Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1993 Dec 1;178(6):2047–2053. doi: 10.1084/jem.178.6.2047

Restitution of superoxide generation in autosomal cytochrome-negative chronic granulomatous disease (A22(0) CGD)-derived B lymphocyte cell lines by transfection with p22phax cDNA

PMCID: PMC2191282  PMID: 8245781

Abstract

The respiratory burst oxidase of phagocytes and B lymphocytes is a multicomponent enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2-production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or physiologic stimuli, such as phagocytosis in phagocytes or cross- linking of surface immunoglobulin in B lymphocytes. The activity of this enzyme is greatly diminished or absent in patients with chronic granulomatous disease (CGD), an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every CGD patient studied so far, an abnormality has been found in a gene encoding one of the four components of the respiratory burst oxidase: the membrane proteins p22phox or gp91phox which together form the cytochrome b558 protein, or the cytosolic proteins p47phox or p67phox. Autosomal recessive cytochrome-negative CGD (A22(0) CGD) is associated with mutations in the gene coding for p22phox. We report here that the capacity for O2- production and cytochrome b558 protein expression were restored to Epstein-Barr virus-transformed B lymphocytes from two A22(0) CGD patients by transfection with an expression plasmid containing a p22phox cDNA. No detectable O2- was generated by untransfected p22phox-deficient lymphocytes. The genetic reconstitution of the respiratory burst in A22(0) CGD B lymphocytes by transfer of the wild-type p22phox cDNA represents a further step towards somatic gene therapy for this subgroup of A22(0) CGD. This system will also be useful for expression of genetically engineered mutant p22phox proteins in intact cells, facilitating the structure-function analysis of cytochrome b558.

Full Text

The Full Text of this article is available as a PDF (901.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abo A., Pick E., Hall A., Totty N., Teahan C. G., Segal A. W. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature. 1991 Oct 17;353(6345):668–670. doi: 10.1038/353668a0. [DOI] [PubMed] [Google Scholar]
  2. Anderson M. L., Spandidos D. A., Coggins J. R. Electroporation of lymphoid cells: factors affecting the efficiency of transfection. J Biochem Biophys Methods. 1991 Apr;22(3):207–222. doi: 10.1016/0165-022x(91)90069-9. [DOI] [PubMed] [Google Scholar]
  3. Canfield V., Emanuel J. R., Spickofsky N., Levenson R., Margolskee R. F. Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector. Mol Cell Biol. 1990 Apr;10(4):1367–1372. doi: 10.1128/mcb.10.4.1367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chanock S. J., Faust L. R., Barrett D., Bizal C., Maly F. E., Newburger P. E., Ruedi J. M., Smith R. M., Babior B. M. O2- production by B lymphocytes lacking the respiratory burst oxidase subunit p47phox after transfection with an expression vector containing a p47phox cDNA. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10174–10177. doi: 10.1073/pnas.89.21.10174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Clark R. A., Malech H. L., Gallin J. I., Nunoi H., Volpp B. D., Pearson D. W., Nauseef W. M., Curnutte J. T. Genetic variants of chronic granulomatous disease: prevalence of deficiencies of two cytosolic components of the NADPH oxidase system. N Engl J Med. 1989 Sep 7;321(10):647–652. doi: 10.1056/NEJM198909073211005. [DOI] [PubMed] [Google Scholar]
  7. Clark R. A., Volpp B. D., Leidal K. G., Nauseef W. M. Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation. J Clin Invest. 1990 Mar;85(3):714–721. doi: 10.1172/JCI114496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cochrane C. G. Cellular injury by oxidants. Am J Med. 1991 Sep 30;91(3C):23S–30S. doi: 10.1016/0002-9343(91)90280-b. [DOI] [PubMed] [Google Scholar]
  9. Dinauer M. C., Orkin S. H., Brown R., Jesaitis A. J., Parkos C. A. The glycoprotein encoded by the X-linked chronic granulomatous disease locus is a component of the neutrophil cytochrome b complex. 1987 Jun 25-Jul 1Nature. 327(6124):717–720. doi: 10.1038/327717a0. [DOI] [PubMed] [Google Scholar]
  10. Dinauer M. C., Pierce E. A., Bruns G. A., Curnutte J. T., Orkin S. H. Human neutrophil cytochrome b light chain (p22-phox). Gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease. J Clin Invest. 1990 Nov;86(5):1729–1737. doi: 10.1172/JCI114898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dinauer M. C., Pierce E. A., Erickson R. W., Muhlebach T. J., Messner H., Orkin S. H., Seger R. A., Curnutte J. T. Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11231–11235. doi: 10.1073/pnas.88.24.11231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heyworth P. G., Curnutte J. T., Nauseef W. M., Volpp B. D., Pearson D. W., Rosen H., Clark R. A. Neutrophil nicotinamide adenine dinucleotide phosphate oxidase assembly. Translocation of p47-phox and p67-phox requires interaction between p47-phox and cytochrome b558. J Clin Invest. 1991 Jan;87(1):352–356. doi: 10.1172/JCI114993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hopkins P. J., Bemiller L. S., Curnutte J. T. Chronic granulomatous disease: diagnosis and classification at the molecular level. Clin Lab Med. 1992 Jun;12(2):277–304. [PubMed] [Google Scholar]
  14. Knaus U. G., Heyworth P. G., Evans T., Curnutte J. T., Bokoch G. M. Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science. 1991 Dec 6;254(5037):1512–1515. doi: 10.1126/science.1660188. [DOI] [PubMed] [Google Scholar]
  15. Leto T. L., Garrett M. C., Fujii H., Nunoi H. Characterization of neutrophil NADPH oxidase factors p47-phox and p67-phox from recombinant baculoviruses. J Biol Chem. 1991 Oct 15;266(29):19812–19818. [PubMed] [Google Scholar]
  16. Leto T. L., Lomax K. J., Volpp B. D., Nunoi H., Sechler J. M., Nauseef W. M., Clark R. A., Gallin J. I., Malech H. L. Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src. Science. 1990 May 11;248(4956):727–730. doi: 10.1126/science.1692159. [DOI] [PubMed] [Google Scholar]
  17. Lomax K. J., Leto T. L., Nunoi H., Gallin J. I., Malech H. L. Recombinant 47-kilodalton cytosol factor restores NADPH oxidase in chronic granulomatous disease. Science. 1989 Jul 28;245(4916):409–412. doi: 10.1126/science.2547247. [DOI] [PubMed] [Google Scholar]
  18. Maly F. E., Cross A. R., Jones O. T., Wolf-Vorbeck G., Walker C., Dahinden C. A., De Weck A. L. The superoxide generating system of B cell lines. Structural homology with the phagocytic oxidase and triggering via surface Ig. J Immunol. 1988 Apr 1;140(7):2334–2339. [PubMed] [Google Scholar]
  19. Maly F. E., Nakamura M., Gauchat J. F., Urwyler A., Walker C., Dahinden C. A., Cross A. R., Jones O. T., de Weck A. L. Superoxide-dependent nitroblue tetrazolium reduction and expression of cytochrome b-245 components by human tonsillar B lymphocytes and B cell lines. J Immunol. 1989 Feb 15;142(4):1260–1267. [PubMed] [Google Scholar]
  20. Maly F. E., Urwyler A., Rolli H. P., Dahinden C. A., De Weck A. L. A single-photon imaging system for the simultaneous quantitation of luminescent emissions from multiple samples. Anal Biochem. 1988 Feb 1;168(2):462–469. doi: 10.1016/0003-2697(88)90344-2. [DOI] [PubMed] [Google Scholar]
  21. Ohno Y., Buescher E. S., Roberts R., Metcalf J. A., Gallin J. I. Reevaluation of cytochrome b and flavin adenine dinucleotide in neutrophils from patients with chronic granulomatous disease and description of a family with probable autosomal recessive inheritance of cytochrome b deficiency. Blood. 1986 Apr;67(4):1132–1138. [PubMed] [Google Scholar]
  22. Parkos C. A., Dinauer M. C., Jesaitis A. J., Orkin S. H., Curnutte J. T. Absence of both the 91kD and 22kD subunits of human neutrophil cytochrome b in two genetic forms of chronic granulomatous disease. Blood. 1989 May 1;73(6):1416–1420. [PubMed] [Google Scholar]
  23. Parkos C. A., Dinauer M. C., Walker L. E., Allen R. A., Jesaitis A. J., Orkin S. H. Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b. Proc Natl Acad Sci U S A. 1988 May;85(10):3319–3323. doi: 10.1073/pnas.85.10.3319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pember S. O., Heyl B. L., Kinkade J. M., Jr, Lambeth J. D. Cytochrome b558 from (bovine) granulocytes. Partial purification from Triton X-114 extracts and properties of the isolated cytochrome. J Biol Chem. 1984 Aug 25;259(16):10590–10595. [PubMed] [Google Scholar]
  25. Quinn M. T., Curnutte J. T., Parkos C. A., Mullen M. L., Scott P. J., Erickson R. W., Jesaitis A. J. Reconstitution of defective respiratory burst activity with partially purified human neutrophil cytochrome B in two genetic forms of chronic granulomatous disease: possible role of Rap1A. Blood. 1992 May 1;79(9):2438–2445. [PubMed] [Google Scholar]
  26. Quinn M. T., Parkos C. A., Walker L., Orkin S. H., Dinauer M. C., Jesaitis A. J. Association of a Ras-related protein with cytochrome b of human neutrophils. Nature. 1989 Nov 9;342(6246):198–200. doi: 10.1038/342198a0. [DOI] [PubMed] [Google Scholar]
  27. Rotrosen D., Yeung C. L., Leto T. L., Malech H. L., Kwong C. H. Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase. Science. 1992 Jun 5;256(5062):1459–1462. doi: 10.1126/science.1318579. [DOI] [PubMed] [Google Scholar]
  28. Smith R. M., Curnutte J. T. Molecular basis of chronic granulomatous disease. Blood. 1991 Feb 15;77(4):673–686. [PubMed] [Google Scholar]
  29. Thrasher A., Chetty M., Casimir C., Segal A. W. Restoration of superoxide generation to a chronic granulomatous disease-derived B-cell line by retrovirus mediated gene transfer. Blood. 1992 Sep 1;80(5):1125–1129. [PubMed] [Google Scholar]
  30. Tucker K. A., Lilly M. B., Heck L., Jr, Rado T. A. Characterization of a new human diploid myeloid leukemia cell line (PLB-985) with granulocytic and monocytic differentiating capacity. Blood. 1987 Aug;70(2):372–378. [PubMed] [Google Scholar]
  31. Verhoeven A. J., Bolscher B. G., Meerhof L. J., van Zwieten R., Keijer J., Weening R. S., Roos D. Characterization of two monoclonal antibodies against cytochrome b558 of human neutrophils. Blood. 1989 May 1;73(6):1686–1694. [PubMed] [Google Scholar]
  32. Volkman D. J., Buescher E. S., Gallin J. I., Fauci A. S. B cell lines as models for inherited phagocytic diseases: abnormal superoxide generation in chronic granulomatous disease and giant granules in Chediak-Higashi syndrome. J Immunol. 1984 Dec;133(6):3006–3009. [PubMed] [Google Scholar]
  33. Volpp B. D., Lin Y. In vitro molecular reconstitution of the respiratory burst in B lymphoblasts from p47-phox-deficient chronic granulomatous disease. J Clin Invest. 1993 Jan;91(1):201–207. doi: 10.1172/JCI116171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Volpp B. D., Nauseef W. M., Donelson J. E., Moser D. R., Clark R. A. Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7195–7199. doi: 10.1073/pnas.86.18.7195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wymann M. P., von Tscharner V., Deranleau D. A., Baggiolini M. Chemiluminescence detection of H2O2 produced by human neutrophils during the respiratory burst. Anal Biochem. 1987 Sep;165(2):371–378. doi: 10.1016/0003-2697(87)90284-3. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES