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.
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 Ad-mediated 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 beta-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 AdIFNgamma vector to secrete detectable human interferon-gamma. 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.
Division of Pulmonary and Critical Care Medicine, The New York Hospital-Cornell Medical Center, New York, New York 10021, USA. nmohamed@mail.med.cornell.edu
R J
RJ
Kaner
S
S
Worgall
P L
PL
Leopold
E
E
Stolze
E
E
Milano
C
C
Hidaka
R
R
Ramalingam
N R
NR
Hackett
R
R
Singh
J
J
Bergelson
R
R
Finberg
E
E
Falck-Pedersen
R G
RG
Crystal
D000256:Adenoviridae / Q000235:genetics*; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D004770:Enterovirus; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D016676:Macrophages, Alveolar / Q000821:virology*; D012333:RNA, Messenger / Q000032:analysis; D011991:Receptors, Virus / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011991:Receptors, Virus; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma; D001616:beta-Galactosidase