Skip to main content
PMC home page
Environmental Health Perspectives logoLink to Environmental Health Perspectives
. 1997 Jul;105(7):706–711. doi: 10.1289/ehp.97105706

Fluorescence imaging of reactive oxygen metabolites generated in single macrophage cells (NR8383) upon phagocytosis of natural zeolite (erionite) fibers.

J F Long 1, P K Dutta 1, B D Hogg 1
PMCID: PMC1470100  PMID: 9294716

Abstract

In this paper we address the phenomenon of reactive oxygen metabolite generation subsequent to phagocytosis of mineral fibers by macrophages. Natural erionite fibers were chosen because of their established toxicity. Macrophages (cell line NR8383) were loaded with the dye 5-(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate and exposed to erionite particles by centrifuging cells and fibers together to effect adherence. Reactive oxygen metabolite generation was examined by monitoring the fluorescence of oxidized dye formed via the reaction with oxygen species produced during phagocytosis. Individual cells were repeatedly scanned for up to 2 hr to monitor the evolution of this fluorescence. It was found that erionite-exposed cells had a mean total fluorescence of three times that of controls during the first 35 min, declining to two times that of controls at 35-60 min and about the same level as that of controls at 60-80 min. Ultrastructural studies of similarly treated aliquots of cells showed marked variation in size and numbers of the phagocytized particles. This study demonstrates that intracellular oxidation can be monitored on a single cell basis over a period of time. Quantitative studies are in progress to establish the relationship between the phagocytized particulate load and the extent of fluorescence.

Full text

PDF
706

Images in this article

706p706-a
on p.706
708Figure 1.
on p.708
708Figure 2.
on p.708
709Figure 3.
on p.709
709Figure 4.
on p.709
710Figure 5.
on p.710
710Figure 6.
on p.710
710Figure 7.
on p.710

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allison A. C., Harington J. S., Birbeck M. An examination of the cytotoxic effects of silica on macrophages. J Exp Med. 1966 Aug 1;124(2):141–154. doi: 10.1084/jem.124.2.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arndt-Jovin D. J., Jovin T. M. Fluorescence labeling and microscopy of DNA. Methods Cell Biol. 1989;30:417–448. doi: 10.1016/s0091-679x(08)60989-9. [DOI] [PubMed] [Google Scholar]
  3. Aucoin M. M., Barhoumi R., Kochevar D. T., Granger H. J., Burghardt R. C. Oxidative injury of coronary venular endothelial cells depletes intracellular glutathione and induces HSP 70 mRNA. Am J Physiol. 1995 Apr;268(4 Pt 2):H1651–H1658. doi: 10.1152/ajpheart.1995.268.4.H1651. [DOI] [PubMed] [Google Scholar]
  4. Bass D. A., Parce J. W., Dechatelet L. R., Szejda P., Seeds M. C., Thomas M. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol. 1983 Apr;130(4):1910–1917. [PubMed] [Google Scholar]
  5. Bender H. S., Chickering W. R. Superoxide, superoxide dismutase and the respiratory burst. Vet Clin Pathol. 1983;12(3):7–14. doi: 10.1111/j.1939-165x.1983.tb00615.x. [DOI] [PubMed] [Google Scholar]
  6. Brown R. C., Carthew P., Hoskins J. A., Sara E., Simpson C. F. Surface modification can affect the carcinogenicity of asbestos. Carcinogenesis. 1990 Oct;11(10):1883–1885. doi: 10.1093/carcin/11.10.1883. [DOI] [PubMed] [Google Scholar]
  7. Burghardt R. C., Barhoumi R., Lewis E. H., Bailey R. H., Pyle K. A., Clement B. A., Phillips T. D. Patulin-induced cellular toxicity: a vital fluorescence study. Toxicol Appl Pharmacol. 1992 Feb;112(2):235–244. doi: 10.1016/0041-008x(92)90193-v. [DOI] [PubMed] [Google Scholar]
  8. Cohen H. J., Chovaniec M. E. Superoxide generation by digitonin-stimulated guinea pig granulocytes. A basis for a continuous assay for monitoring superoxide production and for the study of the activation of the generating system. J Clin Invest. 1978 Apr;61(4):1081–1087. doi: 10.1172/JCI109007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gabig T. G., Babior B. M. The O2(-) -forming oxidase responsible for the respiratory burst in human neutrophils. Properties of the solubilized enzyme. J Biol Chem. 1979 Sep 25;254(18):9070–9074. [PubMed] [Google Scholar]
  10. Garner D. L., Johnson L. A. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod. 1995 Aug;53(2):276–284. doi: 10.1095/biolreprod53.2.276. [DOI] [PubMed] [Google Scholar]
  11. Goodglick L. A., Kane A. B. Role of reactive oxygen metabolites in crocidolite asbestos toxicity to mouse macrophages. Cancer Res. 1986 Nov;46(11):5558–5566. [PubMed] [Google Scholar]
  12. Gormley I. P., Kowolik M. J., Cullen R. T. The chemiluminescent response of human phagocytic cells to mineral dusts. Br J Exp Pathol. 1985 Aug;66(4):409–416. [PMC free article] [PubMed] [Google Scholar]
  13. Halliwell B., Gutteridge J. M., Cross C. E. Free radicals, antioxidants, and human disease: where are we now? J Lab Clin Med. 1992 Jun;119(6):598–620. [PubMed] [Google Scholar]
  14. Hansen K., Mossman B. T. Generation of superoxide (O2-.) from alveolar macrophages exposed to asbestiform and nonfibrous particles. Cancer Res. 1987 Mar 15;47(6):1681–1686. [PubMed] [Google Scholar]
  15. Helmke R. J., Boyd R. L., German V. F., Mangos J. A. From growth factor dependence to growth factor responsiveness: the genesis of an alveolar macrophage cell line. In Vitro Cell Dev Biol. 1987 Aug;23(8):567–574. doi: 10.1007/BF02620974. [DOI] [PubMed] [Google Scholar]
  16. Helmke R. J., German V. F., Mangos J. A. A continuous alveolar macrophage cell line: comparisons with freshly derived alveolar macrophages. In Vitro Cell Dev Biol. 1989 Jan;25(1):44–48. doi: 10.1007/BF02624409. [DOI] [PubMed] [Google Scholar]
  17. Hogg B. D., Dutta P. K., Long J. F. In vitro interaction of zeolite fibers with individual cells (macrophages NR8383): measurement of intracellular oxidative burst. Anal Chem. 1996 Jul 15;68(14):2309–2312. doi: 10.1021/ac960176c. [DOI] [PubMed] [Google Scholar]
  18. Hook G. R., Odeyale C. O. Confocal scanning fluorescence microscopy: a new method for phagocytosis research. J Leukoc Biol. 1989 Apr;45(4):277–282. doi: 10.1002/jlb.45.4.277. [DOI] [PubMed] [Google Scholar]
  19. Kennedy T. P., Dodson R., Rao N. V., Ky H., Hopkins C., Baser M., Tolley E., Hoidal J. R. Dusts causing pneumoconiosis generate .OH and produce hemolysis by acting as Fenton catalysts. Arch Biochem Biophys. 1989 Feb 15;269(1):359–364. doi: 10.1016/0003-9861(89)90118-5. [DOI] [PubMed] [Google Scholar]
  20. Kobzik L., Godleski J. J., Brain J. D. Oxidative metabolism in the alveolar macrophage: analysis by flow cytometry. J Leukoc Biol. 1990 Apr;47(4):295–303. [PubMed] [Google Scholar]
  21. LeBel C. P., Ischiropoulos H., Bondy S. C. Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol. 1992 Mar-Apr;5(2):227–231. doi: 10.1021/tx00026a012. [DOI] [PubMed] [Google Scholar]
  22. Mossman B. T., Marsh J. P., Shatos M. A. Alteration of superoxide dismutase activity in tracheal epithelial cells by asbestos and inhibition of cytotoxicity by antioxidants. Lab Invest. 1986 Feb;54(2):204–212. [PubMed] [Google Scholar]
  23. Reynolds I. J., Hastings T. G. Glutamate induces the production of reactive oxygen species in cultured forebrain neurons following NMDA receptor activation. J Neurosci. 1995 May;15(5 Pt 1):3318–3327. doi: 10.1523/JNEUROSCI.15-05-03318.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rom W. N., Travis W. D., Brody A. R. Cellular and molecular basis of the asbestos-related diseases. Am Rev Respir Dis. 1991 Feb;143(2):408–422. doi: 10.1164/ajrccm/143.2.408. [DOI] [PubMed] [Google Scholar]
  25. Royall J. A., Ischiropoulos H. Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys. 1993 May;302(2):348–355. doi: 10.1006/abbi.1993.1222. [DOI] [PubMed] [Google Scholar]
  26. Ryan T. C., Weil G. J., Newburger P. E., Haugland R., Simons E. R. Measurement of superoxide release in the phagovacuoles of immune complex-stimulated human neutrophils. J Immunol Methods. 1990 Jul 3;130(2):223–233. doi: 10.1016/0022-1759(90)90052-w. [DOI] [PubMed] [Google Scholar]
  27. Shenker B. J., Vitale L. A., Keiba I., Harrison G., Berthold P., Golub E., Lally E. T. Flow cytometric analysis of the cytotoxic effects of Actinobacillus actinomycetemcomitans leukotoxin on human natural killer cells. J Leukoc Biol. 1994 Feb;55(2):153–160. doi: 10.1002/jlb.55.2.153. [DOI] [PubMed] [Google Scholar]
  28. Srivastava V., Miller S., Busbee D. Immunofluorescent evaluation of DNA repair synthesis using interactive laser cytometry. Cytometry. 1993;14(2):144–153. doi: 10.1002/cyto.990140206. [DOI] [PubMed] [Google Scholar]
  29. Szejda P., Parce J. W., Seeds M. S., Bass D. A. Flow cytometric quantitation of oxidative product formation by polymorphonuclear leukocytes during phagocytosis. J Immunol. 1984 Dec;133(6):3303–3307. [PubMed] [Google Scholar]
  30. Vallyathan V., Castranova V., Pack D., Leonard S., Shumaker J., Hubbs A. F., Shoemaker D. A., Ramsey D. M., Pretty J. R., McLaurin J. L. Freshly fractured quartz inhalation leads to enhanced lung injury and inflammation. Potential role of free radicals. Am J Respir Crit Care Med. 1995 Sep;152(3):1003–1009. doi: 10.1164/ajrccm.152.3.7663775. [DOI] [PubMed] [Google Scholar]
  31. Vilím V., Wilhelm J., Brzák P., Hurych J. Stimulation of alveolar macrophages by mineral dusts in vitro: luminol-dependent chemiluminescence study. Environ Res. 1987 Feb;42(1):246–256. doi: 10.1016/s0013-9351(87)80026-9. [DOI] [PubMed] [Google Scholar]
  32. Wiessner J. H., Henderson J. D., Jr, Sohnle P. G., Mandel N. S., Mandel G. S. The effect of crystal structure on mouse lung inflammation and fibrosis. Am Rev Respir Dis. 1988 Aug;138(2):445–450. doi: 10.1164/ajrccm/138.2.445. [DOI] [PubMed] [Google Scholar]

Articles from Environmental Health Perspectives are provided here courtesy of National Institute of Environmental Health Sciences

RESOURCES