Retroviral expression of recombinant p47phox protein by Epstein-Barr
virus-transformed B lymphocytes from a patient with autosomal chronic
granulomatous disease
CS Cobbs, HL Malech, TL Leto, SM Freeman, RM Blaese, JI Gallin and KJ Lomax
Laboratory of Host Defenses, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD 20892.
Patients with chronic granulomatous disease (CGD) have recurrent infections
resulting from a failure of phagocytic cells to produce superoxide. One
third of CGD patients have an autosomal gene defect resulting in absence of
p47phox protein, a cytoplasmic component critical to superoxide production
by phagocytic cells. cDNA encoding p47phox has been cloned and recombinant
p47phox (rp47phox) restores superoxide-generating activity to a cell-free
assay containing cell membranes and cytosol from p47phox-deficient CGD
neutrophils. The goal of the present study was to determine the feasibility
of retrovirus mediated expression of rp47phox in the HL60 and U937 human
hematopoietic cell lines, and in an Epstein-Barr virus transformed B-
lymphocyte cell line (EBV-BCL) derived from a p47phox-deficient CGD
patient. Normal EBV-BCL contain p47phox and generate small amounts of
superoxide, while this CGD EBV-BCL lacks any detectable p47phox protein.
Defective amphotropic retrovirus containing p47phox sequence inserted in
the LXSN vector in sense and antisense orientations were used to transduce
HL60, U937, and CGD EBV-BCL. p47phox mRNA sequence was detected in cells
transduced with either sense or antisense retroviral constructs while
rp47phox protein was detected only with the sense construct. The amount of
rp47phox protein produced within these cells was greater than the native
p47phox present in uninduced HL60 or U937 cells, but substantially less
than that present in normal neutrophils, induced HL60 cells, or even normal
EBV-BCL. Differentiation of transduced HL60 cells and the associated
production of native p47phox in response to dimethyl sulfoxide was not
affected. These studies demonstrate that retrovirus constructs can be used
to mediate stable expression of rp47phox protein in human hematopoietic
cell lines and can restore rp47phox protein within the cytosol of
p47phox-deficient EBV-BCL from patients with CGD.
Volume 79,
Issue 7,
pp. 1829-1835,
04/01/1992
Copyright © 1992 by The American Society of Hematology
