Professional Documents
Culture Documents
From the "Howard Hughes Medical Institute, Department of Microbiology, and tMonteftore
Medical Center, Albert Einstein College of Medicine, Bronx, New York 10461; and
Cenentech, Inc., South San Francisco, California 94080
Summary
Tuberculosis, a major health problem in developing countries, has reemerged in recent years in
many industrialized countries. The increased susceptibility of immunocompromised individuals
to tuberculosis, and many experimental studies indicate that T cell-mediated immunity plays
an important role in resistance. The lymphokine interferon y (IFN-y) is thought to be a principal
mediator of macrophage activation and resistance to intracellular pathogens . Mice have been
developed which fail to produce IFN-y (gko), because of a targeted disruption of the gene for
IFN-y . Upon infection with Mycobacterium tuberculosis, although they develop granulomas, gko
2249 J . Exp. Med . The Rockefeller University Press " 0022-1007/93/12/2249/06 $2 .00
Volume 178 December 1993 2249-2254
Published December 1, 1993
and at 72C for 2 min for 30 cycles, and then at 72C for 5 min. Figure 2. M. tuberculosis CFU recovered from organs of infected gko
The reactions were electrophoresed on 2% agarose gels, transferred (-40-) and +/+ (-4---) mice. Data shown are combined data from three
to nitrocellulose, and probed with a TNF-a or NOS-specific frag- separate experiments. Error bars, SE .
ment amplified from RNA from the murine macrophage cell line
RAW264 .7 .
1001
80-
Figure 1 . Survival curves of mice infected with M. tuber-
60~ culosis. (A) gko (0) and +/+ (") littermates were infected
z intravenously with 106 M. tuberculosis strain Erdman . Data
40- presented are from three separate experiments, and repre-
sent 18 mice in each group. p = < .0001 in paired Stu-
2o- 20- dent's t test . (B) gko mice were implanted subcutaneously
with Alzet osmotic pumps loaded with IFN-.y (104 U/h)
~~ 0
60 (0) or PBS (0), and infected as above. Infected +/+ lit-
0 10 20 30 40 50 60 0 10 20 30 40 50 termates ( ") were used as controls . Each group represents
DAYS POST INFECTION DAYS POST INFECTION six mice. p = .0036 for PBS and IFN-.y-treated gko mice.
from gko mice were consumed by large necrotizing granu- crease in the number of bacilli recovered from the lungs and
lomas filled with AFB, with much of the tissue destroyed livers, respectively, in the IFN-y-treated mice, compared with
(Fig. 3 E) . Necrosis was not seen in any of the organs from the PBS-treated mice at 14 d after infection (lung mean CFU
either +/+ or +/- mice . The survival curves, together with 5 .2 3 .5 x 10 6 vs 5 .1 3.1 x 107 ; liver mean CFU
the CFU and histological data, indicate that IFN-y is a lym- 3.8 2 .7 x 106 vs 1 .9 0.1 x 108) . All control mice
phokine essential to the survival of mice infected with M. (+/+ and +/- littermates) survived the infection, and ad-
tuberculosis. Necrosis, which is generally not observed in im- ministration of IFN-y had no effect on the numbers of bacilli
munocompetent mice, but is observed in human tuberculous recovered from the organs of these mice. When reconstitu-
infections, may be the consequence of the products of the tion was attempted using implantable osmotic pumps to de-
increased bacterial load in gko mice, or of the immune re- liver IFN-y or PBS, a 12 d increase in average survival time
sponse, e.g., possible overproduction of TNF-ci in the in- was seen in gko mice treated with IFN-y compared with
fected mice, or both . PBS-treated mice (28 2 .5 vs 16 0 .4 d, p = .003) (Fig.
Reconstitution of gko Mice with Exogenously Added IFN-y . 1), again with a significant decrease in numbers of bacilli re-
Since the defect in the gko mice was a genetically defined covered from the organs of IFN-y-infused mice at 14 d after
one, the possibility of reconstituting immune function by infection (lung mean CFU 3 .1 1 .6 x 106 VS 1 .6
exogenous recombinant lymphokine was investigated . It was 0.7 x 10 7; liver mean CFU 3 .0 0 .1 x 10 6 vs 3 .3 0.9
reported previously that a relatively susceptible strain of mice, x 108 ). Histological samples from this time point showed
BALB/c, when infected with M. tuberculosis, exhibited increased extensive necrosis with large numbers of AFB in the liver
survival if infused with IFN-y, compared with BALB/c mice and spleen of PBS-infused gko mice, whereas the IFN-y-
given heat-inactivated cytokine (19). We attempted reconsti- infused gko mice did not exhibit necrosis.
tution of gko mice by either of two methods : intramuscular Thus, exogenously added IFN-y reduced the bacterial load
injections of IFN-y or continuous infusion via surgically im- and increased survival of gko mice, demonstrating the im-
planted osmotic pumps . Mice were infected with M. tubercu- portance of IFN-y in the immune response to M. tuberculosis.
losis 2 d after initiation of IFN-y treatment . When IFN-y Nevertheless, treatment with exogenous IFN-y was unable
was given by injection, the gko mice survived 4 d longer to reconstitute fully protective immune function and prevent
on average than gko mice treated with PBS (19 0.7 vs mortality of gko mice using this challenge dose. Although
15 0 .3 d, p = <.0001), and there was a 10-100-fold de- the reconstitution conditions tested were very limited, a
number of possibilities for this outcome require considera- Table 1 . RNI in Sera of M. tuberculosis-infected
tion . Since the circulating half-life of administered IFN-y in gko and +/+ mice
mice is -1 h (Chen, S ., personal communication), it is un-
clear whether the IFN-y was available in effective concentra- Days after
tions, especially in the granulomas . The increased survival Mouse infection Treatment N02
of the mice receiving continuous IFN-y infusion vs IFN-y
injections supports the view that the inability to fully recon- #M
stitute the IFN-y defect may largely be a problem of delivery gko - None <4 .5
and bioavailability. It is also possible that the absence of IFN-y +/+ - None <4 .5
in gko mice has a regulatory effect on the expression of other
gko plus M. tb 8 PBS <4 .5
cytokines that are necessary to control M. tuberculosis infec-
tion, although no differences in the expression levels of TNF-a, gko plus M. tb 8 IFN-y <4 .5
another cytokine involved in macrophage activation, in gko + / + plus M. tb 8 None <4 .5
mice compared with +/+ mice were observed (Fig. 4, and gko plus M. tb 14 PBS 22 .5
our unpublished results). Finally, since exogenous IFN-y gko plus M. tb 14 IFN-y <4 .5
significantly reduced the number of bacilli recovered from + / + plus M. tb 14 None 373 .0
the mice, which probably accounts for the increased time of
survival, more dramatic effects might be seen at lower M.
tuberculosis challenge doses . Sera obtained from mice were treated to reduce N03 to N02, and N02
was measured by the Greiss reagent . 4 .5 uM was the lower limit of de-
To examine the T cell cytokine profiles from gko mice, tection in this assay . Experiment was repeated once, with similar results .
spleens from M. tuberculosis-infected gko and +/+ mice were M. tb, M . tuberculosis .
stimulated in vitro with Con A or tuberculin PPD. Although
splenocytes from all mice tested proliferated to the antigens,
sion of exogenous IFN-y reduced bacillary load and prolonged IFN-y therapy in M. tuberculosis-infected HIV + individuals
survival of infected gko mice, but ultimately failed to protect who retain some level of CD4 T cells, particularly those in-
mice infected with a high challenge dose from death. The fected with multidrug-resistant strains, should be considered .
implications are that effective lymphokine therapy, used as In conclusion, the results presented in this and in the ac-
a single agent, may be difficult to achieve in diseases such companying paper (28) establish IFN-y as a necessary lym-
as tuberculosis which require localization of lymphokines in phokine for a protective immune response to M. tuberculosis
granulomas. However, IFN-y given in combination with ap- infection. Our data further suggest that a key role played by
propriate chemotherapy could contribute significantly to reduc- IFN-y in mediating protection in the mouse is the induction
tion in bacillary numbers and prevention of dissemination. of NOS of macrophages, allowing production of RNI, an
Clearly, gko mice represent an extreme model in which the important microbicidal mechanism for protection against M.
host is unable to produce any IFN-y necessary for activating tuberculosis infection.
microbicidal activities . Thus, the therapeutic possibility of
The authors gratefully acknowledge Susan Kramer for helpful comments and for supplying recombinant
IFN-y and osmotic pumps; Sharon Chen for IFN-y pharmacokinetic data ; Marsha Goldstein, Kathleen
McDonough, Padmini Salgame, and Karen Starko for helpful comments ; Sofia Kuperman for histological
technical assistance; and Andrea Cooper and Ian Orme for kindly sharing their results prior to publication .
References
1. Murray, C.J .L ., K. Styblo, and A. Rouillon . 1992 . In Disease 9. Barnes, RE, S.J. Fong, P.J . Brennan, P.E . Twomey, A.
Control Priorities in Developing Countries. DT Jamison and Mazumder, and R.L . Modlin . 1990. Local production of tumor
WH. Mosley, editors. Oxford University Press for the World necrosis factor and IFN-gamma in tuberculous pleuritis.J Im-
Bank, New York . 50 :233 . munol. 145:149 .
2. Belosevic, M., C.E . Davis, M.S. Meltzer, and C.A . Nary. 1988 . Yamamura, M., K. Uyemura, R.J . Deans, K. Weinberg, TH.
Regulation of activated macrophage antimicrobial activities : Rea, B.R. Bloom, and R.L . Modlin . 1991 . Defining protec-
identification of lymphokines that cooperate with interferon tive responses to pathogens: cytokine profiles in leprosy lesions.
gamma for induction of macrophage resistance to infection . Science (Wash . DC). 254:277 .
J. Immunol. 141 :890 . Nathan, C.F., G. Kaplan, WR. Levis, A. Nusrat, M.D. Witner,
3. Liew, F.Y, S. Millott, C. Parkinson, R.M . Palmer, and S. Mon- S.A . Sherwin, C.K . Job, C.R . Horowitz, R.M. Steinman, and
cada. 1990 . Macrophage killing of Leishmania parasite in vivo Z.A . Cohn . 1986 . Local and systemic effects of intradermal
is mediated by nitric oxide from L-arginine . J. Immunol. recombinant interferon-gamma in patients with lepromatous
144 :4794. leprosy. N. Engl. J. Med. 315:6 .
4. Murray, HW, BY Rubin, and C.D. Rothermel. 1983 . Killing Kaplan, G., A. Nusrat, E.N . Sarno, C.K . Job, J. McElrath,
of intracellular Leishmania donvani by lymphokine-stimulated J.A . Porto, C.F. Nathan, and Z.A . Cohn . 1987 . Cellular re-
human mononuclear phagocytes : evidence that interferon- sponses to the intradermal injection of recombinant human
gamma is the activating lymphokine. J. Clin. Invest. 72:1506. gamma-interferon in lepromatous leprosy patients. Am . J.
5 . Hoover, D.L ., C.A . Nary, and M.S . Meltzer. 1985 . Human Pathol. 128:345 .
monocyte activation for cytotoxicity against intracellular Leish- Orme, I., E. Miller, A. Roberts, S. Furney,J. Griffen, K. Dobos,
mania donovani. Cell . Immunol. 94 :5005. D. Chi, B. Rivoire, and P. Brennan . 1992. T Lymphocytes medi-
6. Kiderlen, A.F., S.H. Kaufmann, and M.L . Lohmann-Matthes. ating protection and cellular cytolysis during the course of
1984 . Protection of mice against the intracellular bacterium Mycobacterium tuberculosis infection. J. Immunol. 148:189 .
Listeria monocytogenes by recombinant immune interferon . Eur. Dalton, D.K., S. Pitts-Meek, S. Keshav, I .S . Figari, A. Bradley,
J. Immunol. 14 :964 . and TA . Stewart. 1993 . Multiple defects of immune cell func-
7. Sasaki, T., M. Mieno, H. Udono, K. Yamaguchi, T. Usui, K. tion in mice with disrupted interferon-gamma genes. Science
Hara, H. Shiku, and E. Nakayama . 1990 . Roles of CD4* and (Wash. DC) . 259:1739 .
CD8* cells, and the effect of administration of recombinant Granger, D.L ., J.B. Hibbs, and L.M . Broadnax . 1991 . Urinary
murine IFN-y in listerial infection. J. Exp. Med. 171:1141. nitrate excretion in relation to murine macrophage activation .
8. Buchmeier, N.A ., and R.D. Schreiber. 1985 . Requirement of Influence of dietary L-arginine and oral NG-monomethyl-
endogenous interferon-gamma production for resolution of L-arginine. J. Immunol . 146:1294.
Listeria monocytogenes infection. Proc. Nad. Acad. Sci. USA . Chan, J., Y Xing, R.S. Magliozzo, and B.R. Bloom. 1992 .
82 :7404 . Killing of virulent Mycobacterium tuberculosis by reactive nitrogen
intermediates produced by activated murine macrophages. J. 23 . Lowenstein, C.J ., C.S. Glatt, D.S . Bredt, and S.H . Snyder. 1992.
Exp. Med. 175:1111. Cloned and expressed macrophage nitric oxide synthase con-
17 . Chomczynski, P., and N. Sacchi . 1987 . Single-step method trasts with the brain enzyme. Proc Natl. Acad. Sci. USA .
of RNA isolation by acid guanidinium thiocyanate-phenol- 89 :6711.
chloroform extraction . Anal. Biochem. 162:156 . 24 . Murray, H.W, BY Rubin, G. Masur, and R.B. Roberts. 1984.
18 . Xie, Q.-W, H.J . Cho, J. Calaycay, R.A . Mumford, K.M . Impaire d production of lymphokines and immune (gamma)
Swiderek, TD. Lee, A. Diny, T Troso, and C. Nathan . 1992 . interferon in the acquired immunodeficiency syndrome . N. Engl.
Cloning and characterization of inducible nitric oxide synthase J. Med. 310:883 .
from mouse macrophages . Science (Wash. DC). 256:225 . 25 . Clerici, M., and G. M. Shearer. 1993 . A TH1 to The switch
19 . Denis, M. 1991 . Involvement of cytokines in determining re- is a critical step in the etiology of HIV infection . Immunol.
sistance and acquired immunity in murine tuberculosis .J. Leu- Today. 14 :107 .
kocyte Biol. 50 :495 . 26 . Hill, A.R ., S. Premkumar, S. Brustein, K. Vaidya, S. Powell,
20 . Denis, M . 1991 . Interferon-gamma-treated murine macro- F.W. Li, and B. Suster. 1991 . Disseminated tuberculosis in the
phages inhibit growth of tubercle bacilli via the generation acquired immunodeficiency syndrome era. Am. Rev. Respir. Dis.
of reactive nitrogen intermediates . Cell. Immunol. 132:150 . 144:1164.
21 . Steuhr, D.J., and M.A . Marletta . 1985 . Mammalian nitratebio- 27 . Abouya, YL ., A. Neaynel, S. Lucas, A. Dago-Akribi, G.
synthesis: mouse macrophages produce nitrite and nitrate in Coulibaly, M. N'Dhatz, J.B. Konan, A. Yapi, and K.M . De
response to Escherichia coli lipopolysaccharide. Proc Natl. Acad. Cock. 1992 . Pneumocystis carinii pneumonia: an uncommon
Sci. USA. 82 :7738. cause of death in African patients with AIDS. Am . Rev. Respir.
22 . Lyons, C.R., G.J . Orloff, andJ.M . Cunningham . 1992 . Mo- Dis. 145:617 .
lecula r cloning and functional expression of an inducible nitric 28 . Cooper, A.M ., D.K . Dalton, TA . Stewart, J.P. Griffin, D.G.
oxide synthase from a murine macrophage cell line. J. BiA Russell, and I.M . Orme. 1993 . Disseminated tuberculosis in
Chem . 267:6370. IFN-,y gene-disrupted mice. J. Exp. Med. 178 :2243 .