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University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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at levels
sufficient to activate macrophages to kill the parasites via induction
of NO3 production (1, 2, 3).
Previous studies have shown that IFN--activated macrophages infected
in vitro with L. major require endogenously produced TNF for
NO production and parasite killing (4). Consistent with a requirement
for TNF are studies showing that in vivo neutralization of TNF using
mAbs enhanced susceptibility to L. major, although such
animals eventually healed (5, 6). However, since the long-term efficacy
of TNF neutralization using mAb is problematic, these experiments left
unresolved the issue of an absolute requirement for TNF in healing.
TNF, which includes homotrimeric TNF-
and lymphotoxin- (also
known as TNF-ß) binds to two different receptors, known as the
TNFRp55 or the TNFR1, and the TNFRp75 or the TNFR2 (7). These receptors
have distinct cytoplasmic domains and mediate different functions.
Studies with TNFR-deficient mice found that many of the responses
associated with TNF are mediated through the TNFRp55, such as
macrophage activation and sensitivity to endotoxin, and involve NF-
B
activation (8, 9). Furthermore, the presence of the p55 receptor is
required for resistance to Listeria monocytogenes, Mycobacterium
tuberculosis, and Toxoplasma gondii (8, 9, 10, 11). In
addition, the TNFRp55 also contains a death domain, homologous to the
Fas death domain, and signaling through the p55 receptor can also
induce apoptosis (12, 13). At present, the factors determining whether
signaling through the TNFRp55 leads to NF-B activation or
the induction of apoptosis are not defined. Signaling through the p75
receptor can also be associated with NF-B activation, although fewer
functions are ascribed to this receptor (14, 15). Similar to wild-type
controls, TNFRp75-/- are susceptible to
endotoxin and resistant to L. monocytogenes. However, in
contrast to control mice, they were found to be resistant to
TNF-induced skin necrosis (14).
We previously reported that mice lacking the TNFRp55 were able to
eliminate L. major parasites at the site of infection,
although, interestingly, they did not heal their lesion (16). The
ability of TNFRp55-/- mice to eliminate L.
major parasites was unexpected, since, in the absence of the
TNFRp55, macrophages do not produce NO in response to stimulation with
IFN- and TNF- (10, 16), suggesting that the TNFRp55 is required
for this pathway of macrophage activation. However, it remained
possible that in the absence of the p55 receptor, the p75 receptor
might compensate for NO induction. Therefore, we examined the course of
L. major infection and in vitro macrophage activation in
TNFRp75-/- and doubly deficient mice generated by
crossing the p55 and p75 deficient mice (TNFRp55p75-/-).
We found that the outcome of L. major infection is not
significantly altered in the absence of the TNFRp75, but that mice
lacking both receptors are unable to heal. Nevertheless, all of the
mice developed a Th1-like response, and all eventually up-regulated
iNOS within, and eliminated parasites from, their lesions. In contrast,
we observed a defect in the activation of resident macrophages from
mice lacking the p55 receptor (both TNFRp55p75-/- and
TNFRp55-/-). To determine whether a TNFR-independent
mechanism for activation of macrophages is invoked during L.
major infection, we elicited peritoneal macrophages from L.
major-infected mice with leishmanial Ag and studied their ability
to kill L. major and produce NO. In contrast to resident
macrophages from uninfected TNFRp55-/- or
TNFRp55p75-/- mice, macrophages specifically elicited
during infection from both of these mouse strains were activated,
demonstrating that L. major infection is associated with
activation of macrophages in vivo in the absence of both TNF
receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Receptor-deficient and control mice were bred and housed at the University of Pennsylvania. Mice were used at 6 and 8 wk of age. TNFRp55 mice were backcrossed onto the C57BL/6 for seven generations (8). The TNFRp75-/- mice and TNFRp55p75-/- mice were maintained on a random C57BL/6 x 129 hybrid background and were initially provided by Dr. Mark Moore (Genentech, South San Francisco, CA) (14). Wild-type (+/+) littermates from the 7th backcross to C57BL/6 (wt (B6(7)) mice and C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were maintained for use as controls for the TNFRp55-deficient animals. No significant differences between wt B6(7) and C57BL/6J (The Jackson Laboratory) were detected, and only data from B6(7) mice is shown. C57BL/6 x 129 hybrids (wt (129/B6)) were used as controls for the TNFRp75-/- and TNFRp55p75-/- mice.
Parasites and Ag
L. major (WHO MHOM/IL-1/80 Freidlin clone) was maintained in a log phase of growth in Grace’s insect cell culture medium (Life Technologies, Grand Island, NY) supplemented with 20% FCS. Stationary phase promastigotes were selected for metacyclic stage organisms by Arachis hypogae agglutinin (Sigma, St. Louis, MO) as described (17). Mice were infected with 2 million metacyclic promastigotes in the hind footpad. Amastigotes for in vitro macrophage experiments were obtained from lesions of infected scid mice, and viable amastigotes were counted by fluorescein diacetate fluorescence as described (18). Soluble leishmanial Ag (SLA) was prepared as described (19).
Course of infection
Lesion size was determined by measuring the diameter of the footpad with a Starret dial caliper and subtracting the diameter of the contralateral uninfected footpad. The total number of parasites in lesions were determined by limiting dilution analysis as previously described (20).
Preparation of cells
Infected mice were sacrificed and the popliteal lymph nodes,
spleen, and feet were collected. Lymph nodes and spleens were disrupted
with a glass tissue homogenizer (Wheaton; Fisher, Pittsburgh, PA), and
the resulting cell suspension was washed and subsequently resuspended
in complete tissue culture medium (CTCM) (4.5 mg/ml glucose DMEM, 10%
FCS, 25 mM HEPES, 5 x 10-5 ß-2-ME (2 ME), 100
U/ml penicillin-6-potassium, 100 µg streptomycin sulfate, 2 mM
glutamine). Ag specific recall responses were determined by stimulation
of cells with SLA (50 µg/ml) and collection of supernatants at
72 h for IFN- and IL-4 analysis (19).
Peritoneal resident or Ag-elicited macrophages (peritoneal exudate
cells, PECs) were harvested by peritoneal lavage, and incubated in
polypropylene tubes in complete tissue culture medium without ß2-ME.
Resident macrophages are derived from naive mice that have not been
injected i.p. with an inflammatory agent. In contrast, Ag-elicited
macrophages are derived from uninfected or L. major-infected
mice that were injected with 1 ml freeze/thawed L. major
(the equivalent of 10 million parasites) into the peritoneal cavity and
were harvested 4 days after injection of Ag. Cells were cultured with
various combinations of the following: IFN- (Genentech), LPS (Sigma,
L5014), and/or neutralizing mAb to TNF (XT22.11). Macrophages were
infected with L. major amastigotes at a 2:1 ratio and
incubated at 37°C for 2 h in suspension cultures (polypropylene
tubes). Nonphagocytosed amastigotes were washed from the cultures by
centrifugation, and the cells were resuspended in medium with cytokine
and Ab. Aliquots were removed at 2 and 72 h and stained for visual
quantitation of the infection. At 72 h supernatants were harvested
and assayed for NO2.
Cytokines, NO, and competitive PCR
IFN- and IL-4 were measured using specific two-site ELISAs as
previously described (21). NO production was assessed by measuring
NO2 in supernatants harvested at 72 h using the
Greiss reagent (22). The expression of iNOS mRNA was measured using a
competitive RT-PCR as previously described (23). Briefly, footpads were
harvested and immediately frozen in liquid nitrogen. The tissue was
homogenized in GITC (guanidium isothiocyanate) lysis buffer, 1/10
volume of 2 M sodium acetate was added, then RNA was extracted using
phenol-chloroform isoamyl alcohol (50:49:1). Samples were precipitated
with an equal volume of isopropanol, and RNA concentration was
calculated by OD at 260 nm. Two micrograms of RNA was reverse
transcribed using Superscript 2 (Life Technologies) and random heximer
primers (Promega, Madison, WI). PCR was done using a multiple-cytokine
competimer construct (23). cDNA was normalized to 0.1 attamoles
polycompetimer for hypoxanthine-quanine phosphoribosyltransferase
(HPRT) (GTT GGA TAC AGG CCA GAC TTT GTT G and GAG GGT AGG CTG GCC TAT
AGG CT). Using normalized cDNA, we performed competitive PCR using iNOS
primers (TGG GAA TGG AGA CTG TCC CAG and GGG ATC TGA ATG TGA TGT TTG).
A titration of the competimer was done to determine the point at which
the amplification of the competimer and sample were equivalent by
ethidium bromide staining. Results are expressed as the ratio of iNOS
message to HPRT.
Statistics
Results are expressed as the mean ± SD or SE as indicated in figure legends. Significance was determined by the Student t test, with p < 0.05 defining significance over control group.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TNFRp55-/- mice fail to heal following L.
major infection, so it was of interest to determine the course of
infection in mice lacking the TNFRp75 or both receptors. Therefore,
TNFRp75-/- and TNFRp75+/+ littermates were
infected with L. major, and the course of infection was
followed (Fig. 1
A). No
significant differences were seen in lesion sizes between control mice
and those lacking the p75 TNF receptor. In contrast,
TNFRp55p75-/- mice exhibited a nonhealing course of
infection similar to that which we previously reported with the
TNFRp55-/- mice (Fig. 1B).
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Using limiting dilution analysis, we found no significant
differences in the ability of TNFRp75-/- mice to
eliminate parasites from the lesions, compared with their wt (129/B6)
controls (Fig. 2, B and
E). In contrast, TNFRp55p75-/- mice
exhibited a delay in parasite clearance compared with their wt controls
(Fig. 2, C and E), with a significantly
higher parasite burden at 6 wk postinfection. Nevertheless, by 14 wk
postinfection, most of the parasites had been eliminated from the
lesions, demonstrating that neither TNF receptor is required for
control of parasites. The results with the TNFRp55p75-/-
mice are similar to those that we previously reported in
TNFRp55-/- mice (16).
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Resistance to L. major infection depends on the
development of a Th1-like response, associated with production of
IFN- but little IL-4 (24, 25, 26, 27). Previous work has suggested that TNF
may play a role in the ability of T cells to differentiate into Th1
cells (28). While our previous studies demonstrated that Th1 cells
develop in the absence of the p55 TNF receptor, we were interested to
determine whether Th1 cells could develop in the absence of both
receptors. Therefore, we measured the levels of IFN- and IL-4
produced by the lymph node and spleen cells taken from mice infected
for 2 and 14 wk. At all time points, IL-4 was below the level of
detection (data not shown). In contrast, IFN- was produced by
Ag-stimulated cells from control and TNFRp55p75-/-, as
well as TNFRp75-/- mice (Table I). Thus, like TNFRp55-/-
mice, the TNFRp75-/- and double deficient mice develop a
Th1 response, demonstrating that TNF signaling through either of these
receptors is not required for Th1 cell development.
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We previously demonstrated that mice lacking the TNFRp55
up-regulated iNOS mRNA in their lesions over time, correlating with
earlier observations that resistant mice show an up-regulation of iNOS
mRNA in their lesions compared with susceptible mice (16, 29). We were
interested in determining what role the p75 receptor might play in iNOS
mRNA expression. Therefore, we examined the expression of iNOS mRNA in
the lesions of TNFR-deficient mice 2 and 6 wk postinfection. As seen in
Figure 3A, iNOS mRNA is
up-regulated in TNFRp55p75-/- and
TNFRp75-/- mice by 6 wk postinfection. While we observed
a slight increase in iNOS mRNA at 2 wk of infection in wt controls
(Fig. 3B), this increase was not significant
(p > 0.06). At 6 wk, we found significant
increases (p < 0.05) in iNOS mRNA in all wt
and TNFR-deficient mice compared with uninfected controls. Thus, it
appears that iNOS gene expression can be induced in vivo in the absence
of signaling through either TNF receptor.
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To determine whether macrophages lacking the TNFRp75 had any
defect in activation, we infected resident macrophages in vitro with
L. major amastigotes in the presence or absence of exogenous
rIFN-. Resident macrophages from TNFRp75-/- and wt
mice controlled the number of parasites per 100 macrophages with equal
efficiency (Fig. 4, A and
D). We also observed NO production by assessing the
amount of NO2 in the culture supernatant (22) in the
presence of IFN- and amastigotes (Fig. 4F),
consistent with previous observations that killing of parasites
correlates with NO production in vitro (4, 30).
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(Fig. 4
C). Similarly, resident macrophages from
TNFRp55p75-/- mice also failed to control parasites or
produce NO following IFN- activation (Fig. 4, B and
F). Consistent with these findings were our results
using neutralizing anti-TNF mAb (XT22.11) (Fig. 4, E and
F). Thus, as previously shown (4), the ability of
macrophages from wt mice (B6(7)) to become activated to produce NO and
kill L. major was blocked when anti-TNF mAb was added.
However, since thioglycollate-elicited macrophages from
TNFRp55-/- mice can be activated with IFN- (16), and
TNFRp55-/- or TNFRp55p75-/- mice were able
to produce NO and eliminate L. major in vivo, these results
suggest that elicitation in vivo may be able to prime macrophages for
NO production in a TNFRp55-independent manner. In vivo activation of macrophages from TNFR-deficient mice
Because we observed a defect in macrophage activation in
TNFRp55-/- and TNFRp55p75-/-
macrophages in vitro, but were able to show increased iNOS gene
expression and parasite elimination during infection, we hypothesized
that during L. major infection other signals could prime
macrophages for activation. To test whether macrophages could be primed
in vivo for activation during the course of L. major
infection, we harvested peritoneal macrophages from L.
major-infected wt and TNFR knockout mice after elicitation with
Leishmania parasites and assessed their ability to produce
NO and kill L. major amastigotes. We found that without
addition of exogenous rIFN-, macrophages elicited from uninfected
mice of wt or knockout genotypes exhibited uncontrolled parasite
replication and no NO production, (Fig. 5, open bars). However, macrophages
harvested from 4-wk-infected wt mice produced NO in response to
infection with amastigotes and controlled parasites over 72 h of
culture (Fig. 5, solid bars). NO production was dependent upon a
triggering stimulus, since incubating the elicited macrophages with
medium alone failed to result in significant NO production (Fig. 5B). Thus, we were able to demonstrate killing by
macrophages harvested from infected wt, TNFRp55-/-, or
TNFRp55p75-/- mice. Interestingly, macrophages elicited
from uninfected mice were similar to resident macrophages, in that the
absence of the TNFRp55 resulted in a defect in NO production and
control of the parasites in the presence of recombinant IFN- over
72 h in culture (data not shown). Thus, the in vivo milieu
provides compensatory signals for macrophage activation in the absence
of the TNFRp55.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is not an absolute requirement for the
development of protective immunity against this parasite and is in
contrast to IFN-, IFN-R, and IL-12p40, which are absolutely
required for resistance (31, 32, 33).
IFN--mediated macrophage activation is a critical part of the
control of many intracellular pathogens, and TNF contributes to such
activation. For example, maximal induction of NO in vitro requires
treatment of macrophages with both TNF and IFN- (34). In
leishmaniasis, it was shown that the ability of IFN--primed
macrophages to kill L. major amastigotes was dependent upon
TNF, since addition of neutralizing anti-TNF mAb abrogated
macrophage activation, results that we reconfirmed in this study (4).
The required receptor for TNF signaling appears to be the p55 receptor,
since resident macrophages from TNFRp55-/- or
TNFRp55p75-/- mice were unable to produce NO or kill
L. major in vitro, while macrophages from
TNFRp75-/- mice were normal. Nevertheless, in contrast to
these in vitro results, we found that mice were able to eliminate
L. major in vivo. One explanation for these findings may be
that macrophage activation is not responsible for parasite elimination
in these animals. However, we were able to show that macrophages
elicited from infected mice lacking TNF receptors were able to produce
NO and control parasite replication. Thus, neither the p55 nor the p75
receptor is absolutely required for macrophage activation when priming
occurs in vivo.
This result raises the obvious question as to how macrophages are
activated in a TNF-independent manner during L. major
infection. One possibility consistent with the data is that macrophage
activation is dependent upon a T cell-macrophage interaction. Activated
T cells, fixed activated T cells, and plasma membranes from activated T
cells can synergise with IFN- to induce NO production by macrophages
(35, 36). One candidate molecule that can transduce an activation
signal is CD40, a member of the TNFR family. In fact, T cell activation
of macrophages can be partially blocked with an antagonistic Ab to
CD40L, and T cells from CD40L knockout mice are defective in their
ability to activate macrophages (35). Interestingly, CD40- and
CD40L-deficient mice exhibit enhanced susceptibility to
Leishmania, although this enhanced susceptibility probably
involves more than simply a lack of macrophage activation (37, 38, 39).
Experiments are ongoing in our laboratory to investigate the role of
CD40-CD40L interactions in TNFR-independent macrophage activation.
We previously reported that thioglycollate-elicited macrophages from
TNFRp55-/- mice could be activated by high doses of
IFN- (16) and postulated that the p75 receptor might compensate for
the absence of the p55 receptor. Our results with macrophages harvested
from TNFRp55p75-/- mice demonstrate that the p75 receptor
is probably not essential in the absence of the p55 receptor and are
similar to those reported by Yap et al. (11), who found that elicited
macrophages from TNFRp55p75-/- mice also could be
activated with high doses of IFN-.
It is useful to compare our findings with those reported following infection of TNFR-deficient mice with other intracellular pathogens. TNFRp55-/- mice infected with M.tuberculosis died by 20 days postinfection, while the wt controls survived (10). TNFRp55-/- mice failed to produce NO early after infection, although by 14 days iNOS levels were equivalent in the wt and the TNFRp55-/- mice. Similarly, infection of TNFRp55p75-/- mice with Toxoplasma was associated with up-regulation of iNOS gene expression, although, during the chronic phase of the disease, TNFRp55p75-/- mice died from severe toxoplasmic encephalitis (11). In the case of Listeria, infected TNFRp55-/- mice died within 5 days of infection (8, 9). Surprisingly, however, these mice had similar up-regulation of iNOS mRNA, compared with wt controls at 24 and 72 h postinfection (40). Reactive oxygen and nitrogen intermediates were found to be similar in the serum of TNFRp55-/- and wt controls, further supporting a TNFRp55-independent pathway for the regulation of these factors. While we found that neither TNF receptor is required for the eventual elimination of L. major, it appears that the TNF-independent pathway of parasite elimination is less efficient in controlling the infection. Thus, both TNFRp55-/- and TNFRp55p75-/- mice maintained a higher parasite burden than control animals at their peak of 6 wk postinfection and took considerably longer to eliminate parasites from the lesions. Macrophage activation contributes to the elimination of all these intracellular organisms, and the differences in the outcome of infection in TNFR-deficient mice with different pathogens might suggest that the delayed kinetics of NO production can be handled better in localized infections where pathology will not be life threatening (such as cutaneous lesions in leishmaniasis), than in systemic infections or those in which vital organs are affected. In some situations, however, severe disease in TNFRp55-/- mice may indicate that other TNF-dependent, iNOS-independent effector mechanisms that are required for eliminating particular pathogens are missing. This may be the case in Listeria (40).
We previously reported that, in spite of eliminating L.
major, TNFRp55-/- mice are unable to resolve their
lesions (16). We now show that TNFRp55p75-/- mice exhibit
a similar phenotype, which eliminates the possibility that this unusual
phenotype was associated with signaling via the p75 receptor.
Similarly, it was recently reported that TNF-deficient mice are
defective in controlling cell infiltration associated with
Corynebacterium parvum infection (41). Signaling via the p55
receptor can lead to NF-B activation or induction of apoptosis (7),
and it is possible that TNF is required for lymphocyte apoptosis
and resolution of the inflammatory lesions associated with certain
infections. TNF has also been shown to suppress T cell responses via
modulation of TCR signaling, which could be another mechanism by which
the absence of the p55 receptor could be associated with
chronic inflammation (42). Studies are ongoing in our laboratory to
investigate this issue.
The discovery that TNF binds to two receptors has generated a large amount of research to define the role that each receptor plays in mediating the effects of TNF, and the availability of genetically modified animals that lack one or both of these receptors has provided the tools to address this issue (8, 9, 14). Our work demonstrates that the p55 receptor plays a role in resident macrophage activation and lesion resolution. In contrast, the TNFRp75 is not required for either of these functions. Nevertheless, TNFRp55-/- and TNFRp55p75-/- mice were able to eliminate parasites following L. major infection, and parasite elimination was associated with NO production and macrophage activation. Future studies to elucidate how this TNF-independent pathway of macrophage activation functions will be important in fully understanding how intracellular pathogens are eliminated by the host’s immune response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Phillip Scott, Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: NO, nitric oxide; iNOS, inducible nitric oxide synthase; HPRT, hypoxanthine-quanine phosphoribosyltransferase; wt, wild-type; SLA, soluble leishmanial Ag.
Received for publication October 31, 1997. Accepted for publication February 4, 1998.
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B by TNF receptor 2 and CD40. Science 269:1424. synergizes with IFN- in mediating killing of Leishmania major through the induction of nitric oxide. J. Immunol. 145:4306.[Abstract]
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S. T. Kanaly, M. Nashleanas, B. Hondowicz, and P. Scott TNF Receptor p55 Is Required for Elimination of Inflammatory Cells Following Control of Intracellular Pathogens J. Immunol., October 1, 1999; 163(7): 3883 - 3889. [Abstract] [Full Text] [PDF]
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L. M. Stamm, A. Raisanen-Sokolowski, M. Okano, M. E. Russell, J. R. David, and A. R. Satoskar Mice with STAT6-Targeted Gene Disruption Develop a Th1 Response and Control Cutaneous Leishmaniasis J. Immunol., December 1, 1998; 161(11): 6180 - 6188. [Abstract] [Full Text] [PDF]
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T. M. Doherty, A. Sher, and S. N. Vogel Paclitaxel (Taxol)-Induced Killing of Leishmania major in Murine Macrophages Infect. Immun., September 1, 1998; 66(9): 4553 - 4556. [Abstract] [Full Text] [PDF]
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 M. Prinz, F. Montrasio, M. A. Klein, P. Schwarz, J. Priller, B. Odermatt, K. Pfeffer, and A. Aguzzi Lymph nodal prion replication and neuroinvasion in mice devoid of follicular dendritic cells PNAS, January 22, 2002; 99(2): 919 - 924. [Abstract] [Full Text] [PDF]
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) of competimer. Top band, competimer.
Bottom band, cDNA. Top row are products resulting from PCR
with iNOS primers; bottom row are products using HPRT primers.
B, iNOS/HPRT ratio from individual wt mice and
TNFR-deficient mice. Increases over uninfected (time 0) compared with 2
wk postinfection were not significant (p >
0.06), whereas increases at 6 wk postinfection were significant
(p < 0.05).
