To determine the relative contributions of respiratory burst-derived reactive oxygen intermediates (ROI) versus reactive nitrogen intermediates (RNI) to macrophage-mediated intracellular host defense, mice genetically deficient in these mechanisms were challenged with Leishmania donovani, a protozoan that selectively parasitizes visceral tissue macrophages. During the
early stage of liver infection at wk 2, both respiratory burst-deficient gp91phox
/
(X-linked
chronic granulomatous disease [X-CGD]) mice and inducible nitric oxide synthase (iNOS)
knockout (KO) mice displayed comparably increased susceptibility. Thereafter, infection was
unrestrained in mice lacking iNOS but was fully controlled in X-CGD mice. Mononuclear cell influx into infected liver foci in X-CGD and iNOS KO mice was also overtly impaired at
wk 2. However, granuloma assembly in parasitized tissue eventually developed in both hosts
but with divergent effects: mature granulomas were functionally active (leishmanicidal) in
X-CGD mice but inert in iNOS-deficient animals. These results suggest that (a) ROI and RNI
probably act together in the early stage of intracellular infection to regulate both tissue recruitment of mononuclear inflammatory cells and the initial extent of microbial replication, (b)
RNI alone are necessary and sufficient for eventual control of visceral infection, and (c) although mature granulomas have traditionally been associated with control of such infections,
these structures fail to limit intracellular parasite replication in the absence of iNOS.
Key words:
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Introduction |
Leishmania donovani, a protozoan that causes visceral
leishmaniasis, selectively parasitizes and initially replicates within resident macrophages in the liver, spleen and
bone marrow. The host capable of controlling this disseminated intracellular infection develops an effective T cell-
dependent immune response mediated by proinflammatory,
largely Th1 cell-associated cytokines including IL-12 and
IFN-
(1-3; for review see reference 4). The net effect of
this complex response, which involves granuloma formation (4, 5), is sufficient activation of both tissue macrophages and
influxing blood monocytes to kill most intracellular L. donovani and induce quiescence in residual infection (4).
In vitro studies suggest that mononuclear phagocytes
possess multiple leishmanicidal mechanisms. Cytokine-activated mouse peritoneal macrophages kill ingested L. donovani amastigotes by secreting either reactive oxygen intermediates (ROI; primarily H2O2 [6, 7]) or reactive nitrogen
intermediates (RNI; derived from nitric oxide [8]). Inhibit
ing either pathway prevents mouse macrophage killing of
L. donovani in vitro (6); thus, these mechanisms may also
act in concert (9, 10). In cytokine-activated human monocyte-derived macrophages, H2O2 also readily induces leishmanicidal effects (11); however, a similar role for RNI has
been difficult to detect in cells from normal donors (12). In
vitro-activated human macrophages can also limit L. donovani replication by degrading extracellular tryptophan (13).
Studies carried out to define the macrophage mechanisms
of intracellular Leishmania killing in vivo have been more
limited. Thorough observations in a separate mouse model
of L. major cutaneous infection largely (14) but not entirely (18, 19) support a critical role for inducible nitric oxide
synthase (iNOS)-generated RNI. RNI are not only leishmanicidal (8, 14) but also regulate immunologic pathways, including endogenous secretion of IL-12 and IFN-
(15, 17). In L. donovani-infected BALB/c mice, iNOS mRNA is induced in parasitized tissues (2, 20) and presumed inhibition of iNOS by aminoguanidine (AG) treatment impairs host defense (2). However, iNOS mRNA induction
(20) and generation of iNOS-derived products in serum and
infected organs (21) do not necessarily correlate with control
over L. donovani (20, 21). In the one published study of parasitized tissue in human visceral leishmaniasis, splenic mononuclear phagocytes from each of 22 untreated Indian patients
showed iNOS immunoreactivity (22) (36 ± 3% of cells
were iNOS positive; Murray, H., A. Ding, S. Sundar, and
C. Nathan, unpublished data).
To directly compare RNI and ROI as macrophage-
derived antimicrobial mediators in vivo, we used L. donovani to challenge gene-disrupted iNOS knockout (KO)
(23) and respiratory burst-deficient gp91phox
/
(X-linked
chronic granulomatous disease [X-CGD]) mice (24).
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Materials and Methods |
Animals.
Mice with a targeted disruption of the gp91phox subunit of the NADPH-oxidase complex (phox), derived from a
C57BL/6 × 129/Sv background and backcrossed six times with
C57BL/6 mice (24), were provided as breeders by Dr. M. Dinauer (Indiana University Medical Center, Indianapolis, IN).
C57BL/6 mice (Charles Rivers Labs.) were used as controls.
iNOS
/
KO mice (C57BL/6 × 129/Sv) were maintained as
originally described (23); wild-type+/+ littermates served as controls. Mice were 8-15 wk old when infected; both males and females were used randomly except for control C57BL/6 mice (all female).
Visceral Infection and Treatment.
Groups of four to five mice
were injected via the tail vein with 1.5 × 107 hamster spleen-
derived L. donovani amastigotes (1 Sudan strain, provided by Dr.
D. Sacks, National Institutes of Health, Bethesda, MD) (2, 3).
Visceral infection was followed microscopically using Giemsa-stained liver imprints by counting the number of amastigotes per
500 cell nuclei × liver weight (g) (Leishman-Donovan units, or
LDU) (2, 3). Granuloma formation was scored using formalin-fixed tissue sections stained with hematoxylin and eosin (1). In
some experiments, starting 1 d after infection AG () was added at 1% (wt/vol) to acidified drinking water changed twice weekly; controls received acidified water alone (2). Differences between mean values were analyzed by a two-tailed Student's t test.
 |
Results and Discussion |
Initial Kinetics of Parasite Replication.
L. donovani replicated in the livers of both strains of control mice during the
first 2 wk after challenge (Fig. 1); thereafter, parasite burdens declined consistent with killing and a self-healing phenotype (1). At wk 2, liver burdens in both X-CGD and
iNOS KO mice were significantly higher (by 1.6- and 2.3-fold, respectively) than in control animals. Since macrophages from X-CGD mice produce RNI normally (24)
and macrophages from iNOS KO mice show intact respiratory burst activity (23), the results at wk 2 suggested that
(a) neither mechanism by itself was sufficient to help limit early L. donovani replication and (b) phox and iNOS may
act in concert to achieve optimal initial activity.

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Fig. 1.
Course of L. donovani liver infection in (A) X-CGD ( ) and
control C57BL/6 mice ( ), and in (B) iNOS KO mice ( ) and wild-type controls ( ). Results are mean ± SEM values for 8-15 mice at each
time point from two to four experiments. Asterisk, significantly higher
(P < 0.05) than value in control mice.
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Acquired Resistance and Resolution of Infection.
After wk 2, C57BL/6 and wild-type control mice acquired resistance
and showed near-resolution of infection by wk 8 (Fig. 1). Although initially more susceptible, X-CGD mice also
controlled infection after wk 2 (Fig. 1 A), indicating that
the phox-derived ROI mechanism was dispensable. In
contrast, liver infection was unrestrained in iNOS KO
mice and increased to high levels by wk 8 (Fig. 1 B and see
Fig. 3). Thus, although an intact respiratory burst was neither required (Fig. 1 A) nor sufficient (Fig. 1 B) for acquired resistance and near-resolution of visceral infection, these responses were iNOS dependent.

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Fig. 3.
Histologic response
in livers of infected iNOS KO
mice and wild-type controls. At
wk 2, granulomas are widespread
and developed in controls (A)
but absent at infected foci (arrows) in iNOS KO mice (B). At
wk 4, granulomas are well-established or empty in control mice
(C and E); in iNOS KO mice (D
and F), granulomas are developing but heavily parasitized. At
wk 8, inflammatory reaction in
controls has involuted (G); in
iNOS KO mice (H), granulomas
are well-formed (mature) but
contain numerous replicating
amastigotes (see also Fig. 4).
Original magnification: A, C,
and D, ×200; B and E-H,
×315.
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Fig. 4.
The ineffective granuloma. Photomicrograph shows well-developed tissue structure in 8-wk-infected iNOS KO liver containing a
striking load of intracellular amastigotes. Original magnification: ×500.
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Antileishmanial Effect of iNOS in the Absence of Phox.
To
determine if the capacity of X-CGD mice to control
L. donovani infection involved iNOS-derived products, AG
was used as an iNOS inhibitor (2, 25). Continuous AG
treatment enhanced initial susceptibility of X-CGD mice at
wk 2 (Table I), pointing to an active iNOS-dependent
mechanism. At wk 4, liver parasite burdens in AG-treated
X-CGD mice remained elevated; nevertheless, visceral infection began to come under control (Table I). Although this latter observation raised the possibility of an antimicrobial pathway independent of both phox and iNOS and although such pathways exist (9), we did not verify the degree of iNOS inhibition induced by AG. Since iNOS KO
mice failed to control L. donovani, incomplete iNOS inhibition may well explain the decrease in parasite load at
wk 4 in treated X-CGD animals.

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Fig. 2.
Histologic response
to L. donovani infection in the
liver in X-CGD versus C57BL/6
mice. 2 wk after challenge, granulomas are developing at infected foci (arrows) in C57BL/6
controls (A); in X-CGD mice
(B), there is little or no reaction
at well-parasitized foci (arrows).
At wk 4, granulomas in C57BL/6
mice (C) in this field are mature
and largely parasite free; (D)
shows developing granulomas in
X-CGD mice. At wk 8, (E)
shows receding granulomas in
C57BL/6 mice, whereas (F) illustrates intense, mature reaction in X-CGD mice. Original
magnification: A and B, ×315;
C-F, ×200.
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Tissue Granulomatous Response.
In the liver, acquired
resistance to L. donovani is expressed by a granulomatous
response that encircles all initially parasitized resident macrophages (Kupffer cells) (1). This reaction is underway
by wk 2 and fully evident at wk 4 as resistance develops. The surrounding mantle of the mature leishmanicidal granuloma is formed by influxing T cells and blood monocytes
(4), a process governed by Th1 cell-derived cytokines (2-
4). During resolution of visceral infection (wk 8), the tissue
reaction involutes and most cellular foci are devoid of visible amastigotes and can be scored as in Table II as microscopically "empty" (5).
The kinetics of the granulomatous response and mature
granuloma assembly were similar in control C57BL/6 (Fig.
2, A, C, and E) and wild-type C57BL/6 × 129/Sv (Fig. 3,
A, C, and E, G) mice (Table II). In contrast, the tissue reaction in both X-CGD and iNOS KO animals was delayed
and incomplete, particularly at the early stages. At wk 2, 70-81% of infected foci in X-CGD and iNOS KO livers
had failed to attract any of the early mononuclear cell reaction already evident at 69-81% of parasitized Kupffer cells in control mice (Table II and Figs. 2, A and B, and 3, A
and B). Thus, both phox-derived ROI and iNOS-derived
RNI, presumably acting together, appear to participate in
the early recruitment of T cells and monocytes into infected tissue. This observation further supports the expanding immunoregulatory spectrum of these molecules, particularly NO (10, 26), and in iNOS KO mice may also
reflect suppressed induction of IL-12 and IFN-
(17), key
antileishmanial cytokines involved in both mononuclear
cell influx and macrophage activation (2).
At wk 4, granuloma formation emerged at 90% of infected sites in X-CGD mice (Fig. 2 D) and at 68% of foci
in iNOS KO mice (Fig. 3, D and F, and Table II). However, further assembly into mature granulomas (present at
31-37% of foci in control mice; Figs. 2 C and 3, C and E)
was retarded and <10% of infected foci in either group of
mutant mice could be scored as mature granulomas (Table
II). In X-CGD mice, AG treatment inhibited early granuloma assembly (Table I), supporting an active role for
iNOS in the initial tissue response. However, granulomas
developed by wk 4 despite AG treatment, suggesting a
mechanism unrelated to phox or iNOS or suboptimal pharmacologic inhibition of iNOS.
Results at wk 8 indicated that neither phox nor iNOS
alone was ultimately required for cell recruitment and
assembly, since granulomas developed in both types of deficient mice (Table II). In X-CGD animals, capable of killing L. donovani, the tissue reaction was widespread, comprised of both mature granulomas and inflammatory foci
devoid of parasites (Fig. 2 F), and as in a prior study (29) was exaggerated at some perivascular areas (data not shown).
However, despite the presence of a similarly well-developed inflammatory reaction, mature-appearing granulomas
in iNOS-deficient mice contained overwhelming numbers
of replicating amastigotes defining a novel structure, the
"ineffective granuloma" (Figs. 3 H and 4).
Together, these results demonstrate clear-cut, probable
interdigitating roles for both the respiratory burst and endogenous iNOS-derived RNI as macrophage antimicrobial
mechanisms in the initial host defense response to L. donovani. However, the activity of the respiratory burst in inflammatory cell recruitment and in limiting intracellular
replication is early and transient, since granulomas formed
and infection resolved in X-CGD mice. Similar findings of
enhanced susceptibility followed by control in X-CGD
mice have been reported in a short-term model of Listeria monocytogenes infection (30).
It is possible that mechanisms related to neither phox nor
iNOS, such as other sources of ROI and RNI, also emerge
to complete the induction of the tissue cellular immune response to L. donovani and contribute additional antileishmanial effects. However, our results clearly illustrate an
obligatory role for iNOS in intracellular killing in vivo and
resolution of visceral infection. The presence in iNOS-deficient livers of numerous granulomas, well-established but
heavily-laden with intracellular parasites (Fig. 4), graphically illustrates the tissue consequences of the absence of
this specific macrophage antimicrobial mechanism.
Address correspondence to Henry W. Murray, Cornell University Medical College, 1300 York Ave., Box
130, New York, NY 10021. Phone: 212-746-6330; Fax: 212-746-6332; E-mail: hwmurray{at}mail.med.cornell.edu
We thank M. Dinauer for providing X-CGD breeders; M. Shiloh, S. Nicholson and S. Potter for their help
in establishing the animal colonies; D. Sacks for providing L. donovani; and A. Delph and S. Delph-Etienne
for technical assistance.
This work was supported by National Institutes of Health grants AI16963 (to H.W. Murray) and HL51967
(to C.F. Nathan).
1.
|
Tumang, M.,
C. Keogh,
L.L. Moldawer,
R.F. Teitelbaum,
J. Hariprashad, and
H.W. Murray.
1994.
The role and effect
of tumor necrosis factor-alpha in experimental visceral leishmaniasis.
J. Immunol.
153:
768-775
[Abstract/Free Full Text].
|
2.
|
Taylor, A., and
H.W. Murray.
1997.
Intracellular antimicrobial activity in the absence of interferon- : effect of interleukin 12 in experimental visceral leishmaniasis in interferon-
gene-disrupted mice.
J. Exp. Med.
185:
1231-1239
[Abstract/Free Full Text].
|
3.
|
Murray, H.W..
1997.
Endogenous interleukin 12 regulates
acquired resistance in experimental visceral leishmaniasis.
J. Infect. Dis.
175:
1477-1479
[Medline].
|
4.
| Murray, H.W. 1999. Granulomatous inflammation: host antimicrobial defense in the tissues in visceral leishmaniasis. In
Inflammation: Basic Principles and Clinical Correlates. J.I.
Gallin, R. Snyderman, D.T. Fearon, B.F. Haynes, and C.F.
Nathan, editors. Lippincott-Raven, Philadelphia. In press.
|
5.
|
Wilson, M.E.,
M. Sandor,
A.M. Blum,
B. Younbag,
A. Metwali,
D. Elliot,
R.G. Lynch, and
J.V. Weinstock.
1996.
Local
suppression of IFN- in hepatic granulomas correlates with
tissue-specific replication of Leishmania chagasi.
J. Immunol.
156:
2231-2239
[Abstract].
|
6.
|
Murray, H.W..
1982.
Cell-mediated immune response in experimental visceral leishmaniasis. II. Oxygen-dependent killing of intracellular Leishmania donovani amastigotes.
J. Immunol.
129:
351-357
[Free Full Text].
|
7.
|
Murray, H.W..
1990.
Effect of continuous administration of interferon-gamma in experimental visceral leishmaniasis.
J. Infect.
Dis.
161:
992-994
[Medline].
|
8.
|
Roach, T.I.,
A.F. Kiderlen, and
J.M. Blackwell.
1991.
Role
of inorganic nitrogen oxides and tumor necrosis factor-alpha
in killing Leishmania donovani amastigotes in gamma-interferon lipopolysaccharide-activated macrophages from Lshs
and Lshr congenic mouse strains.
Infect. Immun.
59:
3935-3944
[Medline].
|
9.
| Shiloh, M.U., J.D. MacMicking, S. Nicholoson, J.E. Brause,
S. Potter, M. Marino, F. Fang, M. Dinauer, and C. Nathan.
1999. Phenotype of mice and macrophages deficient in both
phagocyte oxidase and inducible nitric oxide synthase. Immunity. In press.
|
10.
|
Fang, F.C..
1997.
Mechanisms of nitric oxide-related antimicrobial activity.
J. Clin. Invest.
99:
2818-2825
[Free Full Text].
|
11.
|
Murray, H.W., and
D.M. Cartelli.
1983.
Killing of intracellular Leishmania donovani by human mononuclear phagocytes:
evidence for oxygen-dependent and -independent leishmanicidal activity.
J. Clin. Invest.
72:
32-39
[Medline].
|
12.
|
Murray, H.W., and
R.F. Teitelbaum.
1992.
L-arginine-
dependent reactive nitrogen intermediates and the antimicrobial effect of activated human mononuclear phagocytes.
J. Infect. Dis.
165:
513-517
[Medline].
|
13.
|
Murray, H.W.,
A. Szuro-Sudol,
D. Wellner,
M.J. Oca,
A.M. Granger,
D.M. Libby,
C.D. Rothermel, and
B.Y. Rubin.
1989.
Role of tryptophan degradation in respiratory burst-
independent antimicrobial activity of gamma interferon-stimulated human macrophages.
Infect. Immun.
57:
845-849
[Medline].
|
14.
|
Evans, T.G.,
L. Thai,
D.L. Granger, and
J.B. Hibbs.
1993.
Effect of in vivo inhibition of nitric oxide production in murine leishmaniasis.
J. Immunol.
151:
907-915
[Abstract/Free Full Text].
|
15.
|
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:
408-411
[Medline].
|
16.
|
Stenger, S.,
H. Thuring,
M. Rollinghoff, and
C. Bogdan.
1994.
Tissue expression of inducible nitric oxide synthase is
closely associated with resistance to Leishmania major.
J. Exp.
Med.
180:
783-793
[Abstract].
|
17.
|
Diefenbach, A.,
H. Schindler,
N. Donhauser,
E. Lorenz,
T. Laskay,
J. McMicking,
M. Rollinghoff,
I. Gresser, and
C. Bogdan.
1998.
Type I interferon (IFN / ) and type 2 nitric oxide synthase regulate the innate immune response to
a protozoan parasite.
Immunity.
8:
77-87
[Medline].
|
18.
|
Evans, T.G.,
S.S. Reed, and
J.B. Hibbs.
1996.
Nitric oxide
production in murine leishmaniasis: correlation of progressive
infection with increasing systemic synthesis of nitric oxide.
Am. J. Trop. Med. Hyg.
54:
486-489
[Medline].
|
19.
|
Huang, F.-P.,
D. Xu,
E.-O. Esfandiari,
W. Sands,
X. Wei, and
F.Y. Liew.
1998.
Mice defective in Fas are highly susceptible to Leishmania major infection despite elevated IL-12
synthesis, strong Th1 responses, and enhanced nitric oxide
production.
J. Immunol.
160:
4143-4147
[Abstract/Free Full Text].
|
20.
|
Melby, P.C.,
Y.-Z. Yang,
J. Cheng, and
W. Zhao.
1998.
Regional differences in the cellular immune response to experimental cutaneous or visceral leishmaniasis with Leishmania donovani.
Infect. Immun.
66:
18-27
[Abstract/Free Full Text].
|
21.
|
Bories, C.,
E. Scherman, and
P.N. Bories.
1997.
Serum and
tissue nitrate levels in murine visceral leishmaniasis correlate
with parasite load but not with host protection.
Trans. R. Soc.
Trop. Med. Hyg.
91:
433-436
[Medline].
|
22.
| Nicholson, S., M. da G. Bonecini-Almeida, J.R. Lapa e Silva,
C. Nathan, Q.W. Xie, R. Mumford, J.R. Weidner, J. Calaycay, J. Geng, N. Boechat, et al. 1996. Inducible nitric oxide
synthase in pulmonary alveolar macrophages from patients
with tuberculosis. J. Exp. Med. 183:2293-2302.
|
23.
|
McMicking, J.D.,
C. Nathan,
G. Hom,
N. Chartrain,
D.S. Fletcher,
M. Trumbauer,
K. Stevens,
Q. Xie,
K. Sokol,
N. Hutchinson, et al
.
1995.
Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase.
Cell.
81:
641-650
[Medline].
|
24.
|
Pollock, J.D.,
P.A. Williams,
G. Gifford,
L.L. Li,
X. Du,
J. Fisherman,
S.H. Orkin,
C.M. Doershuck, and
M.C. Dinhauer.
1995.
Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production.
Nat. Genet.
9:
202-208
[Medline].
|
25.
|
Chan, J.,
K. Tanaka,
D. Carroll,
J. Flynn, and
B.R. Bloom.
1995.
Effect of nitric oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis.
Infect. Immun.
63:
736-740
[Abstract].
|
26.
|
Nathan, C..
1997.
Inducible nitric oxide synthase: what difference does it make?
J. Clin. Invest.
100:
2417-2423
[Free Full Text].
|
27.
|
Hogaboam, C.M.,
S.W. Chensue,
M.L. Steinhauser,
G.B. Huffnagle,
N.W. Lukacs,
R.M. Strieter, and
S.L. Kunkel.
1997.
Alteration of the cytokine phenotype in an experimental lung granuloma model by inhibiting nitric oxide.
J. Immunol.
159:
5585-5593
[Abstract].
|
28.
|
Bogdan, C..
1998.
The multiplex function of nitric oxide in
(auto)immunity.
J. Exp. Med.
187:
1361-1365
[Free Full Text].
|
29.
|
Morgenstern, D.E.,
M.A.C. Gifford,
L.L. Li,
C.M. Doerschuk, and
M.C. Dinauer.
1997.
Absence of respiratory burst
in X-linked chronic granulomatous disease mice leads to abnormalities in both host defense and inflammatory response
to Aspergillus fumigatus.
J. Exp. Med.
185:
207-218
[Abstract/Free Full Text].
|
30.
|
Dinauer, M.C.,
M.B. Deck, and
E.R. Unanue.
1997.
Mice
lacking reduced nicotinamide adenine dinucleotide phosphate oxidase activity show increased susceptibility to early
infection with Listeria monocytogenes.
J. Immunol.
158:
5581-5583
[Abstract].
|