Silica binds serum proteins resulting in a shift of the dose-response for silica-induced chemokine expression in an alveolar type II cell line

Our plain-English summary of Silica binds serum proteins resulting in a shift of the dose-response for silica-induced chemokine expression in an alveolar type II cell line, by E.G. Barrett, C. Johnston, G. Oberdörster, et al (Toxicology and Applied Pharmacology, 1999).

The study

• Authors: E.G. Barrett, C. Johnston, G. Oberdörster, J.N. Finkelstein
• Journal: Toxicology and Applied Pharmacology
• Published: 1999;161(2):111-22
• Cited by: 28 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

There is a growing concern about whether the myriad of culture conditions, cell lines, and doses of nonfibrous and fibrous particles used in vitro are truly representative of the complex environment of the in vivo particle exposure situation. The use of serum as a supplement to the growth medium of cultured cells is a widely accepted practice. However, little is known about whether the various serum proteins may interact with the surfaces of particles, consequently altering their toxicity, inflammatory properties, or fibrogenicity, etc. observed in vivo. Using a murine alveolar type II cell line, MLE-15, we measured the early changes in various chemokine mRNA species following exposure of the cells to silica (cristobalite) in the presence or absence of serum. Total mRNA was isolated and assayed using an RNase protection assay after 6 h of particle exposure. We observed that the addition of serum to the culture media reduced the in vitro silica-induced chemokine response (i.e., shift in the dose-response curve) in MLE-15 cells. Further, using Western blot analysis and protein sequencing techniques, we have identified a specific serum component, apolipoprotein-A1 (apo-A1), as a protein in serum that binds selectively to silica, thus leading to the altered chemokine response. We also found that apo-A1 not only binds to silica but also binds to other nonfibrous and fibrous particles such as titanium dioxide and asbestos. These results demonstrate the importance of culture conditions for modifying the outcome of an experiment when performing in vitro particle exposure studies.

Copyright 1999 Academic Press.

In plain English

This was a laboratory (in vitro) study using a murine alveolar type II cell line, MLE-15, and a murine macrophage line, RAW 264.7. The authors exposed cells for 6 hours to cristobalite silica with or without serum and measured chemokine mRNA by RNase protection assay. Adding bovine calf serum eliminated the cristobalite-induced increase in MCP-1 and MIP-2 mRNA, and when particles were pre-incubated with serum, washed and then added to serum-free cultures, the chemokine response was still inhibited. With 1% fetal bovine serum, cristobalite still induced MIP-2 and MCP-1 but only at a higher dose, and the cytotoxicity (LDH) dose-response shifted similarly. A 24-kDa serum protein that bound particles was sequenced and identified as apolipoprotein-A1; purified human apo-A1 produced a dose-dependent decrease in cristobalite-induced MCP-1 and MIP-2. Serum proteins, including the 24-kDa band, were also desorbed from amorphous silica, titanium dioxide, chrysotile and crocidolite. Serum did not significantly alter the response of RAW 264.7 cells. The authors concluded that culture conditions can modify in vitro particle experiments.

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: in vitro experimental study using cultured cell lines, with particle-protein binding, gel electrophoresis, Western blotting and protein sequencing
• Who was studied: Cultured cells: the murine alveolar type II cell line MLE-15 and the murine macrophage cell line RAW 264.7; bronchoalveolar lavage fluid pooled from 12 C57Bl/6J mice was used for particle-binding experiments (University of Rochester, published 1999)
• Exposure: Cristobalite (crystalline silica, mean particle size 0.64 micrometres) at doses of 9, 18, 35 and 70 micrograms per cm2 for 6 h (24 h for cytotoxicity), with or without bovine calf serum or fetal bovine serum; binding experiments also used amorphous silica (Aerosil), titanium dioxide, chrysotile and crocidolite asbestos from the TIMA repository
• Main results:
  • In MLE-15 cells, bovine calf serum abolished the cristobalite-induced MCP-1 and MIP-2 mRNA response
  • When cristobalite particles were pre-incubated with 1% bovine calf serum, isolated and washed, then added to serum-free MLE-15 cultures, the chemokine response was still inhibited
  • With 1% fetal bovine serum, cristobalite still induced MIP-2 and MCP-1 mRNA in MLE-15 cells but a higher particle dose was needed (70 micrograms per cm2 with FBS versus 18 micrograms per cm2 serum-free)
  • Cytotoxicity in MLE-15 cells: under serum-free conditions LDH release rose significantly at 9 (12% total LDH), 18 (30% total LDH) and 35 micrograms per cm2 (52% total LDH); with 1% FBS, 70 micrograms per cm2 was needed (21% total LDH)
  • Four proteins (183, 97, 66 and 24 kDa) were desorbed from cristobalite after incubation with bovine calf serum, the 24-kDa band being the most intense
  • Amorphous silica (Aerosil), titanium dioxide, chrysotile and crocidolite also bound serum proteins; all particles showed bands at 183, 66 and 24 kDa, and all except chrysotile appeared to bind the 24-kDa protein with the highest affinity
  • The 24-kDa particle-bound protein was identified as apolipoprotein-A1 by peptide sequencing and confirmed by immunoblotting
  • Purified human apo-A1 caused a dose-dependent decrease in cristobalite-induced MCP-1 and MIP-2 expression in MLE-15 cells, though the authors noted the inhibition was less than expected if apo-A1 were solely responsible
  • In RAW 264.7 macrophage cells, 5% bovine calf serum did not significantly change the cristobalite-induced chemokine response seen under serum-free conditions
  • Mouse lung lavage proteins bound to cristobalite gave mostly bands different from those seen with serum, apart from the 97 and 66 kDa bands
  • Albumin alone bound cristobalite dose-dependently but had only a slight inhibitory effect on MCP-1 and MIP-2 mRNA levels in MLE-15 cells
• The authors' conclusion: The authors concluded that serum, through binding of serum proteins such as apo-A1 to particles, shifted the cristobalite-induced chemokine dose-response in MLE-15 cells, and that in vitro culture conditions must be critically considered when interpreting particle exposure studies
• Limitations the authors state:
  • Relative amounts of protein bound to different particle types could not be compared because particle doses were not matched for surface area
  • Lavage proteins were more dilute than serum, so a more concentrated lavage sample might have shown additional binding proteins
  • The mechanism of apo-A1 binding to the particles was not determined, and other serum proteins may also contribute
  • An apparent decrease in MIP-1b with increasing cristobalite dose in serum-free RAW 264.7 cultures could not be reproduced consistently
  • Whether the serum effect extends to other cell types, type II cell lines or primary type II cells remains to be tested

Topics

Medical terms in this study, explained in our Lexicon: Dose-response relationship.

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