Cytokine Activation Leads to Acidification and Increases
Maturation of Mycobacterium avium-Containing Phagosomes in
Ulrich E. Schaible,2* Sheila Sturgill-Koszycki,* Paul H. Schlesinger,†and David G. Russell3*
Mycobacterium avium (MAC) organisms multiply in phagosomes that have restricted fusigenicity with lysosomes, do not acidify
due to a paucity of vacuolar proton-ATPases, yet remain accessible to recycling endosomes. During the course of mycobacterial
infections, IFN-?-mediated activation of host and bystander macrophages is a key mechanism in the regulation of bacterial
growth. Here we demonstrate that in keeping with earlier studies, cytokine activation of host macrophages leads to a decrease in
MAC viability, demonstrable by bacterial esterase staining with fluorescein diacetate as well as colony-forming unit counts from
infected cells. Analysis of the pH of MAC phagosomes demonstrated that the vacuoles in activated macrophages equilibrate to pH
5.2, in contrast to pH 6.3 in resting phagocytes. Biochemical analysis of MAC phagosomes from both resting and activated
macrophages confirmed that the lower intraphagosomal pH correlated with an increased accumulation of proton-ATPases. Fur-
thermore, the lower pH is reflected in the transition of MAC phagosomes to a point no longer accessible to transferrin, a marker
of the recycling endosomal system. These alterations parallel the coalescence of bacterial vacuoles from individual bacilli in single
vacuoles to communal vacuoles with multiple bacilli. These data demonstrate that bacteriostatic and bactericidal activities of
activated macrophages are concomitant with alterations in the physiology of the mycobacterial phagosome.
Immunology, 1998, 160: 1290–1296.
acteristics that support the intracellular survival and growth of
these pathogens in professional phagocytes. Previous studies es-
tablished that phagosomes containing MAC4and Mycobacterium
tuberculosis have restricted fusigenicity with lysosomes (1–6) and
do not acidify (2, 7, 8) due to a block in accumulation of vacuolar
proton-ATPase (2). Despite the apparent sequestration of MAC
and M. tuberculosis vacuoles outside the endosomal/lysosomal
continuum, recent studies have shown that these vacuoles are rel-
atively dynamic, maintaining access to glycosphingolipids and
glycoconjugates (9) from the host cell plasmalemma.
Immunoelectron microscopical studies by Clemens and Horwitz
(4) on M. tuberculosis-infected MO revealed the presence of MHC
class II and transferrin receptor in mycobacteria-containing phago-
The Journal of
ollowing phagocytosis, Mycobacterium species reside and
multiply in phagosomes of the host’s macrophages (1).
Mycobacteria-containing phagosomes have unique char-
somes. Indeed, we demonstrated recently that MAC phagosomes,
although restricted in acquisition of proton-ATPases, have access
to cathepsins B, L, and D which enter phagosomes early in their
maturation (10). In spite of this, the high phagosomal pH limits
processing and activation of cathepsin D. The hypothesis that my-
cobacterial phagosomes represent early endosomes stabilized in
this stage was given further credence in two studies showing their
accessibility to transferrin, a marker for the recycling endosomal
system (10, 11).
Several independent studies published during the past 10 years
have highlighted the role of certain cytokines in mycobacterial
infections. It is accepted that activation of MO by T cell-, NK cell-,
or macrophage-derived cytokines such as IFN-?, IL-1, granulo-
cyte-macrophage-CSF, and TNF-?, alone or in concert, can con-
tribute to the antimycobacterial potential of these cells, resulting in
control of the infection in vitro (12–19) and in vivo (20, 21). Here,
we have studied the influence of MO activation by IFN-? and LPS
on the maturation of MAC phagosomes and correlated these
changes with mycobacterial survival. The data presented suggest
that MAC phagosomes are shifted from an early to a late endoso-
mal stage of phagosome maturation by MO activation, which is
concomitant with a reduction in mycobacterial growth and
Materials and Methods
The following Abs were used in this study: the mAb ID4B against
LAMP-1 was obtained from the Developmental Hybridoma Bank, Iowa
City, IA; mouse mAbs E11 and H9 against the vacuolar proton-ATPase E
subunits were generous gifts from Dr. S. Gluck (Washington University,
St. Louis, MO); rat mAb against transferrin receptor (R17/18, Tib217)
were obtained from American Type Culture Collection, mouse mAb anti-
digoxigenin was purchased from Boehringer Mannheim, Indianapolis, IN.
The rabbit polyclonal Ab to cathepsin D was a generous gift from Dr. S.
Kornfeld (Washington University). Secondary species-specific Abs labeled
*Departments of Molecular Microbiology and†Physiology and Cell Biology, Wash-
ington University, School of Medicine, St. Louis, MO 63110
Received for publication May 21, 1997. Accepted for publication October 9, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was in part supported by a postdoctoral fellowship of the stipend program
for infectious diseases by the Deutsche Bundesministerium fu ¨r Bildung, Wissen-
schaft, Forschung, und Technologie, Germany (U.E.S.) and by Grants AI33348 and
HL55936 (D.G.R.). D.G.R. is a recipient of the Burroughs Wellcome Scholar award
in Molecular Parasitology.
2Current address: MPI fur Infektionsbiologie, Monbijoustrasse 2, D-10117 Berlin,
3Address correspondence and reprint requests to Dr. David G. Russell, Department
of Molecular Microbiology, Washington University School of Medicine, 660 S. Eu-
clid Avenue, St. Louis, MO 63110. E-mail address: email@example.com
4Abbreviations used in this paper: MAC, Mycobacterium avium complex; LAMP 1,
lysosome-associated membrane protein 1; NHS, N-hydroxysuccinimide; MO, bone
marrow-derived macrophages; ?2m, ?2-macroglobulin; NO, nitric oxide; iNOS, in-
ducible nitric oxide synthase.
Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00
7. Oh, Y.-K., and R. M. Straubinger. 1995. Intracellular fate of Mycobacterium
avium: use of dual label spectrofluorometry to investigate the influence of bac-
terial viability and opsonization on phagosomal pH and phagosome-lysosome
interaction. Infect. Immun. 64:319.
8. Crowle, A., R. Dahl, E. Ross, and M. H. May. 1991. Evidence that vesicles
containing living Mycobacterium tuberculosis or Mycobacterium avium in cul-
tured human macrophages are not acidic. Infect. Immun. 59:1823.
9. Russell, D. G., J. Dant, and S. Sturgill-Koszycki. 1996. Mycobacterium avium-
and Mycobacterium tuberculosis-containing vacuoles are dynamic, fusion-com-
petent vesicles that are accessible to glycosphingolipids from the host cell plas-
malemma. J. Immunol. 156:4764.
10. Sturgill-Koszycki, S., U. E. Schaible, and D. G. Russell. 1996. Mycobacterium-
containing phagosomes are accessible to early endosomes and reflect a transi-
tional state in normal phagosome biogenesis. EMBO J. 15:6960.
11. Clemens, D. L., and M. A. Horwitz. 1996. The Mycobacterium tuberculosis
phagosome interacts with early endosomes and is accessible to exogenously ad-
ministered transferrin. J. Exp. Med. 184:1349.
12. Rook, G. A. W., J. Steele, M. Ainsworth, and B. R. Champion. 1986. Activation
of macrophages to inhibit proliferation of Mycobacterium tuberculosis: compar-
ison of the effects of recombinant gamma-interferon on human monocytes and
murine peritoneal macrophages. Immunology 59:333.
13. Flesch, I. E. A., and S. H. E. Kaufmann. 1987. Mycobacterial growth inhibition
by interferon-?-activated bone marrow macrophages and differential susceptibil-
ity among strains of Mycobacterium tuberculosis. J. Immunol. 138:4408.
14. Flesch, I. E. A., and S. H. E. Kaufmann. 1988. Attempts to characterize the
mechanisms involved in mycobacterial growth inhibition by gamma-interferon-
activated bone marrow macrophages. Infect. Immun. 56:1464.
15. Denis, M. 1991. Interferon-? treated murine macrophages inhibit growth of tu-
bercle bacilli via the generation of reactive nitrogen intermediates. Cell. Immunol.
16. Flesch, I. E. A., and S. H. E. Kaufmann. 1991. Mechanisms involved in myco-
bacterial growth inhibition by gamma interferon-activated bone marrow macro-
phages: role of reactive nitrogen intermediates. Infect. Immun. 59:3213.
17. Chan, J., Y. Xing, R. S. Magliozzo, and B. R. Bloom. 1992. Killing of virulent
Mycobacterium tuberculosis by reactive nitrogen intermediates produced by ac-
tivated macrophages. J. Exp. Med. 175:1111.
18. Appelberg, R., and I. M. Orme. 1993. Effector mechanisms involved in cytokine-
mediated bacteriostasis of Mycobacterium avium infections in murine macro-
phages. Immunology 80:352.
19. Sarmento, A., and R. Appelberg. 1996. Involvement of reactive oxygen interme-
diates in tumor necrosis factor alpha-dependent bacteriostasis of Mycobacterium
avium. Infect. Immun. 64:3224.
20. Cooper, A. M., D. K. Dalton, T. A. Stewart, J. P. Griffin, D. G. Russell, and
I. M. Orme. 1993. Disseminated tuberculosis in interferon-? gene disrupted mice.
J. Exp. Med. 178:2243.
21. Flynn, J. L., M. M. Goldstein, J. Chan, K. J. Triebold, K. Pfeffer,
C. J. Lowenstein, R. Schreiber, T. W. Mak, and B. R. Bloom. 1995. Tumor
necrosis factor-alpha is required in the protective immune response against My-
cobacterium tuberculosis in mice. Immunity 2:561.
22. McDonough, K. A., and Y. Kress. 1995. Cytotoxicity for lung epithelial cells is
a virulence associated phenotype of Mycobacterium tuberculosis. Infect. Immun.
23. Schlesinger, P. H. 1994. Measuring the pH of pathogen-containing phagosomes.
Methods Cell. Biol. 45:289.
24. Nathan, C. 1990. Nitric oxide as a secretory product of mammalian cells. FASEB
25. Meng, F., and C. A. Lowell. 1997. Lipopolysaccharide (LPS)-induced macro-
phage activation and signal transduction in the absence of Src-family kinases
Hck, Fgr, and Lyn. J. Exp. Med. 185:1661.
26. Denis, M., and E. Gregg. 1990. Recombinant tumor necrosis factor-alpha de-
creases whereas recombinant interleukin-6 increases growth of a virulent strain of
Mycobacterium avium in human macrophages. Immunity 71:139.
27. Flesch, I. E. A., J. H. Hess, I. P. Oswald, and S. H. E. Kaufmann. 1994. Growth
inhibition of Mycobacterium bovis by IFN-? stimulated macrophages: regulation
by endogenous tumor necrosis factor-? and by IL-10. Int. Immunol. 6:693.
28. Chan, J., K. Tanaka, D. Carroll, J. Flynn, and B. R. Bloom. 1995. Effects of nitric
oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis.
Infect. Immun. 63:736.
29. MacMicking, J. D., C. Nathan, G. Horn, N. Chartrain, D. S. Fletcher,
M. Trumbauer, K. Stevens, Q. W. Xie, K. Sokol, N. Hutchinson, H. Chen, and
J. S. Mudgett. 1995. Altered responses to bacterial infection and endocytic shock
in mice lacking inducible nitric oxide synthase. Cell 81:641.
30. Wei, X. Q., I. G. Charles, A. Smith, J. Ure, G. J. Feng, F. P. Huang, D. Xu,
W. Muller, S. Moncada, and F. Y. Liew. 1995. Altered immune responses in mice
lacking inducible nitric oxide synthase. Nature 375:408.
31. Pacelli, R., D. A. Wink, J. A. Cook, M. C. Krishna, W. DeGraff, N. Friedman,
M. Tsokos, A. Samuni, and J. B. Mitchell. 1995. Nitric oxide potentiates hydro-
gen peroxide-induced killing of Escherichia coli. J. Exp. Med. 182:1469.
32. Gobin, J., C. H. Moore, J. R. Reeve, D. K. Wong, B. W. Gibson, and
M. A. Horwitz. 1995. Iron acquisition by Mycobacterium tuberculosis: isolation
and characterization of a family of iron-binding exochelins. Proc. Natl. Acad. Sci.
33. Andrew, P. W., P. S. Jackett, and D. B. Lowrie. 1985. Killing and degradation of
microorganisms by macrophages. In Mononuclear Phagocytes: Physiology and
Pathology, R. T. Dean and W. Jessup, eds. Elsevier/North-Holland Biomedical
Press, Amsterdam, p. 311.
34. Douvas, G. S., M. H. May, and A. J. Crowle. 1993. Transferrin, iron and serum
lipids enhance or inhibit Mycobacterium avium replication in human macro-
phages. J. Infect. Dis. 167:857.
35. O’Brien, L., B. Roberts, and P. W. Andrew. 1996. In vitro interaction of Myco-
bacterium tuberculosis and macrophages: activation of anti-mycobacterial activ-
ity of macrophages and mechanisms of anti-mycobacterial activity. Curr. Top.
Microbiol. Immunol. 215:97.
36. Wayne, L. G. 1994. Dormancy of Mycobacterium tuberculosis and latency of
disease. Eur. J. Clin. Microbiol. Infect. Dis. 13:908.
1296 PHAGOSOME MODULATION IN ACTIVATED M. AVIUM-INFECTED MACROPHAGES