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Controls Intracellular Mycobacterial Growth by Both Inducible Nitric Oxide Synthase-Dependent and Inducible Nitric Oxide Synthase-Independent Pathways1
*
The Rockefeller University, New York, NY 10021;
Department of Immunology, Medical School, University of Cape Town, Groote Schuur Hospital, South Africa; and
Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in the control of mycobacterial growth in
murine macrophages was studied in vitro. Infection of macrophages from
TNF- gene disrupted (TNF-knockout (KO)) mice with recombinant
Mycobacterium bovis bacillus Calmette
Guérin (BCG) expressing the vector only (BCG-vector)
resulted in logarithmic growth of the intracellular bacilli. Infection
with BCG-secreting murine TNF- (BCG-TNF) led to bacillary killing.
Killing of BCG-TNF was associated with rapid accumulation of inducible
NO synthase (iNOS) protein and the production of nitrite. The
uncontrolled growth of BCG-vector was associated with low iNOS
expression but no nitrite production. Thus, iNOS expression appears to
be TNF- independent but iNOS generation of NO requires TNF-. In
cultures of TNF-KO macrophages infected with BCG-TNF, inhibition of
iNOS by aminoguanidine (AMG) abolished the killing of the bacilli.
However, the growth of the organisms was still inhibited, suggesting an
iNOS-independent TNF--mediated growth inhibition. To confirm this,
macrophages from iNOS-KO mice were infected with either BCG-vector or
BCG-TNF. As expected, no nitrite was detected in the culture medium.
TNF- was detected only when the cells were infected with BCG-TNF. In
the iNOS-KO macrophages, the growth of BCG was inhibited only in the
BCG-TNF infection. These results suggest that in the absence of iNOS
activity, TNF- stimulates macrophages to control the growth of
intracellular BCG. Thus, there appears to be both a
TNF--dependent-iNOS-dependent killing pathway as well as a
TNF--dependent-iNOS-independent growth inhibitory pathway for the
control of intracellular mycobacteria in murine
macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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plays a major role in the control of mycobacterial
infection both in vivo and in vitro. The cytokine is required for the
inhibition of growth of Mycobacterium tuberculosis in mice
and for the development of the granulomatous response in the infected
tissues (1, 2). The experimental addition of TNF- to
the culture medium of human monocytes infected in vitro with M.
tuberculosis results in a reduction in the number of intracellular
organisms (3). Moreover, when cultures of human alveolar
macrophages infected with M. tuberculosis H37Ra are treated
with polyclonal neutralizing Ab to TNF-, intracellular mycobacterial
growth in the macrophages increases (4). Although TNF-
is clearly necessary, how this cytokine contributes to the control of
mycobacterial growth is not fully understood.
TNF- in combination with IFN-
has been shown to activate
macrophages, rendering the cells better able to control the growth of
and/or kill intracellular organisms. This activation has been
associated with the generation of reactive oxygen intermediates
(ROI)4
(5, 6, 7). Cytokine-activated murine macrophages are also
capable of generating reactive nitrogen intermediates (RNI). RNI have
been shown to mediate the killing of mycobacteria within murine cells
in vitro (8). Indeed, the results of Akaki et al.
(9) suggest that RNI may be more important effectors than
ROI in the control of intracellular mycobacteria infection in mouse
macrophages in vitro. Pretreatment of mouse peritoneal macrophages with
IFN- and TNF- before infection potentiates the production of
nitrites and is correlated with the antimycobacterial activity of the
macrophages (10). In vivo, when the inducible NO synthase
(iNOS) gene is disrupted, mice are unable to generate RNI and
are highly susceptible to M. tuberculosis infection
(11).
To directly examine the role of TNF- in these pathways, we have
established an in vitro infection model using murine macrophages and
recombinant bacillus Calmette Guérin (BCG) secreting murine
TNF-. Macrophages were prepared from the peritoneal cavity or the
lungs of either wild-type mice or from mice with specific gene
disruptions (TNF-, iNOS, or IFN-). The macrophages were infected
in vitro with recombinant BCG secreting murine TNF- (BCG-TNF) and
bacillary growth and mycobacterial survival were compared. In addition,
the ability of the cultured macrophages to produce TNF-, to express
iNOS protein, and to release nitrites into the culture medium was
evaluated. The studies reported here suggest that RNI are important for
the killing of intracellular BCG in murine macrophages. However, RNI
are not sufficient for maximal control of the survival and growth of
the intracellular mycobacteria. The results indicate that even in the
absence of any iNOS activity, intracellular mycobacterial growth in
vitro can be controlled by TNF-.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to 10-wk-old C57BL/6 x 129 mice and homozygous
TNF- gene-disrupted (TNF-knockout (KO)) and homozygous iNOS
gene-disrupted (iNOS-KO) mice on a C57BL/6 x 129 genetic
background were used (12, 13). In addition, 8- to
10-wk-old BALB/c mice and homozygous IFN- gene-disrupted (IFN-KO)
mice were used (14). The mice were kept under specific
pathogen-free conditions at the University of Cape Town animal facility
or at The Rockefeller University Laboratory Animal Research Center.
Macrophage preparation
Alveolar macrophages were obtained by a modification of the method described by Holt (15). Briefly, after a lethal dose of anesthetic, the animal was pinned onto a cork dissection board and the skin area overlying the trachea was dissected away. Overlying muscles and membranes were removed carefully by blunt dissection, taking care to avoid lateral neck vasculature. Once the trachea was fully exposed, it was cannulated using a sterile disposable 24-gauge i.v. cannula (0.7-mm diameter; Introcan, B Braun, Melsungen, Germany). The inner needle was removed and the cannula was sutured lightly into place using 4.0 silk suture on a round needle (Clinisutl; Sasurel, Port Elisabeth, Republic of South Africa). The dissection board was then tilted slightly so that the animal was in the head up position. Initially, 0.8 ml of DMEM (Life Technologies, Grand Island, NY) supplemented with lidocaine hydrochloride (lavage fluid; Sigma, St. Louis, MO) was instilled into the lung via the cannula. The fluid was left in situ for 3 min and then gently aspirated. The process was repeated five times with an increase in volume of lavage fluid to 1 ml. The lavage fluids from repeated washes were pooled.
Peritoneal macrophages were obtained by lavage of the peritoneal cavity using a standard procedure (16). Briefly, the skin overlying the abdomen was dissected away and the peritoneal cavity was flushed with 5 ml of cold DMEM using a heparinized syringe and a 26-gauge needle. The lavage fluid was removed with a sterile glass pasteur pipette.
Both alveolar and peritoneal lavage fluids were centrifuged (1000 rpm) for 8 min at 4°C. Cells were washed in cold DMEM and then suspended in DMEM supplemented with 10% FCS (Gemini, Calabasas, CA), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete medium; Life Technologies) and plated in 48-well plates at 5 x 105 cells/well. After 2 h of incubation at 37°C to allow adherence, the cells were washed with warm complete medium and then incubated overnight. Before infection, cells were washed with complete medium without antibiotics. All reagents used are LPS free.
Mycobacteria
Recombinant Mycobacterium bovis BCG Montreal strain
secreting murine TNF- (BCG-TNF), or secreting murine IFN-
(BCG-IFN) or containing the vector only (BCG-vector) were a kind gift
from Richard Young (Whitehead Institute, MIT, Cambridge, MA). Murine
cDNA for TNF- or IFN- was cloned into the plasmids pRBD3 and
pRBD4 as described elsewhere (17). The expression vectors
contained a kanamycin resistance gene. The BCG-TNF, BCG-IFN, and
BCG-vector were grown to mid-log phase in Middlebrook 7H9 medium
containing kanamycin (18 µg/ml; Difco, Detroit, MI) with minimal
agitation for 7 days (18). These three recombinant strains
had similar rates of growth in liquid medium (generation times of
37–38.5 h).
In vitro macrophage infection
BCG-vector, BCG-IFN, or BCG-TNF were suspended in complete medium without antibiotics at 37°C and added to 18-h macrophage cultures at a multiplicity of infection of 1 mycobacterium per macrophage (multiplicity of infection of 1:1). Bacilli were phagocytosed fully with no organisms remaining in the extracellular medium, by 6 h of culture as evaluated in pilot studies. The cultures were therefore not washed after addition of the mycobacteria to the cells.
CFU assay
To assess intracellular growth of recombinant BCG, infected macrophage monolayers were probe sonicated (model 60 sonic dismembrator; Fisher Scientific, Springfield, NJ) four times at 5-s pulses/well to release intracellular bacilli. Bacterial suspensions were serially diluted and plated on kanamycin-supplemented 7H11 agar plates. The number of viable bacilli was evaluated by counting individual colonies using a magnifying microscope after 2–3 wk of growth at 37°C. Each dilution was plated in multiples of six and a mean colony count was obtained. Generation time of mycobacterial growth from day 1–5 or 6 was determined using the equation TD = (log2/slope) x 24 h as described previously(19).
Cytokine analysis
Macrophage culture supernatants were removed and stored at
-70°C before cytokine analysis by ELISA. TNF-, IL-12, IL-10, and
IFN- were measured in the culture supernatant using commercial kits
(Endogen, Boston, MA) according to the manufacturer’s
specifications.
Immunoblotting for macrophage proteins
Cell lysates were prepared from peritoneal macrophage monolayers at 8, 24, and 48 h after infection with recombinant BCG as previously described (20). Cell lysates were subjected to electrophoresis on a 4–15% SDS-polyacrylamide gel and transferred to nitrocellulose. Immunoblots were probed for IRF-1 (48 kDa) using a rabbit anti-IRF-1 Ab (1:2000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and for iNOS (130 kDa) using a monoclonal anti-iNOS Ab (1:1000; Transduction Laboratories, Lexington, KY). HRP-conjugated anti-rabbit or anti-mouse secondary Abs (Pierce, Rockford, IL) were used to visualize the bands by chemiluminescence (21).
Nitrite production
The production of nitrite by peritoneal macrophages was measured in culture supernatants at 24 h by the Griess assay (22). One hundred microliters of culture supernatants was mixed with 100 µl of modified Griess reagent (Sigma) and incubated for 10 min. Absorbance was measured at 570 nm in an ELISA microreader (Opsys MR; Dynatech, Chantilly, VA). A standard curve of NaNO2 was used to establish the NO2- concentration in the samples.
iNOS inhibition
iNOS activity was inhibited using aminoguanidine (AMG; Sigma). A stock solution of AMG (1 M) in sterile normal saline was prepared. AMG was added to each well at a final concentration of 1 mM at the time of infection with recombinant BCG (23).
Electron microscopy
Peritoneal or alveolar macrophages from both TNF-KO and wild-type control mice were seeded into wells on plastic coverslips and infected either with BCG-TNF or BCG-vector at a multiplicity of infection of 1:1. Coverslips were removed on day 1 after infection, fixed in 1.0% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 h at 37°C, and postfixed in 1.0% osmium tetraoxide in 0.1 M cacodylate buffer (pH 7.4). After washing in buffer, the cells were stained en bloc with 2.0% uranyl acetate and then subjected to dehydration in ascending alcohol series, followed by embedding in Epon (Fluka, Buchs, Switzerland). Poststained sections were examined and photographed in a JEOL electron microscope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the role of TNF- in the control of intracellular
mycobacterial growth, peritoneal macrophages from TNF-KO mice were
infected in vitro with either BCG-vector or a recombinant BCG secreting
murine TNF- (BCG-TNF). Electron microscopic examination of the
infected TNF-KO macrophages revealed that both BCG-vector and BCG-TNF
were phagocytosed efficiently by the peritoneal and by the alveolar
macrophages. At 8 h after infection, single organisms were found
within phagosomes in the cytoplasm of the cells (Fig. 1
). The double membrane of the phagocytic
vacuole was tightly apposed to the mycobacterial cell wall of either
recombinant strain. No obvious morphologic differences were noted
between the phagocytosed BCG-vector (Fig. 1A) and BCG-TNF
(Fig. 1B). Similarly, no differences in the morphology of
the macrophages were noted following phagocytosis of BCG-vector
compared with BCG-TNF.
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). Thereafter, the number of
BCG-vector, evaluated daily by CFU assay, increased steadily for the
next 5 days. The number of bacilli during this period increased
10-fold with a generation time of 30 h (Fig. 2 and Table I). In contrast, when peritoneal
macrophages from TNF-KO mice were infected with BCG-TNF, the number of
viable mycobacteria decreased from the time of infection. A
statistically significant difference in intracellular bacillary numbers
between the two recombinant strains was noted by day 3 after infection
(p = 0.005; Fig. 2). When wild-type peritoneal
macrophages were infected with recombinant BCG, both BCG-vector and
BCG-TNF were killed at similar rates (Fig. 2 and Table I). Thus, the
presence of TNF-, whether of host or pathogen origin, was required
for the intracellular control of growth and killing of the
organisms.
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, inset). The generation time in this case was 34
h. However, the TNF-KO alveolar macrophages killed BCG-TNF as did the
alveolar macrophages from wild-type mice infected with either strain
(Fig. 2, inset). These observations suggest that murine
peritoneal and alveolar macrophages control intracellular growth of BCG
similarly and that TNF- is required for control of mycobacterial
infection. iNOS expression in peritoneal macrophages infected with BCG
To examine the role of iNOS expression during growth or killing of
BCG in infected macrophages, Western blot analysis of cell lysates was
conducted at 8, 24, and 48 h after infection. Uninfected
peritoneal macrophages from wild-type mice or from TNF-KO mice did not
express iNOS at any time point (data not shown). These cells did,
however, express the IFN--induced protein IRF-1. Infection of
macrophages from TNF-KO mice with BCG-vector resulted in delayed low
levels of iNOS protein expression compared with protein expression
following infection with BCG-TNF. Thus, at 8 h after infection,
iNOS protein was clearly detectable only in macrophages infected with
BCG-TNF (Fig. 3, lane 3). By
24 h, low levels of iNOS protein were also detected in macrophages
infected with BCG-vector (Fig. 3, lane 1) while higher
levels of the protein were induced by infection with BCG-TNF (Fig. 3, lane 3). When macrophages from wild-type mice infected with
BCG-vector (Fig. 3, lane 5) were compared to infection with
BCG-TNF (Fig. 3, lane 7), iNOS expression was similar to
that seen when macrophages from TNF-KO mice were infected. By 48 h
postinfection, iNOS was undetectable in any of the cell lysates (data
not shown). IRF-1 was expressed in all infected cultures at 8 h,
reduced by 24 h (Fig. 3), and undetectable by 48 h (data not
shown). These observations suggested that iNOS induction in the
macrophages did not require TNF-. However, if TNF- was present,
iNOS protein was expressed earlier and at higher levels.
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, lanes 2, 4, 6, and
8). AMG in the culture supernatants did not affect
substantially the expression of IRF-1. iNOS activity in peritoneal macrophages infected with BCG
To investigate whether TNF- affected iNOS activity in
macrophages infected with recombinant BCG, nitrite accumulation in the
culture medium was evaluated. TNF-KO peritoneal macrophages infected
with BCG-vector in vitro produced no nitrite (Fig. 4 and Table I). However, when the cells
were infected with BCG-TNF, nitrite production was observed. The
presence of AMG in the culture supernatants fully inhibited this
activity. When peritoneal macrophages from wild-type mice were
infected, both BCG-vector and BCG-TNF induced nitrite production (Fig. 4 and Table I). AMG added to these cultures significantly reduced
nitrite production. These results suggested that TNF- is required
for iNOS enzymatic activity although it does not appear to be required
for iNOS expression (Fig. 3).
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The effect of inhibition of iNOS activity by AMG on BCG growth and
survival was studied next. The presence of AMG in the cultures had no
effect on the growth of BCG-vector in the TNF-KO peritoneal macrophages
(Fig. 5 and Table I). However, the drug
rendered these macrophages less capable of controlling the BCG-TNF
infection. Under these inhibitory conditions, the number of BCG-TNF in
the infected TNF-KO macrophages were not reduced. Rather, the number of
bacilli increased very slowly, resulting in less efficient growth
compared with the BCG-vector (generation time of 89 h vs 33
h, respectively). In macrophages from wild-type mice infected in vitro
with either BCG-vector or BCG-TNF, the addition of AMG abolished the
killing of the bacilli and rendered these macrophages more permissive
to mycobacterial growth (Fig. 5 and Table I). However, in the presence
of excess TNF- (infection with BCG-TNF) the organisms grew more
slowly than BCG-vector (66-h vs 34-h generation times). Thus, when
TNF- is present, inhibition of nitrite production only partially
restores the growth of the bacilli.
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production by BCG-infected macrophages
The cytokines produced by the peritoneal macrophages infected with
recombinant BCG were measured by ELISA. Macrophages from wild-type mice
infected in vitro with BCG-vector or BCG-TNF secreted TNF- into the
culture medium. In contrast, when peritoneal macrophages from TNF-KO
mice were infected, TNF- was found in the culture medium only if the
cells were infected with BCG-TNF. The addition of AMG to the culture
medium reduced the amount of TNF- present in the medium whether the
cytokine was of host or bacterial origin (Table I).
The association among NO, IFN-, TNF-, and control of BCG
growth
To directly examine the role of NO production in the control of
mycobacterial infection in vitro, peritoneal macrophages from iNOS-KO
mice were infected in vitro with either BCG-vector or BCG-TNF (Fig. 6 and Table I). As expected, no nitrite
was detected in the culture medium of the BCG-vector infected cells.
TNF- was detected only when the cells were infected with BCG-TNF.
The growth of the BCG was inhibited in the BCG-TNF infection
(generation time, 98.9 ± 7.9 h) compared with the BCG-vector
infection (46.9 ± 6.5 h; p = 0.001). These
results suggested that even in the absence of iNOS, TNF- stimulates
macrophages to partially control the intracellular BCG.
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gene disrupted (IFN-KO) mice were infected in vitro with either
BCG-vector or BCG-TNF. Again, no nitrite was detected in the culture
medium of the cells infected by either recombinant (Table I). TNF-
was detected only when the cells were infected with BCG-TNF. IFN-KO
macrophages partially controlled the growth of BCG-TNF (generation
time, 72 h) but not of BCG-vector (34 h). Surprisingly, when the
IFN-KO macrophages were infected with BCG-IFN-, the presence of
IFN- in the culture medium (19.7 ± 1.6 pg/ml) did not restore
the production of nitrite by the macrophages (Table I). TNF- was not
detected in these cultures. The presence of IFN- did not affect
growth of the recombinant BCG (generation time of 30 h for
BCG-IFN- compared with 27 h for BCG-vector). Thus, TNF-
appeared to be required for control of intracellular growth of BCG and
the presence of IFN- could not compensate for the absence of
TNF-. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the killing
of mycobacteria (iNOS dependent) and in the control of mycobacterial
growth (iNOS independent) in murine macrophages in vitro. Although both
recombinant strains of BCG used here were phagocytosed similarly as
shown morphologically by electron microscopy, the subsequent fates of
the strains were very different. In the absence of any TNF-, whether
of macrophage origin or from recombinant BCG-TNF, the macrophages from
TNF-KO mice could neither control the growth of nor kill BCG. However,
when TNF- was added back by infection with BCG-TNF, the replication
of the infecting organisms was reduced and the number of viable
bacteria was decreased. This was also noted in vivo: when TNF-KO mice
were infected with recombinant BCG-TNF, the number of infecting BCG-TNF
was reduced whereas BCG-vector grew in all organs tested
(24). Similarly, in human macrophages infected in vitro
with M. tuberculosis, it has been shown that when TNF- is
added, the cells are more efficient at curbing the growth of the
bacilli than when other cytokines or cytokine combinations, including
IFN-, are added (3). The relative lack of effect of
IFN- may be explained by recent findings of Ernst and colleagues
(25, 26), who have shown that IFN- fails to activate
human macrophages to kill M. tuberculosis. They have
recently found that M. tuberculosis infection blocks
macrophage responses to IFN- via the direct binding of cell wall
components to the nuclear transcription factor CBP/p300, thereby
sequestering it and preventing its interaction with STAT1, precluding
further transcription (25, 26).
The mycobacteriocidal effect of TNF- observed in the present study
appeared to be associated with iNOS-mediated production of NO.
Interestingly, when AMG was added to these cultures to block nitrite
production, the antimicrobial activity was less efficient, although not
fully inhibited. The bacilli were not killed but their growth was
inhibited. This result suggested the presence of an iNOS-independent
TNF--dependent antimycobacterial activity. Furthermore, infection of
macrophages prepared from iNOS-KO mice confirmed the observation of an
iNOS-independent but TNF--dependent mycobacterial growth inhibitory
activity.
The iNOS-independent but TNF--dependent antimycobacterial activity
could be mediated by ROI. The ROI pathway has been studied using
respiratory burst-deficient gp91 (phox-/-) mice
(27). These mice are particularly susceptible to
mycobacterial infection early in the course of infection. In vitro, the
oxidative burst has been shown to be important in the control of
intracellular infection with other pathogens (28). Recent
reports suggest that cytokine treatment of host macrophages facilitates
maturation and acidification of the vacuoles (29, 30). We
have recently shown that acidification of the phagocytic vacuole
inhibits the growth of intracellular M. tuberculosis
(31). Thus, TNF- may facilitate acidification of the
late phagosome-lysosome vacuole via an oxidative mechanism leading to
the M. tuberculosis growth inhibition seen in this study. It
is also possible that the role of the oxidative pathway in
mycobacterial killing may have been underestimated in previous studies
in which AMG was used as a specific inhibitor of RNI (23).
Recently we have observed that AMG inhibited the ROI pathway as well as
the RNI pathway (our unpublished observations). Tian et al.
(32) have also shown that an inhibitor of the ROI pathway
(e.g., catalase) affects NO production in an arginase-dependent manner.
These results suggest caution in interpreting results of experiments
with inhibitors (32).
Some investigators have suggested that RNI are more important than ROI
in the control of mycobacterial growth by murine macrophages
(9). In the mouse model, macrophage activation results in
iNOS induction (11). iNOS then catalyzes two sequential
monooxygenase reactions to convert L-arginine to
L-citrulline resulting in the production of NO. It is NO
and other RNI that are toxic to mycobacteria. In the present studies,
iNOS protein was expressed in the macrophages even in the absence of
TNF-. However, the enzyme appeared to be inactive, since nitrite was
not detected in the culture supernatant of infected cells, and the
intracellular mycobacteria were not killed. Similarly, iNOS protein has
been shown to be expressed in the macrophages of mycobacteria-infected
TNF- receptor gene-disrupted mice (TNFR-KO) (1) and
TNF-KO mice (2, 24), even in the absence of TNF- or
TNF- signaling. However, since the infection was not controlled in
all of these in vivo studies, either the enzyme was not active or it
was not activated efficiently enough or early enough. Only when TNF-
was present were high levels of nitrites found in the culture
supernatant and the organisms killed. Thus, the enzymatic function of
iNOS rather than its expression, appears to require TNF-. Why the
enzyme appears to be inactive in the absence of TNF- is as yet
unclear.
How may BCG infection induce the expression of iNOS in the absence of
TNF-? Recent studies have demonstrated that mycobacteria activate
macrophages via the Toll-like receptor 2 (TLR2) (33, 34),
leading to activation of NF-
B, which in turn results in the
up-regulation of genes for cytokines such as TNF- and enzymes such
as iNOS. TLR2 may thus be utilized by BCG to induce iNOS in the TNF-KO
macrophages (35). Taken together with the previous
observations, our results suggest that both the TLR2 pathway and the
TNF- signaling pathway are required for killing of mycobacteria by
murine macrophages. In this study, we have observed both a
TNF--dependent-iNOS-dependent and a
TNF--dependent-iNOS-independent antimycobacterial mechanism. Since
iNOS has been difficult to demonstrate in human mononuclear phagocytes,
the TNF--dependent-iNOS-independent pathway may be a significant
pathway in controlling human mycobacterial infection.
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Acknowledgments |
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Footnotes |
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2 Current address: Infectious Disease Clinical Research Unit, University of Cape Town, Lung Institute, P.O. Box 34560, Groote Schuur 7937, Cape Town, South Africa.
3 Address correspondence and reprint requests to Dr. Gilla Kaplan, Laboratory of Cellular Physiology & Immunology, The Rockefeller University, 1230 York Avenue, New York, NY 10021. E-mail address: kaplang{at}rockvax.rockefeller.edu
4 Abbreviations used in this paper: ROI, reactive oxygen intermediate; RNI, reactive nitrogen intermediate; iNOS, inducible NO synthase; BCG, bacillus Calmette Guérin; KO, knockout; AMG, aminoguanidine; TLR2, Toll-like receptor 2.
Received for publication October 16, 2000. Accepted for publication March 19, 2001.
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is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2:561.[Medline]
(IFN-) and tumour necrosis factor- (TNF-). Clin. Exp. Immunol. 112:63.[Medline]
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S Ehlers Role of tumour necrosis factor (TNF) in host defence against tuberculosis: implications for immunotherapies targeting TNF Ann Rheum Dis, November 1, 2003; 62(90002): ii37 - 42. [Abstract] [Full Text] [PDF]
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T. Parish, D. A. Smith, S. Kendall, N. Casali, G. J. Bancroft, and N. G. Stoker Deletion of Two-Component Regulatory Systems Increases the Virulence of Mycobacterium tuberculosis Infect. Immun., March 1, 2003; 71(3): 1134 - 1140. [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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A. P. Gobert, B. D. Mersey, Y. Cheng, D. R. Blumberg, J. C. Newton, and K. T. Wilson Cutting Edge: Urease Release by Helicobacter pylori Stimulates Macrophage Inducible Nitric Oxide Synthase J. Immunol., June 15, 2002; 168(12): 6002 - 6006. [Abstract] [Full Text] [PDF]
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R. Lang, R. L. Rutschman, D. R. Greaves, and P. J. Murray Autocrine Deactivation of Macrophages in Transgenic Mice Constitutively Overexpressing IL-10 Under Control of the Human CD68 Promoter J. Immunol., April 1, 2002; 168(7): 3402 - 3411. [Abstract] [Full Text] [PDF]
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T. Musso, R. Badolato, D. Ravarino, S. Stornello, P. Panzanelli, C. Merlino, D. Savoia, R. Cavallo, A. N. Ponzi, and M. Zucca Interaction of Bartonella henselae with the Murine Macrophage Cell Line J774: Infection and Proinflammatory Response Infect. Immun., October 1, 2001; 69(10): 5974 - 5980. [Abstract] [Full Text] [PDF]
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