDL, and cellular accumulation of lipids derived from modified LDL.     

Deficient in vitro and in vivo phagocytosis of apoptotic T cells by resident murine alveolar macrophages

Apoptotic lymphocytes are readily identified in murine lungs, both during the response to particulate Ag and in normal mice. Because apoptotic lymphocytes are seldom detected in other organs, we hypothesized that alveolar macrophages (AMphi) clear apoptotic lymphocytes poorly. To test this hypothesis, we compared in vitro phagocytosis of apoptotic thymocytes by resident AMphi and peritoneal macrophages (PMphi) from normal C57BL/6 mice. AMphi were deficient relative to PMphi both in percentage containing apoptotic thymocytes (19.1 +/- 1% vs 96 +/- 2.6% positive) and in phagocytic index (0.23 +/- 0.02 vs 4.2 +/- 0.67). This deficiency was not due to kinetic differences, was seen with six other inbred mouse strains, and was not observed using carboxylate-modified polystyrene microbeads. Annexin V blockade indicated that both Mphi types cleared apoptotic T cells by a mechanism involving phosphatidylserine expression. By contrast, neither mAb blockade of a variety of receptors (CD11b, CD29, CD51, and CD61) known to be involved in clearance of apoptotic cells, nor the tetrapeptide RGDS (arginine-glycine-aspartic acid-serine) blocked ingestion by either type of macrophage. To confirm these studies, apoptotic thymocytes were given intratracheally or i.p. to normal mice, and then AMphi or PMphi were recovered 30-240 min later. Ingestion of apoptotic thymocytes by AMphi in vivo was significantly decreased at all times. Defective ingestion of apoptotic lymphocytes may preserve AMphi capacity to produce proinflammatory cytokines in host defense, but could contribute to development of autoimmunity by failing to eliminate nucleosomes.     

Nonspecificity of the phenomenon of influenza virus phagocytosis by murine macrophages

Peritoneal macrophage cultures from intact mice and those immune to influenza virus A/PR/8/34 (HON1) were infected with homologous virus or influenza virus A/England/42/72 (H3N2) whereupon virus was isolated from chick embryos. It was established that in intact macrophages, both viruses duplicated similarly. Macrophages immune to virus HON1 equally disintegrated both in homologous virus and heterologous influenza virus H3N2.     

Porphyromonas gingivalis with either Tannerella forsythia or Treponema denticola induces synergistic IL-6 production by murine macrophage-like J774.1 cells

BackgroundChronic periodontitis is caused by mixed bacterial infection. Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are frequently detected in deep periodontal pockets. We demonstrate that these bacteria induce proinflammatory cytokine production by the mouse macrophage-like cell line J774.1.Materials and methodsJ774.1 cells were incubated with and without bacteria for 24 h in 96-well flat-bottomed plates. The culture supernatants were analyzed by enzyme-linked immunosorbent assay for secreted mouse interleukin (IL)-6, monocyte chemoattractant protein-1, IL-23, IL-1β and tumor necrosis factor-α. The cytokine concentrations were determined using a standard curve prepared for each assay.ResultsMixed infection with P. gingivalis and either T. forsythia or T. denticola at 10⁵ CFU/ml acted synergistically to increase IL-6 production, but not monocyte chemoattractant protein-1, IL-23, IL-1β or tumor necrosis factor-α production. Gingipain inhibitors KYT-1 and KYT-36 inhibited IL-6 production by J774.1 cells incubated with 10⁵ CFU/ml of mixed bacteria.ConclusionThese results suggest that P. gingivalis with either T. forsythia or T. denticola directly induces synergistic IL-6 protein production and that gingipains play a role in this synergistic effect.     

In vitro phagocytosis of several candida berkhout species by murine leukocytes

In vitro phagocytosis of thirteen Candida berkhout species by rat leukocytes was studied to assess a possible correlation between pathogenicity and phagocytosisYeast-leukocyte suspensions were mixed up for 3 h and phagocytic index, germ-tube formation and leukocyte candidacidal activity were evaluated. Highest values for phagocytosis were reached in all cases at the end of the first hour. Leukocyte candidacidal activity was absent. Only C. albicans produced germ-tubes.The various phagocytosis indices were determined depending on the Candida species assayed. Under these conditions, the more pathogenic species presented the lower indices of phagocytosis.It is determined that the in vitro phagocytic index may bear a close relationship with the pathogenicity of the Candida berkhout.     

Theiler's murine encephalomyelitis virus subgroup strain-specific infection in a murine macrophage-like cell line.

We compared infection of a murine macrophage-like cell line, J774-1, with two Theiler's murine encephalomyelitis virus subgroup strains. The GDVII strain, which is highly virulent and produces acute polioencephalomyelitis in mice, did not actively replicate in J774-1 cells, although there was a significant inhibition in cellular protein synthesis. In contrast, the DA strain, which is less virulent and causes demyelination with a persistent virus infection, productively infected J774-1 cells; however, there was less virus produced than in BHK-21 cells, and there was little if any cellular protein shutoff. These in vitro data may provide some explanation for the biological activities that are observed between both subgroup strains.     

Ornithine-containing lipids stimulate CD14-dependent TNF-alpha production from murine macrophage-like J774.1 and RAW 264.7 cells

The ornithine-containing lipids (OL)-induced cytokine production pattern in macrophage-like J774.1 and RAW 264.7 cells was different from that in the peritoneal macrophages previously reported. OLs, as well as lipopolysaccharide (LPS) of Escherichia coli, strongly induced tumor necrosis factor (TNF) alpha but not interleukin (IL)-1beta in J774.1 cells. In the RAW cells, IL-1beta, TNF-alpha and prostaglandin E(2) were strongly induced by the OLs and LPS. OL- and serine-glycine-containing lipid (SGL)-induced TNF-alpha production in J774.1 and RAW 264.7 cells required serum. However, in CD14-deficient LR-9 cells, TNF-alpha was not induced by the OLs in the presence or absence of serum. OLs and a SGL almost completely inhibited the binding of (125)I-LPS to J774.1 cells. These results suggested that OLs and SGL activate macrophages via the CD14-dependent pathway.