Flow cytometric measurement of ceroid accumulation in macrophages

The exposure of proteins to high concentrations of glucose in vitro is widely considered a relevant model of the functional degeneration of tissue occurring in diabetes mellitus. In particular, the enhanced atherosclerosis in diabetes is often discussed in terms of glycation of low-density lipoprotein (LDL), the non-enzymic attachment of glucose to apolipoprotein amino groups. However, glucose can undergo transition-metal-catalysed oxidation under near-physiological conditions in vitro, producing oxidants that possess a reactivity similar to the hydroxyl radical. These oxidants can fragment protein, hydroxylate benzoic acid and induce lipid peroxidation in human LDL. In this study, glycation of LDL in vitro is accompanied by such oxidative processes. However, the oxidation of LDL varies with glucose concentration in a manner which does not parallel changes in protein glycation. Glycation increases in proportion to glucose concentration, whereas in our studies maximal oxidation occurs at a glucose concentration of approx. 25 mM. The modification of LDL resulting from exposure to glucose alters macrophage ceroid accumulation, a process which occurs in the human atherosclerotic plaque. The accumulation of ceroid in macrophages is shown to be related to LDL oxidation rather than LDL glycation, per se, as it too occurs at a maximum of approx. 25 mM. Oxidative sequelae of protein glycation appear to be a major factor in LDL-macrophage interactions, at least with respect to ceroid accumulation. Our observations are discussed in the context of the observed increase in the severity of atherosclerosis in diabetes.

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“…Resident peritoneal macrophages were isolated from male Balb/c mice aged 6-8 weeks as previously described [42][43][44].…”

Section: Methodsmentioning

confidence: 99%

Glucose oxidation and low-density lipoprotein-induced macrophage ceroid accumulation: possible implications for diabetic atherosclerosis

Hunt

Bottoms

Clare

et al. 1994

Biochemical Journal

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“…Lipoproteins were prepared as previously described (4, 14). The molar ratio of lipid to BSA was 60:l. During preparation, BSA was used a t 10 mg/ml (150 pM) in phosphate-buffered saline (PBS) and the cholesteryl linoleate (9 mM) was first dissolved in acetone (BDH-Analar), added to the BSA while vortexing and then sonicated for 1 min.…”

Section: Artificial Lipoprotein Preparationmentioning

confidence: 99%

“…Murine peritoneal macrophages (MPM) were isolated from male BALB/c mice, aged 6-8 weeks, as previously described (4, 14). Cells were resuspended in RPMI 1640 medium containing 10% lipoprotein-deficient fetal calf serum (LPDFCS) and plated at the desired cell number.…”

Section: Cell Culturementioning

confidence: 99%

“…Cells were then collected and any adherent cells remaining were scrape-harvested in 3 ml Hanks balanced salt solution (HBSS). A cell pellet was prepared by centrifugation as previously described (4, 14). For flow cytometric analysis, cells were resuspended in 0.25 ml FACS fixative (1.5% formaldehyde, 0.1% BSA in PBS) by vortexing.…”

Section: Harvesting Of Cellsmentioning

confidence: 99%

“…We have investigated flow cytometry as a method of monitoring ceroid accumulation more accurately in macrophages cultured with lipoproteins. In previous studies, excitation by 351.1-363.8nm lines of an argon ion laser and emission at 490 nm and above were chosen for the measurements (14). The choice of these parameters of fluorescence were based upon experience with the fluorescence microscopy of ceroid in human lesions and in macrophages in vitro (4, 19).…”

mentioning

confidence: 99%

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Measurement of ceroid accumulation in macrophages by flow cytometry

Hunt¹,

Bottoms²,

Skamarauskas³

et al. 1994

Cytometry

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The accumulation in macrophages of ceroid, an sutofluorescent polymer composed of oxidised protein and lipid, can be monitored seiniquantitatively by staining techniques. However, such methods are crude and give little infor mation about the amount of ceroid within cells. Flow cytometry, however, can give a quantitative assessment of cellular ceroid accumulation in vitro. Recently, flow cytometry was explored as a method for measurement of the accumulation in macrophages of ceroid. The accumulation appeared to be diminished in the presence of the antioxidant, α‐tocopherol. This is consistent with the role of lipoprotein oxidation in ceroid accumulation. Here the optimum wavelengths of emission and excitation, using both conventional fluorescence spectroscopy of cellular ceroid and w cytometric measurements with a number of optical filters, are determined. The use of optimal wavelengths determined in these studies enhances overall sensitivity. The findings are discussed in the context of future investigation of cell‐mediated lipid oxidation and its potential antagonists. © 1994 Wiley‐Liss, Inc.

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Characterization of viable autofluorescent macrophages among cultured peripheral blood mononuclear cells

et al. 2001

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Background Inflammatory macrophages that demonstrate intense autofluorescence have been isolated directly from alveolar and peritoneal tissues, but their generation in vitro remains vague. We use flow cytometry to identify a population of autofluorescent macrophages as they arise among nonadherent populations of cultured blood mononuclear cells. Methods Cells were obtained from donated blood buffy coats and placed in culture for 14 days. Unstained populations from the cells remaining in suspension were sampled daily using flow cytometry. During the first 5 culture days, a distinct population of autofluorescent cells arose and comprised an average of ≤14% of the total cell population. This population declined to less than 6% by culture day 8. Results The cells were identified as viable macrophages expressing CD68, lysozyme, and HLA‐DR. Quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) demonstrated a unique cytokine profile with IL‐1α expression levels 138‐fold higher than those measured in uncultured monocytes. No significant elevation in the levels of other cytokines was identified. Upon replating, the sorted populations became readherent, were able to ingest plastic beads, and remained viable for 6 or more additional weeks in culture without evidence of proliferation or multinucleation. Conclusion Viable autofluorescent macrophage populations arising among cultured peripheral blood may be easily identified and isolated for further study using flow cytometry. Cytometry 44:38–44, 2001. Published 2001 Wiley‐Liss, Inc.

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Flow cytometric measurement of ceroid accumulation in macrophages

Cited by 9 publications

References 26 publications

Glucose oxidation and low-density lipoprotein-induced macrophage ceroid accumulation: possible implications for diabetic atherosclerosis

Glucose oxidation and low-density lipoprotein-induced macrophage ceroid accumulation: possible implications for diabetic atherosclerosis

Measurement of ceroid accumulation in macrophages by flow cytometry

Characterization of viable autofluorescent macrophages among cultured peripheral blood mononuclear cells

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