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Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
as macrophages from
wild-type mice. In vivo, the infection progressed at similar rates in
wild-type and NO-deficient mice during the first 2 mo of infection, but
the latter mice were subsequently more efficient in clearing the
mycobacteria than the former. The increased resistance of iNOS-/-
mice was associated with higher IFN- levels in the serum and
following in vitro restimulation of spleen cells with specific Ag,
increased formation of granulomas and increased survival of
CD4+ T cells. We show that NO is not involved in the
antimycobacterial mechanisms of M. avium-infected
macrophages and, furthermore, that it exacerbates the infection by
causing the suppression of the immune response to the
pathogen. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF- (3, 4). The
antimicrobial mechanisms used by the activated macrophages to control
M. avium growth are not clear. The oxygen-reactive
intermediates produced by activated macrophages do not seem to play a
major role in the control of bacterial proliferation, with the
exception of a few strains with limited virulence (5). Recently, it has
been argued that the acidification of M. avium-containing
phagosomes following stimulation of these phagocytes with IFN- and
LPS was underlying the killing of the infecting mycobacterium (6).
Following the observations by Stuehr and Marletta (7, 8) that
macrophages can secrete large amounts of nitrite and nitrate in
response to bacterial products and cytokines, there have been countless
reports implicating NO in the antimicrobial activity of macrophages
against a wide variety of microorganisms (reviewed in Refs. 9 and 10).
In the case of mycobacteria, NO is claimed to play a crucial role in
the control of M. tuberculosis and M. bovis
bacillus Calmette-Guérin (BCG). Chan et al. (11) have shown that
the restriction of M. tuberculosis growth induced in
macrophages by IFN- plus LPS or TNF- was partially reverted by
the addition of an inducible NO synthase
(iNOS)3 inhibitor. The same
authors used two NO synthase inhibitors in vivo to treat mice after
infection with M. tuberculosis. Mice rendered incapable of
producing NO during infection showed increased mortality along with
increased numbers of mycobacteria in their organs, as compared with
control untreated mice (12). These results were more recently confirmed
by MacMicking et al. (13) using iNOS gene-disrupted mice. Studying the
mechanisms of microbial growth inhibition in IFN--activated
macrophages, Flesch and Kaufmann (14) have also found a role for NO in
the inhibition of growth of M. bovis BCG.
In the case of M. avium, the role of NO is not so clearly
established. When studying the interaction of M. avium with
bone marrow-derived macrophages (BMM
) in vitro, we saw that most
strains are not eliminated inside the macrophages, but rather persist
and grow. The growth of a wide panel of M. avium strains was
shown to be inhibited if the macrophages were treated with IFN-
and/or TNF- (3, 4). However, this restriction of mycobacterial
growth was not reversed in the presence of the NO synthase inhibitor
NG-monomethyl-L-arginine (3). Similar
observations were also reported by Bermudez (15). Observations by other
groups on the resistance of M. avium to nitrite or NO
generated in vitro (16, 17) further suggested that the production of
nitrogen-reactive species was not a key factor in the control of
M. avium infection in the mouse. More recently, Doherty and
Sher (18), using iNOS gene-disrupted mice, showed that M.
avium growth was not increased in the absence of iNOS and
suggested a negative role for NO in the suppression of T cell function.
We used iNOS-deficient mice to assess the role of NO in M.
avium infection and found that NO strongly exacerbated M.
avium infection at late time points, probably by inhibiting the
production of IFN- by Ag-specific T cells. While this manuscript was
being prepared, Karupiah et al. (19) reported that iNOS gene-disrupted
mice showed increased resistance to infection by influenza A virus.
These authors showed that this increased resistance was associated with
and dependent on an increased production of IFN- by lung cells and
that iNOS gene-disrupted mice had less inflammation-related lung
pathology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. avium strain 25291, smooth transparent (SmT) variant, was obtained from the American Type Culture Collection (Manassas, VA). Strains 2447 SmT and 2-151 SmD were isolated from AIDS patients and given to us by F. Portaels (Institute of Tropical Medicine, Antwerp, Belgium) and J. Belisle (Colorado State University, Fort Collins, CO), respectively.
All mycobacteria were grown in Middlebrook 7H9 broth (Difco, Detroit, MI) with 0.04% Tween 80 (Sigma, St. Louis, MO). Cultures were harvested during log phase, centrifuged, washed in saline with Tween 80, briefly sonicated, and stored in aliquots at -70°C until used.
Animals
iNOS-deficient mice were bred in our facilities from a breeding pair kindly provided by Drs. J. D. MacMicking and C. Nathan (Cornell University, New York, NY) and J. Mudgett (Merck Research Laboratories, Rahway, NJ) (20). These mice were kept in HEPA-filter-bearing cages and fed sterilized chow and water. From the initial breeders, males were chosen to start the backcrossing with C57BL/6 females. Backcrossing was monitored by performing PCR of the markers for the disrupted gene on DNA samples from the progeny as described below. In one experiment, the progeny of the seventh backcross were used in an in vivo infection experiment.
C57BL/6 mice were purchased from the Gulbenkian Institute (Oeiras, Portugal), and 129Sv mice were purchased from Harlan (Bicester, U.K.). These mice were kept under standard hygiene conditions.
PCR screening for natural resistance-associated macrophage protein 1 (Nramp1) allele
Genomic DNA samples were obtained from each mouse by treating a
portion of the ear with proteinase K (Sigma). The amplification of the
Nramp1 gene was performed using Taq DNA
polymerase (Ampligène-Oncor, Gaithersburg, MD) and primers
specific for the Nramp1 gene, one oligonucleotide being
common to both alleles and the other being specific for either R or S
allele, as described elsewhere (21). The amplification was done in a
Gene Amp PCR System 9600 (Perkin-Elmer-Roche, Branchburg, NJ). Results
for the iNOS-/- mice, as well as 129Sv, C57BL/6, and (129Sv x
C57BL/6)F1 mice, are shown in Fig. 1
. According to the results of this
screening, 129Sv mice were used in all experiments as iNOS+/+ controls,
except for the one experiment where the mice had been backcrossed to
C57BL/6.
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Macrophages were derived from the bone marrow as follows. Each femur was flushed with 5 ml of HBSS. The resulting cell suspension was centrifuged and the cells resuspended in DMEM (Life Technologies, Paisley, U.K.) containing 10% FBS (Life Technologies) and 10% L929 cell-conditioned medium (LCCM) as a source of M-CSF. The cells were distributed in 24-well plates and incubated at 37°C in a 7% CO2 atmosphere. Three days after seeding, another 0.1 ml of LCCM was added. On the 7th day, the medium was renewed.
On the 10th day of culture, when cells were completely differentiated into macrophages, they were infected with M. avium. To each well, about 106 CFU of M. avium were added, in 0.2 ml of DMEM. Cells were incubated for 4 h at 37°C in a CO2 atmosphere and then washed with warm HBSS to remove noninternalized bacteria and reincubated in DMEM, with 10% FBS and 10% LCCM. In some of the wells, the macrophages were immediately lysed and the number of viable intracellular bacteria counted as described below (time zero). The other cells were incubated during 7 days to measure the intracellular growth of the bacteria.
The measurement of mycobacterial growth was done by counting CFU. Briefly, 7 days after infection, the cells were lysed by adding 0.1% saponin to each well. The resulting bacterial suspension was serially diluted 1:10 in water containing 0.04% Tween 80. The dilutions were plated on Middlebrook 7H10 agar (Difco) and the number of colonies counted 8–10 days later. For each condition tested, three culture wells were used. The results presented correspond to the mean and SD of these three wells.
In some of the experiments, macrophages were treated from day 0 to day
4 of infection with recombinant murine IFN- (100 U/well/day), alone,
or in combination with recombinant murine TNF- (50 U/well/day) (both
cytokines from Genzyme, Cambridge, MA)
In vivo infection
Each mouse was infected i.v. with 106 CFU of
M. avium, either strain 2447 SmT or strain 25291 SmT (two
independent experiments). At different time points, animals were
sacrificed and their livers, spleens, and lungs collected. These organs
were homogenized, and serial dilutions of the resulting suspensions
were plated in Middlebrook 7H10 agar medium, as described above for
BMM cultures, and the bacterial colonies counted after culture for
10 days at 37°C.
In one of the experiments, endogenous IFN- activity was blocked by
treating mice with anti-IFN- IgG1, obtained from the hybridoma
XMG1.2 (DNAX, Palo Alto, CA). Control mice were treated with normal rat
Ig. Abs were given i.p., 2 mg per animal every 2 wk.
Characterization of splenic populations by flow cytometry
A single cell suspension was prepared from half of each spleen and the total cell number determined by counting in a hemocytometer. To assess the proportions of CD3+, CD4+, and CD8+ cells present, cells were stained with FITC-conjugated anti-CD3 (17A2) and PE-conjugated anti-CD4 (L3T4) or FITC-anti-CD4 and PE-anti-CD8 (Ly-2) Abs (all from PharMingen, San Diego, CA) and analyzed in a FACSort Apparatus (Becton Dickinson, Mountain View, CA).
In vitro stimulation of splenic cells
Cells obtained from the spleens of each mouse were washed with
HBSS, and the erythrocytes were lysed using a hemolytic solution (155
mM NH4Cl, 10 mM KHCO3 (pH 7.2)). Cells were
then distributed in 96-well plates and incubated in triplicate in
DMEM/10% FCS either with no further stimulus or in the presence of
mycobacterial Ag (4 µg/ml) or Con A (4 µg/ml; Sigma). Supernatants
from the cultures were collected after 3 days in culture, and
quantification of the IFN- produced was done by ELISA. Mycobacterial
Ag was prepared as described elsewhere (22). Briefly, the culture
supernatant of M. avium 25291 SmT grown in Sauton medium was
concentrated by ultrafiltration, precipitated with ammonium sulfate,
and extensively dialysed against PBS.
Detection of IFN- in the serum and in culture supernatants
The measurement of IFN- was done using an ELISA method.
R4-6A2 and biotinylated AN18 anti-IFN- mAbs were used as the
capture and detection Abs, respectively. Recombinant IFN- from
Genzyme was used as standard.
Histology
Portions of the livers of the infected mice were fixed in buffered formaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most strains of M. avium grow exponentially inside
unstimulated murine BMM, the growth rate being dependent on the
particular strain of the bacterium. We have previously shown that when
BMM were treated with IFN- and/or TNF-, the intracellular
growth of M. avium was restricted (3). However, we have not
identified the mechanism underlying this cytokine-induced
bacteriostasis. Since NO is a well-known antimicrobial mechanism of
macrophages, we addressed the question of whether the IFN-- and
TNF--induced mycobacteriostasis seen in BMM was mediated by NO.
We took BMM from mice that do not express the iNOS (iNOS-/- mice)
and infected them with different strains of M. avium, as
described in Materials and Methods. The infected macrophages
were then treated with 100 U of IFN- or 50 U of TNF- per culture
well and per day, both individually and in combination, during the
first 4 days of infection. At different time points after infection,
the cells were lysed and the number of bacteria quantified as CFU in
agar medium. As control, iNOS-expressing macrophages we used BMM
from the 129Sv strain. IFN- treatment led to growth inhibition of
all three strains of M. avium tested in both types of
macrophages (iNOS+/+ or iNOS-/-). TNF- led to a more modest
inhibitory effect, while the combination of the two cytokines was the
most efficient stimulus to induce mycobacteriostasis. The results of
treatment with IFN- plus TNF- are shown in Fig. 2 and compared with untreated
macrophages. While wild-type macrophages treated with IFN- plus
TNF- produced 55.3 ± 5.6, 50.1 ± 2.1, and 47.9 ±
1.6 nmol of NO2- per mg of macrophage protein
during infection by strains 25291 SmT, 2447 SmT, and 2-151 SmD,
respectively, no nitrite was detectable in the culture supernatants of
similarly treated macrophages from iNOS-/- mice. It is clear from
these data that macrophages can inhibit intracellular M.
avium growth even if they are unable to produce NO.
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Since our in vitro results indicated that production of NO was not
a key factor for the control of M. avium growth inside
murine macrophages, we decided to investigate the capacity of
iNOS-deficient mice to control an in vivo infection by M.
avium. As a control for iNOS-/- mice, we used 129Sv mice, the
strain used for the generation of the mutants. We infected them with
two different strains of M. avium, namely strain 25291 SmT,
which is virulent for mice, and strain 2447 SmT, which has a low
virulence. At different time points after infection, we sacrificed four
or five mice of each group and determined the number of CFU in their
organs. The results are shown in Fig. 3.
Mice lacking iNOS not only did not show an increased susceptibility to
infection, but even controlled it more efficiently at late time points
of infection. We also used a small cohort of iNOS-/- that had been
backcrossed to C57BL/6 background infecting them with M.
avium 25291 SmT and using C57BL/6 mice as controls. At 10 wk of
infection, the mycobacterial loads in the spleens, livers, and lungs of
the iNOS-/- were 8.2 ± 0.4, 9.1 ± 0.5, and 7.2 ±
0.3 log 10, respectively, as compared with 10.8 ± 0.1, 10.7
± 0.1, and 8.5 ± 0.4 log 10 in the C57BL/6 organs.
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is required for in vivo control of M. avium
growth in wild-type mice (4, 23), we tested whether this cytokine was
playing a role in the control of the M. avium infection in
iNOS-/- mice. Mice infected with M. avium 25291 SmT were
treated with anti-IFN- mAbs or normal rat Ig and were sacrificed
4 mo after inoculation. As shown in Fig. 4A, mice treated with
anti-IFN- Abs showed increased bacterial multiplication as
compared with control mice, particularly in the spleen and the lung,
confirming that IFN- may mediate bacteriostasis independently of the
secretion of NO.
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is needed for that control. As the
in vitro results had suggested, IFN- is probably activating the
macrophages for intracellular growth restriction or killing of the
bacteria, in an NO-independent way. Differences in the immune response of iNOS-/- and iNOS+/+ mice
Since IFN- was shown to play such a critical role in the growth
of M. avium in the mouse, we wanted to know if the
difference in resistance between iNOS-/- and control mice could be
due to a different capacity to produce IFN- in vivo. To assess that,
we measured the amount of IFN- in the sera of infected mice
throughout the course of infection. In mice infected with M.
avium 25291 SmT, a clear difference in the amounts of IFN-
present in the serum was seen, with iNOS-/- mice showing at least 10
times more cytokine in the serum than their controls throughout the
experiment (Fig. 4
B). Uninfected mice of either strain had
no detectable IFN- in their sera (data not shown).
To relate the differences in IFN- with the function and number of
the producing cells, we initially studied the cell populations in the
spleen during the course of infection with the 25291 SmT strain of
M. avium. As shown in Table I,
the total cell numbers in the spleen were significantly higher in
iNOS-/- mice than in iNOS+/+ mice during the first 2 mo of infection.
This difference was also found both among the CD4+ and
CD8+ T cells, as assessed by FACS analysis, although the
results were not statistically significant. By month 4 of infection,
however, the numbers of cells decreased markedly in the two strains of
mice. At this late time point, the iNOS-deficient mice had
significantly lower numbers of CD4+ and CD8+ T
cells as compared to the wild-type controls. Throughout the time course
of infection, there was no difference in the relative proportions of
CD3+, CD4+, and CD8+ cells in the
spleens between the two strains of mice. To assess the state of
differentiation of the T cells present in the spleens of infected mice,
cell suspensions were prepared that were stimulated in vitro either
with M. avium-secreted Ag or Con A, as described in
Materials and Methods. After 3 days in culture, the
supernatants were collected and assayed for IFN-. Splenic cells from
iNOS-/- mice produced two to four times more IFN- than cells from
iNOS+/+ mice (Fig. 4C), a difference which could not be
accounted for by alterations in the percentage of CD4+ T
cells in the cultures (Table I). Ag stimulation of splenic cells from
uninfected mice of either strain led to no detectable secretion of
IFN- (data not shown).
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, both the number of granulomas
and their size were considerably higher in iNOS-/- as compared with
their controls. Granulomas of the iNOS-deficient mice consisted of a
well-structured core of epithelioid macrophages surrounded by an
extense mantle of lymphoid cells, whereas the organization of
granulomas in wild-type mice was very incipient at both time points. No
granulomatous inflammation or signs of any infectious disease were
found in uninfected mice of either strain.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One could argue that the differences in mycobacterial growth between the two groups of animals in our experiments could be related to differences in genetic background other than the iNOS gene. We find this very unlikely for the following reasons. First, the Nramp1 gene is the only gene identified so far to have a proven role in determining resistance to M. avium in murine models of infection. We screened our breeders of iNOS-/- mice and found them to have the wild-type (resistant) allele of Nramp1 gene in homozygosity. Therefore, our in vivo data were obtained by comparing infections of iNOS-/- mice with the 129Sv strain, also harboring the wild-type allele of Nramp1. Second, 129Sv mice are naturally resistant to infection, and, therefore, the iNOS-deficient mice would be expected to be equally resistant or more susceptible to infection rather than more resistant as observed here. Third, the kinetics of the infection showed that the increased resistance of the knock-out mice was a late event, and there have been no such differences in resolution of infection reported for M. avium infections when comparing different strains of mice. However, this evidence cannot exclude the possibility that unidentified genes playing a role in determining resistance to M. avium, and present in distinct allelic forms in the strains used to obtain the iNOS-deficient animals, could underlie the different susceptibilities to infection observed here. Therefore, we started to backcross the iNOS-/- strain to the C57BL/6 background, and, from a limited cohort of animals, we were able to confirm the data previously generated. It should, however, be stressed that great care should be taken when using recently generated gene-disrupted animals, as there is always a risk that data generated with those animals may depend as much on the genetically engineered deficiency as on unrelated genetic differences between the strains used in the studies.
Our data are consistent with the interpretation that NO produced during
infection hampers the development or maintenance of the protective
immune response. The numbers of lymphocytes present in the spleen were
higher in iNOS-/- mice than in the wild-type controls during the
first mo of infection, including the CD4+ T cells, which
are the cells required for the development of protective immunity
against M. avium (4). When the latter cells were stimulated
in vitro with the specific Ag, a higher production of IFN- was found
among cells from infected iNOS-/- mice than from the controls
suggesting that there was a loss or reduction of differentiation of
Ag-specific Th1 cells when mice had the ability to produce NO in
response to infection. Previous work done with T cell clones had
suggested that NO has an inhibitory effect on IFN- production by Th1
cells (24). The fact that IFN- responses to a mitogen were not
affected in the same way as the Ag-specific responses in our
experiments means that the results with the mycobacterial Ag are not
due to in vitro inhibitory effects of NO and suggests that the in vivo
effects are most prominent on cells that are responding specifically to
Ag rather than a general effect on the whole CD4+ T cell
population. Furthermore, there was an in vivo correlate for these
results, namely the amounts of serum IFN- detected throughout the
infection, which were higher in the iNOS-deficient mice as compared
with the controls. These results are consistent with those obtained by
MacMicking et al. (13), who found higher levels of IFN- in the
plasma of iNOS-/- vs iNOS+/+ mice infected with M.
tuberculosis at early time points of infection, when there were no
significant differences in bacterial burdens in the organs of infected
mice. The negative effect on protective immunity mediated by NO may be
particularly evident in our model of infection due to the high
resistance of M. avium to NO in contrast to other microbes.
Data similar to ours have been recently reported for an influenza model
of infection (19). In that study, both increased resistance and
increased IFN- responses were found in iNOS-/- mice as compared
with control animals, showing that this scenario may be more than an
odd occurrence.
At 4 mo of infection, we found a drastic reduction in the number of T
cells in both strains of mice, showing that there are other mechanisms
affecting the survival of these cells during an M. avium
infection. However, despite the marked decrease in total
CD4+ cells, the amount of IFN- induced by Ag stimulation
remained high, suggesting that Ag-specific CD4+ T cells
could survive this latter mechanism of T cell depletion or,
alternatively, that these cells further differentiated into very high
producers of the cytokine, allowing for a certain loss among them.
Work by other groups has already implicated NO in immunosuppression.
Secondary immunosuppression following infection with attenuated
Salmonella typhimurium was shown to be mediated by
NO-dependent mechanisms associated with IFN--activated macrophages
(25). Likewise, activated macrophages from Corynebacterium
parvum-treated rats were shown to inhibit lymphocyte proliferation
driven by a mitogen through the production of NO (26). Similar
NO-mediated inhibitory mechanisms were described for mouse resident
peritoneal macrophages responsible for reducing mitogen-stimulated T
cell proliferation (27). On the other hand, NO may regulate macrophage
function in an autocrine/paracrine way, as illustrated by the
down-modulation of IL-6 secretion in IFN--treated, Legionella
pneumophila-infected macrophages (28). Gregory et al. (29) showed,
in addition, that NO could have a deleterious effect on the development
of protective immune responses to Listeria monocytogenes,
although there is still no consensus as to whether NO may be required
for the control of experimental listeriosis (20, 31, 32, 33). A tight
regulation of NO production appears to be fundamental for the host
response. With M. avium, however, having no direct role in
the killing of the mycobacteria, NO’s role is just a deleterious one.
It is still not clear whether its effects are due to a direct toxicity
on T cells or to a more subtle regulatory role, such as the
determination of the balance between the type of immune response (24).
We have already described the state of unresponsiveness to unrelated
Ags that develops during M. avium infection and the role
played by the activated macrophages in this mechanism (33). We
speculate that NO may be involved in such nonspecific
immunosuppression.
Two of the immunological outcomes of the lack of NO synthesis were the
increased production of IFN- and the increase in size and number of
granulomas. The latter effect of NO depletion has been described in
other models, such as following pulmonary embolization with latex beads
coupled to purified protein derivative in sensitized animals (34). It
is unclear at the moment if the increased resistance to M.
avium in iNOS-/- mice is due to either of these effects or
whether additional T cell-associated mechanisms may be the culprits of
the late killing of M. avium seen in the iNOS-deficient
mice. The organization of granulomata in mice, namely during
mycobacterial infections, has been found different from that in humans.
Our results suggest that NO may be related to this. In fact, NO
production in human macrophages has been shown to be of a much lesser
magnitude and, maybe for that reason, granulomas in man are better
organized. We should also stress that we were able to detect
differences in granuloma formation in iNOS-/- mice as compared with
the controls because they were matched for the allele of
Nramp1 they express. Otherwise, when comparing granuloma
formation in the iNOS-deficient mouse strain (naturally resistant,
expressing the G169 allele) to mice harboring the susceptible allele
(D169), one would not have found such dramatic differences as bacterial
growth would be faster in the wild-type but naturally susceptible
animals as compared with the resistant ones, leading to bacterial loads
able to trigger a more vigorous granuloma response.
In summary, we report here a counterprotective role for NO in an infectious model, the basis of which probably being related to its immunosuppressive effects. These data illustrate the double-edged sword nature of NO and the delicate equilibrium that should underlie its production. Also, the opposite effects of NO on the resistance of mice to two different mycobacterial species, namely M. avium and M. tuberculosis, emphasize the distinct nature of the strategies adopted by these pathogens to survive the host’s antimicrobial machinery. Of relevance to human disease is the fact that M. avium infection is especially frequent in HIV-infected individuals, in whom it could cause, through NO production, a further deterioration of the already impaired T cell function.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rui Appelberg, Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology, Rua do Campo Alegre 823, 4150-171 Porto, Portugal. E-mail address:
3 Abbreviations used in this paper: iNOS, inducible NO synthase; BMM, bone marrow-derived macrophage; LCCM, L-929 cell conditioned medium; Nramp1, natural resistance-associated macrophage protein 1; SmT, smooth transparent; SmD, smooth-domed.
Received for publication January 4, 1999. Accepted for publication March 17, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interferon and tumor necrosis factor during T-cell-independent and -dependent phases of Mycobacterium avium infection. Infect. Immun. 62:3962.. J. Immunol. 139:518.[Abstract]
-dependent clearance of influenza A virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice. J. Exp. Med. 188:1541. in the induction of the nitric oxide-synthesizing pathway. J. Immunol. 147:144.[Abstract]
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C. G. Feng, C. M. Collazo-Custodio, M. Eckhaus, S. Hieny, Y. Belkaid, K. Elkins, D. Jankovic, G. A. Taylor, and A. Sher Mice Deficient in LRG-47 Display Increased Susceptibility to Mycobacterial Infection Associated with the Induction of Lymphopenia J. Immunol., January 15, 2004; 172(2): 1163 - 1168. [Abstract] [Full Text] [PDF]
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J. P. Lopez, E. Clark, and V. L. Shepherd Surfactant protein A enhances Mycobacterium avium ingestion but not killing by rat macrophages J. Leukoc. Biol., October 1, 2003; 74(4): 523 - 530. [Abstract] [Full Text] [PDF]
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 M. Poljakovic and K. Persson Urinary tract infection in iNOS-deficient mice with focus on bacterial sensitivity to nitric oxide Am J Physiol Renal Physiol, January 1, 2003; 284(1): F22 - F31. [Abstract] [Full Text] [PDF]
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 Y.-J. Jung, R. LaCourse, L. Ryan, and R. J. North Virulent but not Avirulent Mycobacterium tuberculosis Can Evade the Growth Inhibitory Action of a T Helper 1-dependent, Nitric Oxide Synthase 2-independent Defense in Mice J. Exp. Med., October 7, 2002; 196(7): 991 - 998. [Abstract] [Full Text] [PDF]
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M. S. Gomes and R. Appelberg NRAMP1- or cytokine-induced bacteriostasis of Mycobacterium avium by mouse macrophages is independent of the respiratory burst Microbiology, October 1, 2002; 148(10): 3155 - 3160. [Abstract] [Full Text] [PDF]
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M. L. Olleros, R. Guler, N. Corazza, D. Vesin, H.-P. Eugster, G. Marchal, P. Chavarot, C. Mueller, and I. Garcia Transmembrane TNF Induces an Efficient Cell-Mediated Immunity and Resistance to Mycobacterium bovis Bacillus Calmette-Guerin Infection in the Absence of Secreted TNF and Lymphotoxin-{alpha} J. Immunol., April 1, 2002; 168(7): 3394 - 3401. [Abstract] [Full Text] [PDF]
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S. I. Mannering and C. Cheers Interleukin-2 and Loss of Immunity in Experimental Mycobacterium avium Infection Infect. Immun., January 1, 2002; 70(1): 27 - 35. [Abstract] [Full Text] [PDF]
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S. Ehlers, J. Benini, H.-D. Held, C. Roeck, G. Alber, and S. Uhlig {alpha}{beta} T Cell Receptor-positive Cells and Interferon-{gamma}, but not Inducible Nitric Oxide Synthase, Are Critical for Granuloma Necrosis in a Mouse Model of Mycobacteria-induced Pulmonary Immunopathology J. Exp. Med., December 17, 2001; 194(12): 1847 - 1859. [Abstract] [Full Text] [PDF]
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S. S. Bohlson, J. A. Strasser, J. J. Bower, and J. S. Schorey Role of Complement in Mycobacterium avium Pathogenesis: In Vivo and In Vitro Analyses of the Host Response to Infection in the Absence of Complement Component C3 Infect. Immun., December 1, 2001; 69(12): 7729 - 7735. [Abstract] [Full Text] [PDF]
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 G.A.W. Rook, G. Seah, and A. Ustianowski M. tuberculosis: immunology and vaccination Eur. Respir. J., March 1, 2001; 17(3): 537 - 557. [Abstract] [Full Text] [PDF]
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A. M. Cooper, J. E. Pearl, J. V. Brooks, S. Ehlers, and I. M. Orme Expression of the Nitric Oxide Synthase 2 Gene Is Not Essential for Early Control of Mycobacterium tuberculosis in the Murine Lung Infect. Immun., December 1, 2000; 68(12): 6879 - 6882. [Abstract] [Full Text] [PDF]
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L. B. Adams, C. K. Job, and J. L. Krahenbuhl Role of Inducible Nitric Oxide Synthase in Resistance to Mycobacterium leprae in Mice Infect. Immun., September 1, 2000; 68(9): 5462 - 5465. [Abstract] [Full Text] [PDF]
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 C. Nathan and M. U. Shiloh Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens PNAS, August 1, 2000; 97(16): 8841 - 8848. [Abstract] [Full Text] [PDF]
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P. Mastroeni, A. Vazquez-Torres, F. C. Fang, Y. Xu, S. Khan, C. E. Hormaeche, and G. Dougan Antimicrobial Actions of the NADPH Phagocyte Oxidase and Inducible Nitric Oxide Synthase in Experimental Salmonellosis. II. Effects on Microbial Proliferation and Host Survival In Vivo J. Exp. Med., July 17, 2000; 192(2): 237 - 248. [Abstract] [Full Text] [PDF]
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T. F. Pais and R. Appelberg Macrophage Control of Mycobacterial Growth Induced by Picolinic Acid Is Dependent on Host Cell Apoptosis J. Immunol., January 1, 2000; 164(1): 389 - 397. [Abstract] [Full Text] [PDF]
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