Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the coxsackie/adenovirus receptor

Robert J. Kaner, Stefan Worgall, Philip L. Leopold, Eric Stolze, Eric Milano, Chisa Hidaka, Ramu Ramalingam, Neil R. Hackett, Ravi Singh, Jeffrey Bergelson, Robert Finberg, Erik Falck-Pedersen, Ronald Crystal

Research output: Contribution to journalArticle

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Abstract

Robust expression of genes transferred by adenovirus (Ad) vectors depends upon efficient entry of vectors into target cells. Cells deficient in the coxsackie/adenovirus receptor (CAR) are difficult targets for Admediated gene transfer. We hypothesized that low levels of CAR expression may be responsible, in part, for the relative inefficiency of Ad-mediated gene transfer to human alveolar macrophages (AMs). CAR gene expression was detected in human AMs by reverse transcription - polymerase chain reaction and at low levels by Northern analysis. Indirect immunofluorescence showed specific, low-intensity surface staining for CAR, but at levels below those found on the positive-control A549 human lung epithelial cell line. Consistent with this, AMs expressed Ad vector transgenes 100 to 1,000-fold less efficiently than A549 cells, as assessed using the β-galactosidase reporter (chemiluminescence assay) and green fluorescent protein (fluorescence microscopy and flow cytometry). At high multiplicity of infection, AMs from an HIV+ individual could be transduced with an AdIFNγ vector to secrete detectable human interferon-γ. Ad transgene expression by AMs was blocked by capsid fiber protein, suggesting that CAR is required in the pathway for productive Ad entry into alveolar macrophages. To confirm that Ad transgene expression by AMs is limited by low levels of CAR expression, cells were infected with an Ad vector containing the CAR complementary DNA (cDNA). Enhanced expression of CAR protein was demonstrated by indirect immunofluorescence, and the CAR cDNA-transduced cells showed 5-fold enhancement of subsequent Ad transgene expression. These observations demonstrate that human AMs can be targets for Ad-mediated gene transfer, but that efficiency of transgene expression is limited, at least in part, by low levels of CAR expression.

Original languageEnglish
Pages (from-to)361-370
Number of pages10
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume20
Issue number3
DOIs
Publication statusPublished - 1 Jan 1999
Externally publishedYes

Fingerprint

Coxsackie and Adenovirus Receptor-Like Membrane Protein
Medical Genetics
Alveolar Macrophages
Adenoviridae
Transgenes
Gene transfer
Indirect Fluorescent Antibody Technique
Complementary DNA
In Vitro Techniques
Galactosidases
Genes
Gene Expression
Chemiluminescence
Flow cytometry
Fluorescence microscopy
Polymerase chain reaction
Capsid Proteins
Transcription
Luminescence
Green Fluorescent Proteins

ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

Cite this

Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the coxsackie/adenovirus receptor. / Kaner, Robert J.; Worgall, Stefan; Leopold, Philip L.; Stolze, Eric; Milano, Eric; Hidaka, Chisa; Ramalingam, Ramu; Hackett, Neil R.; Singh, Ravi; Bergelson, Jeffrey; Finberg, Robert; Falck-Pedersen, Erik; Crystal, Ronald.

In: American Journal of Respiratory Cell and Molecular Biology, Vol. 20, No. 3, 01.01.1999, p. 361-370.

Research output: Contribution to journalArticle

Kaner, Robert J. ; Worgall, Stefan ; Leopold, Philip L. ; Stolze, Eric ; Milano, Eric ; Hidaka, Chisa ; Ramalingam, Ramu ; Hackett, Neil R. ; Singh, Ravi ; Bergelson, Jeffrey ; Finberg, Robert ; Falck-Pedersen, Erik ; Crystal, Ronald. / Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the coxsackie/adenovirus receptor. In: American Journal of Respiratory Cell and Molecular Biology. 1999 ; Vol. 20, No. 3. pp. 361-370.
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AU - Worgall, Stefan

AU - Leopold, Philip L.

AU - Stolze, Eric

AU - Milano, Eric

AU - Hidaka, Chisa

AU - Ramalingam, Ramu

AU - Hackett, Neil R.

AU - Singh, Ravi

AU - Bergelson, Jeffrey

AU - Finberg, Robert

AU - Falck-Pedersen, Erik

AU - Crystal, Ronald

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