An adherent cell perifusion technique to study the overall and sequential response of rat alveolar macrophages to toxic substances

Our plain-English summary of An adherent cell perifusion technique to study the overall and sequential response of rat alveolar macrophages to toxic substances, by G. Forget, M.J. Lacroix, A. Cadieux, et al (Environmental Health Perspectives, 1983).

The study

• Authors: G. Forget, M.J. Lacroix, A. Cadieux, R. Calvert, J.H. Grose, P. Sirois
• Journal: Environmental Health Perspectives
• Published: 1983;51:131-40
• Cited by: 12 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Essentially pure (97%) alveolar macrophages were isolated by bronchoalveolar lavage of rats with warm (37 degrees C) PBS solution. These cells were allowed to adhere to the inside walls of open-ended glass cylinders which were closed off at each end by three-way stopcocks. The adhering cells were perifused with RPMI-1640 medium supplemented with 5% fetal bovine serum for 18 hr at the rate of 1 mL/hr, and the effluent medium was collected automatically in 2-mL aliquots. Cell recoveries and viabilities did not differ from those found for Petri cultures treated similarly, indicating that the perifusion method under study offered an adequate milieu for short-term primary cultures. The alveolar macrophages in culture were subjected to the presence of particulate (chrysotile asbestos) and soluble (phorbol myristate) toxicants, and their response was monitored in the effluent medium by measuring the release of prostaglandins (PGE) by radioimmunoassay. A significant increase in the sequential release of PGE was observed in the presence of asbestos (100 micrograms/mL) or phorbol myristate (200 ng/mL). Treatment of the cells with indomethacin (20 microM) completely abolished the release of PGE stimulated with phorbol myristate. A cumulative response to the toxicants was also observed when cells were harvested manually from the chambers: asbestos caused a 2-fold increase in cell mortality relative to control, while phorbol myristate brought about a 3-fold increase in the number of dead cells. This effect was not prevented by the presence of indomethacin. Cell aggregation was also observed when cells were perifused in the presence of phorbol myristate, whether indomethacin was present or absent. Our results indicate that the cell perifusion system combines the advantages of conventional adherent cell cultures (viability, aggregation) with those of perifusion techniques (sequential metabolism studies).

In plain English

In this laboratory study, the authors described a method for keeping rat alveolar macrophages (immune cells collected by washing out rat lungs) alive in glass chambers through which fresh culture medium flowed continuously for 18 hours, so that substances released by the cells could be measured over time. Cells kept this way stayed in the chamber and survived about as well as cells in ordinary Petri dishes. The authors then exposed the cells to Canadian chrysotile B asbestos (100 micrograms per mL) or to a chemical, phorbol myristate. Asbestos-exposed cells released more prostaglandin E, an inflammation-related substance, than control cells, and the increase was statistically significant between 10 and 16 hours. After 18 hours, 44% of asbestos-exposed cells were dead, compared with 21.9% in the control chamber. Phorbol myristate also raised prostaglandin release and cell death. The drug indomethacin blocked that prostaglandin release but not the cell death. The authors concluded that the method combines the advantages of conventional cell cultures with those of flow-through techniques. This was an experiment on rat cells, not a study of people.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) experiment developing and testing a flow-through (perifusion) culture method for rat alveolar macrophages
• Who was studied: Alveolar macrophages obtained by lung lavage of rats; mean cell yield reported from 28 preparations; cells were 97% alveolar macrophages
• Exposure: Canadian chrysotile B asbestos (UICC) at 100 micrograms per mL; for comparison, the soluble compound phorbol myristate (200 ng/mL), alone or with indomethacin, a prostaglandin synthesis inhibitor
• Main results:
  • Without any toxicant, the perifusion chambers retained a number of cells (1.17 ± 0.04 x 10^6, 6 chambers) that did not differ significantly from Petri dishes (1.24 ± 0.06 x 10^6, 7 dishes). Mortality also did not differ significantly (17.1 ± 1.7% vs 16.6 ± 1.5%).
  • In cells perifused without toxicant, undialyzed serum preserved cell viability better than dialyzed serum (mortality 14.5 ± 1.2% vs 22.9 ± 0.6%, p less than 0.01)
  • In the asbestos-treated chamber, prostaglandin E release rose over the first 6 hours to a plateau of about 1.8 ng per million cells per 2 hours. Untreated cells averaged less than 1 ng per 2 hours. The increase was significant between 10 and 16 hours.
  • Cell mortality after 18 hours was 44% in the asbestos chamber versus 21.9% in the control chamber (p less than 0.01)
  • Phorbol myristate increased prostaglandin E release, and indomethacin blocked that increase. Mortality was 32.9% with phorbol myristate alone and 33.9% with phorbol myristate plus indomethacin, versus 10.4% in the control chamber (p less than 0.01).
  • Phorbol myristate caused marked cell clumping (aggregation) with or without indomethacin, compared with 9% in control chambers
• The authors' conclusion: The authors concluded that the perifusion system preserves cell viability as well as traditional cultures. They found it usable with both particulate (asbestos) and soluble compounds. They also stated that their asbestos findings confirm earlier reports of asbestos-induced prostaglandin release from macrophages.
• Limitations the authors state:
  • Cell aggregation was not measured in the asbestos experiments, because the cells clumped onto the asbestos fibers and this prevented easy measurement of aggregate size
  • The authors found it difficult to explain why indomethacin did not lower baseline prostaglandin E output, even though it blocked the release stimulated by phorbol myristate

Topics

Medical terms in this study, explained in our Lexicon: Alveolar Macrophage, Bronchoalveolar Lavage (BAL), Chrysotile.

Related studies

• Response of perifused alveolar macrophages to glass fibers: effect of exposure duration and fiber length — G. Forget, M.J. Lacroix, et al. (1986)
• Response of bovine alveolar macrophages in vitro to welding fume particles — L.R. White, A.B. Marthinsen, et al. (1983)
• Asbestos-induced alveolar injury. Evidence for macrophage-derived PDGF as a mediator of the fibrogenic response — J.C. Bonner, A.R. Brody (1991)
• In vitro reactivity of alveolar macrophages and red blood cells with asbestos fibres treated with oxalic acid, sulfur dioxide and benzo-3,4-pyrene — M.C. Jaurand, L. Magne, et al. (1981)
• Sialic acid mediates the initial binding of positively charged inorganic particles to alveolar macrophage membranes — J.E. Gallagher, G. George, et al. (1987)
• In vitro biodegradation of chrysotile fibres by alveolar macrophages and mesothelial cells in culture: comparison with a pH effect — M.C. Jaurand, A. Gaudichet, et al. (1984)

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