Human Blood Monocyte-derived Macrophages Research Articles

Cytokine Regulation of the Myeloid Glycoprotein CD14

Monocyte membrane CD14 (mCD14) antigen expression was measured in normal human monocytes and blood monocyte-derived macrophages. Seven-day culture of monocytes in serum-containing medium lead to an increase in mCD14. Addition of interferon-gamma (IFN gamma) or interleukin-4 (IL-4) to monocytes caused a dose-dependent reduction in mCD14 within 3 or 45 h respectively. These effects were strong in monocytes, weak in macrophages, and they were blocked by anti-IFN gamma and anti-IL-4 antibodies, respectively. Interleukin-2 and interferon-alpha produced a decrease in mCD14 in mononuclear leukocyte cultures but not in purified monocytes. Their effect was abolished in the presence of anti-IFN gamma. Other cytokines (TFN alpha, Ill beta, IL-6, IL-3, IL-5, GM-CSF, TGF beta 1) did not change mCD14. In conclusion, IFN gamma and IL-4 are revealed to be the only cytokines which directly affect monocyte CD14 expression.

Human non-transformed monocyte-derived macrophage cell lines

We have demonstrated that human blood monocyte-derived macrophages can be passed from primary cultures and replicate. Passaged cells have typical macrophage characteristics: they are non-specific esterase positive, phagocytic, and respond to 3 days of treatment with interferon-γ with enhanced production of superoxide on stimulation with PMA. The passaged cells express Fc, CR1, CR3 and FMLP receptors. Both primary and passaged cultures constitutively produce CSF-1 after 3 weeks in culture. Cultures studied between 7 and 16 weeks in culture produce 3712 ± 478 U of CSF-1 per 10 6 cells. Randomly selected lines were examined to look for cell proliferation by looking at numbers of cells over time and by labelling cells with tritiated thymidine to determine the number of cells synthesizing DNA. In addition, the cells can be frozen at the time of isolation and stored for at least 1 year, and then thawed and shown to retain functional activity. Human monocyte-derived macrophages can be cultured as finite cell lines.

Regulation of interleukin-6 expression in cultured human blood monocytes and monocyte-derived macrophages

A culture system that allows human blood monocytes to differentiate into macrophages in vitro was used to study B-cell stimulatory factor- 2/interleukin-6 (interferon-beta 2/26 kd protein) expression in mononuclear phagocytes. Using B-cell stimulatory factor-2 (BSF-2) cDNA and a polyclonal, monospecific antibody directed against human BSF-2, we find that strong interleukin-6 (IL-6) expression is initiated in cultured monocytes on stimulation with endotoxin. Maximally induced monocytic BSF-2/IL-6 synthesis (1% to 2% of total proteins secreted by monocytes) is more than ten times stronger than in terminally differentiated macrophages (approximately 0.1% of total secretory proteins). BSF-2/IL-6 mRNA was detectable as early as one hour after stimulation with endotoxin, reaching maximum levels three hours after stimulus. Interleukin-1 (IL-1) was able to stimulate IL-6 synthesis in monocytes, but not in macrophages. Tumor necrosis factor, interferon- gamma and interleukin-2 (IL-2) had no effect on IL-6 synthesis in monocytes or macrophages. We found five molecular weight forms of BSF- 2/IL-6 to be secreted by monocytes of 21.5 kd, 23.5 kd, 24 kd, 26 kd, and 28 kd apparent molecular weight. The 26 kd and 28 kd forms were found to represent N-glycosylated molecules, which were not detectable on treatment of the cells with the N-glycosylation inhibitor tunicamycin. The 21.5 kd, 23.5 kd, and 24 kd BSF-2/IL-6 forms were unaffected by tunicamycin treatment. We conclude from our data that cells of the mononuclear phagocyte lineage are one of the main sites of BSF-2/IL-6 (interferon-beta 2/26 kd protein/HSF) synthesis.

Degradation of lipoproteins by human monocyte-derived macrophages. Evidence for two distinct processes for the degradation of abnormal very-low-density lipoprotein from subjects with type III hyperlipidaemia.

Human blood monocyte-derived macrophages that had been cultured in medium containing human serum for 7 days degraded the abnormal very-low-density lipoproteins (VLDL) from the plasma of subjects with type III hyperlipoproteinaemia by two distinct saturable processes. One process was stimulated when cells from normal subjects were preincubated with lipoprotein-free medium, was inhibited by excess unlabelled low-density lipoproteins (LDL) and was absent from cells from subjects with homozygous familial hypercholesterolaemia; on these criteria it was identified as an LDL-receptor-dependent process. Degradation by the second process was of equal magnitude in both cell types and was unaffected by excess unlabelled LDL or acetylated LDL. The activity of this process was reduced when the cells were preincubated in lipoprotein-free medium. The abnormal VLDL from the plasma of cholesterol-fed rabbits were also degraded by this process, which was similar to that in mouse peritoneal macrophages mediated by the receptor for VLDL of beta-electrophoretic mobility [Goldstein, Ho, Brown, Innerarity & Mahley (1980) J. Biol. Chem. 255, 1839-1848].

Degradation by cultured monocyte-derived macrophages from normal and familial hypercholesterolaemic subjects of modified and unmodified low-density lipoproteins.

Human blood monocyte-derived macrophages that had been cultured for 7 days in the presence of 20% whole human serum exhibited saturable degradation of low-density lipoprotein (LDL). This degradation could be abolished by pre-incubating the cells with a high concentration of LDL in the medium and increased by pre-incubating the cells in medium containing lipoprotein-deficient serum. Cells obtained from the blood of homozygous familial-hypercholesterolaemic (FH) patients only exhibited a low rate of non-saturable degradation of LDL, even when pre-incubated without lipoproteins. Thus the saturable degradation of LDL by normal cells was mediated by the LDL receptors that are defective in FH patients and little LDL was taken up and degraded through any of the other endocytotic processes present in macrophages. Degradation by normal cells pre-incubated with lipoprotein-deficient serum had a higher apparent affinity for LDL than that of cells maintained in whole serum, which suggests that incubation with lipoprotein-deficient serum may not only induce the formation of LDL receptors but may also have a direct effect on the receptors themselves. Monocyte-derived macrophages from normal and FH subjects showed similar saturable degradation of acetylated LDL and also of LDL complexed with dextran sulphate. Maximal degradation of each was in the same range as the degradation of unmodified LDL by normal cells, and was not increased if the cells were pre-incubated with lipoprotein-deficient serum.