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Dependent1
Department of Bacteriology, University of Wisconsin, Madison, WI 53706
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and other cytokines in host resistance, we determined the
resistance phenotypes and Th cell responses of IFN- and IL-4
knockout mice. Infected C57BL/6-IFN- knockout mice were as
susceptible as C57BL/6-scid mice and made an IL-2, but
not an IL-4, cytokine response to VSG, while C57BL/6-IL-4 knockout mice
were as resistant as the wild-type strain and exhibited both IL-2 and
IFN- cytokine responses. Passive transfer of spleen cells from
wild-type mice to IFN- knockout mice resulted in enhanced survival.
Both wild-type and IFN- knockout mice controlled parasitemia with
VSG-specific Ab responses, although parasitemias were higher in the
IFN- knockout mice. Overall, this study demonstrates for the first
time that relative resistance to African trypanosomes is associated
with a strong Th1 cell response to parasite Ags, that IFN-, but not
IL-4, is linked to host resistance, and that susceptible animals do not
make compensatory Th2 cell responses in the absence of Th1 cell
cytokine responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activation of macrophages are able to destroy the
parasites and localize the infection, while mice producing IL-4, a Th2
cell cytokine, exhibit exacerbated disease and disseminated infection
(reviewed in Refs. 6–10); these experimental studies have been
mirrored at the clinical level as well (11, 12). Additional work in the
Leishmania model system has demonstrated that in the absence
of IFN-, resistant mice are unable to control infection, and
neutralization of IL-4 allows susceptible mice to resolve infection
(13). Similar findings in experimental infections with Candida
albicans (14, 15), Borrelia burgdorferi (16),
Listeria monocytogenes (17), Trichuris muris
(18), and influenza virus (19) as well as in human infections with
Mycobacterium tuberculosis (20) and M. leprae
(21), support the idea that the nature of the emerging or predominant
Th cell response may dramatically affect the outcome of
disease.
African trypanosome infections are partially controlled by host B cell
responses to variant surface glycoprotein
(VSG)3 determinants expressed
by trypanosome variants appearing in the bloodstream (reviewed in Refs.
22–25). However, it is clear that VSG-specific B cell responses and
control of parasitemia are not functionally or genetically linked alone
to overall host resistance, and that other immune elements contribute
to resistance (25, 26, 27). Recent work in this laboratory has demonstrated
that mice with the BL/10 or BL/6 genetic backgrounds, which are
relatively resistant to Trypanosoma brucei rhodesiense,
mount polarized VSG-specific Th1 cell responses following infection
(28, 29, 30). This response consists of IFN- and IL-2 secretion in
response to parasite Ags and includes a degree of tissue
compartmentalization in which peritoneal CD4+ T cells
produce the strongest cytokine responses. Because Th cell responses
have been associated with resistance to other parasitic organisms, we
asked whether Th1 cell-associated cytokine responses are associated
with host resistance to African trypanosomes. This question assumes a
controversial dimension because of studies suggesting that IFN- is a
requisite growth factor for trypanosomes rather than a factor that
contributes to host resistance (31, 32, 33, 34).
In the present study, therefore, we have examined parasite Ag-specific
Th cell responses in relatively resistant B10.BR and susceptible C3H
mice as well as in resistant wild-type and IFN- or IL-4 cytokine
gene knockout (KO) mice. We found that while infected B10.BR and
C57BL/6 mice produced strong Th1 cell cytokine responses upon
stimulation with trypanosome VSG, C3H mice produced weak or no
detectable Th1 cell cytokine responses. No infected mouse strain
exhibited evidence of Th2 cell cytokine responses to parasite Ag. Using
C57BL/6 wild-type and cytokine gene KO mice, we showed that IFN-
production associated with the Th1 cell responses is a critical factor
in determining the relative level of host resistance, and that the
ability to produce IL-4 is neither detrimental nor helpful to the host.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Age-matched female B10.BR/SgSnJ, C3HeB/FeJ, C57BL/6J
(wild-type), C57BL/6J-Ifg (IFN- KO) (35), C57BL/6J-Il4 (IL-4 KO)
(36), and C57BL/6J-scid/SzJ (scid) mice
were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were
8–12 wk old at the time of infection. Outbred Swiss mice (retired
breeders; Harlan Sprague-Dawley, Madison, WI) were used for propagation
of trypanosome stabilates. All animals were housed in
university-approved facilities.
Trypanosomes and VSG
Trypanosoma brucei rhodesiense clone LouTat 1 was used in all experiments. Frozen stabilates of the parasite were thawed and used to infect Swiss mice that had been immunosuppressed with cyclophosphamide (300 mg/kg body weight; Cytoxan, Mead Johnson and Co., Evansville, IN) (37). After 4–5 days of infection, blood was collected and diluted in PBS and 1% glucose (PBSG), and the trypanosomes were counted in a hemocytometer. Experimental infections were established by injecting 1 x 105 trypanosomes i.p. in a total volume of 0.3 ml of PBSG.
For parasitemia studies, trypanosomes in the blood were enumerated by the method of Kolmer (38, 39). Briefly, 10 µl of tail blood was collected into a heparinized capillary tube and expelled into 90 µl of trypanosome staining buffer (2% formalin, 2% glacial acetic acid, and 2% Ziehl-Neelson’s carbol fuchsin in water). Appropriate dilutions of the suspension were made in trypanosome staining buffer, and the trypanosomes were counted using a hemocytometer.
Trypanosomes used for VSG isolation were separated from blood
components by passage over Selectacel DEAE (Polysciences, Warrington,
PA), and the VSG was purified by a modification of procedures
previously described (28, 40). Briefly, trypanosomes were resuspended
in 0.3 mM zinc acetate (1 x 109 trypanosomes/ml), and
incubated on ice for 5 min, followed by 10 min of centrifugation at
1000 x g. The supernatant fluid was decanted, the
protease inhibitors
N-
-p-tosyl-L-lysine chloromethyl
ketone (0.1 mM; Sigma Chemical Co. Chemical, St. Louis, MO) or
aprotinin (0.01 mg/ml; Sigma, St. Louis, MO) and leupeptin (0.04 mg/ml;
Sigma) were added, and the mixture was placed on ice. The pellet from
the same centrifugation step was resuspended in PB with PMSF (0.05 mM;
BRL, Rockville, MD) and
N--p-tosyl-L-lysine chloromethyl
ketone (0.1 mM). The suspension was incubated at 37°C for 20 min,
cooled on ice, and then centrifuged at 5,000 x g for
15 min. From this second centrifugation, the pellet was discarded; the
supernatant fluid (in PB) was saved but handled separately from the
earlier supernatant (in zinc acetate). Both supernatant fluids
subsequently were centrifuged at 300,000 x g for 70
min at 4°C, collected, and kept on ice. The zinc acetate fraction was
dialyzed in 50,000 MWC dialysis tubing against 100 vol of phosphate
buffer for 5–6 h (4°C). Both fractions were pooled and concentrated
using a Centriprep-30000 concentrator (Amicon, Beverly, MA). The
resulting concentrate was applied to a DEAE-Sephadex column (Sigma)
equilibrated with PB. Fractions containing protein (as determined by
absorbance at 280 nm) were pooled. The buffer was changed to PBS, and
the protein was concentrated using a Centriprep-30000. The VSG was
filter-sterilized through a 0.2-µm filter and stored at -70°C. The
concentration of the purified protein was determined using the BCA
Protein Assay System (Pierce, Rockford, IL) compared with a BSA
standard (Pierce), and the purity of VSG was determined by SDS-PAGE as
we previously reported (41, 42).
Cell culture
Cells for in vitro culture were collected from infected mice at different times postinfection. Mice were exsanguinated by retro-orbital bleeding under anesthesia (Metofane, Mallinckrodt Veterinary, Mundelein, IL). Peritoneal cells (PC) were subsequently collected by injecting 5 ml of tissue culture medium (RPMI 1640; JRH Biosciences, Lenexa, KS) containing gentamicin (50 µg/ml; Life Technologies, Grand Island, NY), HEPES (16 mM; JRH Biosciences), and heparin (2 U/ml; Amersham, Arlington Heights, IL) without FBS i.p. and mechanically agitating the mice for 4 min at 3500 rpm on an orbital shaker. The PC were then withdrawn aseptically using an 18-gauge needle and syringe, and washed in tissue culture medium (c-RPMI) supplemented with 10% FBS and L-glutamine (JRH Biosciences). Spleens were removed and dissociated to single cell suspensions (SPC) in c-RPMI. RBC and debris removal was achieved by hypotonic lysis and subsequent passage of the cell suspension over a glass-wool column. Cells were washed in c-RPMI and counted with trypan blue and a hemocytometer. Splenocytes for adoptive transfer experiments were prepared in the same manner, except cells were washed an additional two times in PBS and resuspended in PBS immediately before i.v. injection.
Cultures of SPC (1.2 x 107 cells/well) or PC (3 x 106 cells/well) were incubated for 24 h at 37°C in 7% CO2 in six-well plates (6 ml of medium/well). Cells were cultured in c-RPMI alone, with Con A (2.5 µg/ml, Sigma), or with VSG (50 µg/ml) for 24 h. At the end of the incubation period, supernatant fluids were collected, centrifuged to remove cells, and stored at -20°C until analysis. RNA was isolated from cultured cells using Ultraspec (Biotecx, Houston, TX) according to the manufacturer’s specifications.
Cytokine assays
Levels of IFN-, IL-2, and IL-4 in culture supernatant fluids
were determined by sandwich ELISA. Immulon 4 plates (Dynatech,
Chantilly, VA) were coated by incubating plates at 4°C overnight with
capture Ab diluted in PBS. Plates were washed four times with PBS and
0.05% Tween-20 (PBST; Sigma) and blocked by incubation for 30 min at
37°C with PBS and 2% BSA. One hundred microliters of culture
supernatant fluid or a dilution of supernatant fluid in medium was
added to each well and incubated for 2 h at 37°C. The plate was
again washed four times with PBST before addition of biotinylated
detection Ab (diluted in PBS and 2% BSA). Following additional
incubation (1 h at 37°C) and washing (four times with PBST) steps,
avidin-conjugated alkaline phosphatase was prepared and used according
to the manufacturer’s instructions (Vectastain ABC-AP, Vector
Laboratories, Burlingame, CA). The substrate
p-nitrophenylphosphate disodium (Sigma) was prepared at a
concentration of 2 mg/ml in 0.1 M sodium carbonate/bicarbonate buffer,
pH 9.6, and 0.01 M MgCl2. The absorbance at 405 nm was
measured with a microtiter plate reader (EL311, BioMetallics,
Princeton, NJ), and the concentration of cytokine in each well was
calculated by comparison with a standard curve of recombinant cytokine
(IL-2, IL-4 (PharMingen, San Diego, CA), or IFN- (Genzyme,
Cambridge, MA)) using Deltasoft II software (BioMetallics).
Anti-IFN- Ab (R4-6A2 ascites fluid; 1/8000) was used as the capture
Ab in the IFN- ELISA. Other Abs used in ELISA were purchased from
PharMingen (San Diego, CA) and used at the concentrations noted:
IFN- detection, 0.5 µg/ml; IL-2 capture, 0.5 µg/ml; IL-2
detection, 0.25 µg/ml; IL-4 capture, 0.25 µg/ml; and IL-4
detection, 0.5 µg/ml.
IL-2 and IL-4 activities were also assessed using the CTLL-2 bioassay system (43). The cells were grown in c-RPMI supplemented with 5% T-Stim (culture supplement, rat, with Con A; Collaborative Biomedical Products, Bedford, MA) and were routinely passaged or used for assays upon reaching stationary growth. Cells were washed in unsupplemented culture medium three times before the assay to remove residual growth factors provided by the culture supplement. Standard curves were prepared with rIL-2 and rIL-4 (PharMingen). Cultures of 5 x 103 cells/well with the test culture supernatant fluids were set up in triplicate in 96-well plates. To determine which cytokine was inducing proliferation, blocking concentrations of Ab to IL-2 (S4B6 ascites), IL-4 (11B11 ascites), or both were added to replicate cultures. The plates were incubated at 37°C in 5% CO2 in air. After 24 h of incubation, [3H]thymidine (1 µCi/well) was added, and the plates were incubated for an additional 24 h. The cultures were harvested onto nylon-backed glass-fiber filters (Packard, Meriden, CT) using an Inotech cell harvester (Inotech Biosystems International, Lansing, MI) and counted with a Matrix 9600 direct beta counter (Packard).
Quantitation of cytokine transcripts
IFN- and IL-4 transcript levels in cultured cells were
determined by quantitative competitive PCR (QC-PCR) as we have
previously described (30). Total RNA in each cell culture sample was
quantitated by measuring absorbance at 260 nm after purification. cDNA
was generated in a reverse transcription reaction (AMV Reverse
Transcription Kit, Promega, Madison, WI) according to the directions of
the manufacturer and was then used in QC-PCR reactions using the method
described by Clontech (Palo Alto, CA) and others (44, 45). Reactions
containing cDNA were set up with fourfold dilutions of a known amount
of competing DNA fragment (MIMIC) that contained 5' and 3'
regions complementary to sequences of the gene-specific primers. To
calculate the number of copies of a given gene transcript within an RNA
sample, an internal standard for RNA quantitation and efficiency of the
RT reaction, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), was also
determined. Primers used in quantitation reactions are as follows:
G3PDH upstream, 5'-TGA AGG TCG GTG TGA ACG GAT TTG GC-3'; G3PDH
downstream, 5'-CAT GTA GGC CAT GAG GTC CAC CAC-3'; IFN- upstream,
5'-CAT CTT GGC TTT GCA GCT CTT CCT CAT GGC-3'; IFN- downstream,
5'-TGG ACC TGT GGG TTG TTG ACC TCA AAC TTG GC-3'; IL-4 upstream, 5'-GAG
ATC ATC GGC ATT TTG AAC-3'; IL-4 downstream, 5'-GCT CTT TAG GCT TTC CAG
GAA GTC-3'; IL-2 upstream, 5'-ATG TAC AGC ATG CAG CTC GCA TC-3'; IL-2
downstream, 5'-GGC TTG TTG AGA TGA TGC TTT GAC A-3'. Following a "hot
start" protocol (94°C for 3 min, cooling to 42°C), Taq polymerase
(Promega) was added, and the reactions were cycled in a thermocycler
(MJ Research, Watertown, MA) under the following conditions (G3PDH,
IFN-, IL-2): initial denaturation at 94°C for 1 min, 30 cycles of
denaturation at 94°C for 45 s, annealing at 60°C for 45
s, and extension at 72°C for 2 min. A final extension step of 5 min
at 72°C was performed. Conditions for IL-4 were the same, except the
annealing temperature was 55°C, and 33–35 cycles were run.
Ab isotype ELISAs
Sera from normal and infected mice were tested in Ab isotype-specific ELISAs for VSG reactivity as we have previously described (30). Briefly, Immulon-4 (Dynatech) plates were coated with 4 µg/ml purified VSG overnight at 4 C. The plates then were washed, blocked, and washed again as described for the cytokine ELISAs. A dilution series of individual serum samples from different time points of infection was added to wells in triplicate and incubated for 2 h at 37 C followed by another wash. The VSG-specific Ab ELISA plates then were incubated with horseradish peroxidase (HRP)-conjugated rat anti-mouse IgM or IgG1 or with HRP-conjugated rabbit anti-mouse IgG3 (Zymed, South San Francisco, CA) or alkaline phosphatase-conjugated sheep anti-mouse IgG2a (The Binding Site, Birmingham, U.K.) for 1 h at 37 C. Plates were washed four times, then the substrate o-phenylenediamine dihydrochloride (Sigma) for the HRP conjugates or p-nitrophenylphosphate disodium for the alkaline phosphatase conjugate was added, and the assay was developed for 10 min at 25 C in the absence of light. The OD490 (IgM, IgG1, and IgG3) or OD405 (IgG2a) reading for each well was determined using an automated plate reader.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether there were differences in the Th cell
responses of trypanosome-infected mice with different resistance
phenotypes, supernatant fluids from VSG-stimulated lymphocyte cultures
were assayed for the presence of IFN-, IL-2, and IL-4 (Fig. 1
). Cytokines present in VSG-stimulated
cultures were compared with those in unstimulated cultures to
differentiate Ag-specific cytokine secretion from spontaneous cytokine
release. As shown in Fig. 1A, VSG-stimulated PC from
relatively resistant B10.BR mice (MST, >60 days) secreted significant
amounts of IFN-, whereas little, if any, IFN- was detectable in
supernatant fluids derived from cells of relatively susceptible C3H
mice (MST, <20 days). Secretion of IFN- by SPC of both mouse
strains in response to parasite Ag was less than that of PC (data not
shown), consistent with our earlier observations of relatively
compartmentalized Th cell responses during trypanosome infection (28, 30).
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B). VSG-stimulated PCs from
B10.BR mice produced measurable IL-2, while no IL-2 was detectable in
VSG-stimulated supernatant fluids of cells from C3H mice. In comparison
and contrast, no IL-4 was detected by ELISA (Fig. 1C) in
culture fluids from either mouse strain. Previously we have shown that
the VSG-specific Th cell response is the major component of total
parasite Ag-specific Th cell responses occurring during infection
(28, 29, 30, 46). Therefore, taken together, these results support our
earlier findings that B10.BR mice produce a partially compartmentalized
Th1 cell cytokine response and demonstrate, in addition, that C3H mice
produce quantitatively weaker parasite Ag-specific Th1 cell responses
without producing a qualitative shift to Th2 cell responses.
To confirm that VSG-specific cytokine responses were exclusively of the
Th1 type, and that the absence of IL-4 in culture supernatant fluids
was not due to an inability to detect low levels of IL-4 product,
levels of IFN- and IL-4 mRNA in cultured cells were quantitated by
QC-PCR. SPC and PC from uninfected B10.BR or C3H mice cultured in
medium exhibited no background levels of either IFN- or IL-4 mRNA,
confirming what we have shown previously (30). After stimulation with
VSG, SPC and PC from infected B10.BR mice showed a marked increase in
IFN- transcripts compared with cells cultured in medium alone, while
a smaller increase was observed in PC from C3H mice (data not shown);
little or no increase in IFN- mRNA was detected in SPC of infected
C3H mice. Similar analysis of IL-4 transcript levels showed no
VSG-induced IL-4 mRNA in SPC or PC cultured from either mouse strain,
although very low levels of IL-4 transcript were occasionally detected
in freshly isolated cells from these mice, similar to our previous
observations (30). Together, these findings confirm the results of the
ELISA and CTLL assays, showing that while the relatively resistant
B10.BR mice produce a strong Th1 cytokine response, this response is
much weaker in the relatively susceptible C3H mouse strain.
Furthermore, there is no evidence, by measurement of either IL-4
product or transcript, for Ag-specific Th2 cell responses in either the
resistant or susceptible mouse strains. The absence of IL-4 was
indicative of the absence of other Th2 cell cytokine responses, such as
IL-5 (data not shown), as we have previously demonstrated, and the
cytokine profiles presented here represent the peak responses observed
in response to VSG following infection (28, 30, 46).
Role of IFN- in determining host resistance
Because we detected quantitative, but not qualitative, differences
in the parasite Ag-specific Th1 cytokine responses of resistant B10.BR
and susceptible C3H mice, we asked whether Th cell cytokines played any
role in determining host resistance. To specifically address the
potential roles of IFN- and IL-4 in host resistance, we used IFN-
and IL-4 KO mice with the C57BL/6 genetic background (35, 36).
Wild-type C57BL/6 mice are relatively resistant to T.
brucei rhodesiense LouTat 1 and have a MST of 44 days or
more (47). In this study C57BL/6 mice were found to survive an average
of 46 days, while the IFN- KO mice survived only 19 days, similar to
the MST of scid mice (Fig. 2)
and genetically susceptible C3H mice (27, 48), indicating that IFN-
is critical for host resistance. Survival times of the IL-4 KO mice did
not differ significantly from the wild-type mice indicating that, in
contrast to IFN-, IL-4 has a neutral impact on host resistance.
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than the somewhat more resistant B10.BR
strain (47). IFN- and IL-2 were secreted by VSG-stimulated PC (Fig. 3, A and B), while
Ag-stimulated SPC populations secreted relatively lower levels of
IFN- and IL-2 (data not shown). As expected, cells from IFN- KO
mice did not secrete IFN-. Some biologically active IL-2 was
secreted by VSG-stimulated PCs, but levels were markedly reduced
compared with those in the wild-type mice (data not shown). However,
neither wild-type nor IFN- KO mice produced detectable IL-4, as
determined by both CTLL bioassay and ELISA (Fig. 3C).
Analysis of mRNA from the same cultured cells confirmed that induction
of IFN- transcripts occurred in VSG-stimulated cultures of PC from
wild-type mice, while there was no VSG-specific increase in levels of
IL-4 mRNA (Fig. 4). IL-4 KO mice had
cytokine responses similar to those of the wild-type mice, although
levels of IFN- and IL-2 secretion were higher in the IL-4 KO mice
compared with those in the wild-type mice (Fig. 5). Susceptible scid mice made
no detectable cytokines in response to VSG (data not shown), lending
additional support to the idea that Th1 cells are mediating resistance
as a result of Ag-induced IFN- secretion.
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Because IFN- was shown to be important for determining the
relative level of host resistance in this study, yet studies by others
have suggested that IFN- is a growth factor for trypanosomes, we
next asked whether the absence of IFN- in the IFN- KO mice had
any effect on patterns of parasitemia. For this purpose, parasitemia
was determined on a daily basis in blood sampled from the tail vein
(Fig. 6). For the first 7 days of
infection, all wild-type and IFN- KO mice displayed similar patterns
of parasitemia; typically, the first wave of parasites reached a peak
level in the blood on day 4 or 5 of infection and was completely
cleared by day 7. All mice displayed a period of remission before the
return of detectable numbers of trypanosomes in the blood. In wild-type
mice, the second wave of parasitemia was not as high as the first wave,
and three of five mice went on to clear this second wave completely by
day 21, while two mice had very low, but detectable, levels of
trypanosomes in the blood at that time. In contrast, the period of
remission between waves was slightly shorter in the IFN- KO mice
compared with that in the wild-type mice, and IFN- KO mice
experienced a second wave of parasitemia that was 3–10 times higher
than the first wave. This second wave of parasitemia was never cleared
by the IFN- KO mice before they succumbed to infection. Although the
pattern of parasitemia in experimental trypanosome infections is known
to be somewhat heterogeneous in timing and magnitude after the first
wave (49), the overall pattern that emerges from this study is one of
earlier and higher relapsing parasitemia in the IFN- KO mice
compared with that in wild-type mice.
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was affecting host resistance and patterns of
parasitemia by altering Ab isotype switch patterns, we compared the
isotypes and quantities of VSG-specific Abs in wild-type and IFN- KO
mice. Levels of VSG-specific IgM and IgG1 were similar in serum from
representative 2-wk-infected wild-type and IFN- KO mice, while the
level of IgG3 was slightly higher in wild-type mice (Fig. 7). There was, however, a complete
absence of VSG-specific IgG2a in IFN- KO mice compared with
that in wild-type mice. This is consistent with the dependency of the
Ig Cµ to C2a switch response on IFN- and confirms our recent
findings on cytokine-induced Ig switching events in trypanosomiasis
(30).
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To determine whether the presence of VSG-specific IgG2a was
associated with the relative resistance of wild-type mice, we pooled
the serum from uninfected or 2-wk-infected wild-type and IFN- KO
mice and administered 1 ml of serum i.p. to IFN- KO mice on day 7 of
infection, just after clearance of the first peak of parasitemia. The
mice were then monitored for survival. There was no difference in the
MST of mice receiving serum from infected wild-type mice compared with
that of mice receiving serum from normal or infected IFN- KO mice or
serum from normal wild-type mice (Table I). These results suggest that
VSG-specific Abs produced by wild-type mice are not responsible for the
enhanced resistance of these mice compared with IFN- KO mice.
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-producing lymphocytes mediate host
resistance we transferred splenocytes from uninfected wild-type or
IFN- KO mice to IFN- KO mice on days -1 and +14 of infection. As
shown in Fig. 2, IFN- KO mice receiving cells from wild-type mice
survived an average of 28 days, while control mice receiving cells from
IFN- KO mice showed no enhanced resistance compared with
nonmanipulated mice. Reconstituted IFN- KO mice did not survive as
long as wild-type mice, however, presumably due to the lower numbers of
IFN--producing cells in reconstituted mice compared with wild-type
mice. Regardless, these results clearly show that resistance to
trypanosomes in IFN- KO mice can be significantly enhanced by the
passive administration of cells capable of producing IFN- in
response to parasite Ag. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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KO mice both
made Ab responses to trypanosome VSG and controlled parasitemias, yet
differed markedly in their susceptibility to infection.
The discovery of T cell responses to trypanosome Ags, including VSG,
opened a new door in the analysis of host resistance to trypanosomes
(28, 29, 30, 46). In the present study we have revealed for the first time
a contributing role for VSG-specific Th1 cell IFN- production in
determining host resistance to African trypanosomes. Using B10.BR/SgSnJ
and C3HeB/FeJ mice as prototypical resistant and susceptible mouse
strains (27, 47, 48, 57), respectively, we have shown that there are
quantitative differences in the levels of parasite Ag-induced Th1 cell
cytokines produced by these two mouse strains. There was no evidence
for Ag-specific Th2 cell responses occurring in either mouse strain,
however; this finding was supported by an analysis of Ag-induced
cytokine mRNA. Together these results suggest that the difference in
resistance to trypanosomes displayed by these mice may be due to
quantitative and not qualitative differences in Th cell activation.
Results of cytokine analyses and survival studies performed with
wild-type, IFN- KO, IL-4 KO, and scid mice with the
relatively resistant C57BL/6 genetic background provided direct
evidence that IFN- production associated with Th1 cell responses is
an important component of protective immunity, and that Th2 cell
cytokine responses are not involved in determining host resistance or
susceptibility.
These results reveal several interesting features of experimental
trypanosomiasis. First, unlike other model systems of infectious
disease where qualitative differences in Th cell responses may
determine disease outcome, trypanosomes appear to elicit quantitative
differences in Th1 cell responses that are associated with the level of
host resistance. For example, the emerging pattern in one parasitic
model system, Leishmania major (58), has been that a
dominant Th1 cell response leads to resistance, whereas a dominant Th2
cell response leads to susceptibility. The opposite pattern is true for
the helminths Trichuris muris (59) and Nippostrongylus
brasiliensis (60) as well as for organisms such as Borrelia
burgdorferi (16), where Th2 cell responses lead to resolution of
the infection, and Th1 cell responses are associated with
susceptibility. In either case, the focus has been on qualitative
differences (Th1 vs Th2) in response to a pathogen and the association
of these qualitative differences with resistance or susceptibility. In
contrast to this paradigm, we observed quantitative differences in Th1
cell cytokine profiles between resistant and susceptible mice, and we
found no evidence for Th2 cell responses in any infected mouse strain.
A similar difference in the development of Th cell responses has been
reported during infection with Yersinia enterocolitica.
Resistant C57BL/6 mice infected with Y. enterocolitica were
strong Th1 cell responders and produced high levels of IFN-, while
susceptible BALB/c mice produced a delayed IFN- response; there was
no evidence for Th2 cell cytokine production (61). Thus, a mounting
body of evidence, including our findings with T. brucei
rhodesiense infections, challenges the general idea that
differences in resistance and susceptibility are due to qualitative
differences in Th cell cytokine responses. Rather, we observed that the
magnitude of the Th1 cell response is important in determining host
resistance, and that susceptibility is not associated with outgrowth of
a Th2 cell response.
A legitimate question is whether T cell priming with VSG occurs during
trypanosome infection of susceptible mouse strains, in that such
strains may be incapable of recognizing parasite Ags. However, we
recently demonstrated that infected susceptible IFN- KO mice are
defective in suppressor macrophage activity, which is associated with
depressed proliferative T cell responses (46), and such mice made
proliferative T cell responses to
VSG.4 Thus, this finding
demonstrates the underlying existence of VSG-reactive T cells in
susceptible mice.
The mechanism by which Th1, but not Th2, cell cytokine responses are
preferentially generated during trypanosome infection appears to be due
to two distinct effects.5
First, IFN--independent production of IL-12 occurs within hours of
infection and is necessary for the early development of VSG-specific
Th1 cell responses in resistant mice. Second, in the absence of IL-12
or IFN-, Th2 cell responses do not develop and appear to be
inhibited, directly or indirectly, by infection or by the trypanosomes
themselves. Thus, the induction of IL-12 appears to play an important
role in enhancing the development of Th1 cell responses, but the
parasites also appear to specifically depress the outgrowth of Th2 cell
responses by an unknown mechanism. Since we have observed that
susceptible strains of mice produce lower levels of IL-12 during the
early time points of infection compared with more resistant
strains,5 it may be that the stimulus for Th1 cell
outgrowth is weak, while the inhibitory effect in terms of Th2 cell
outgrowth is strong, resulting in the T cell phenotype seen in
susceptible mice.
Regardless of the mechanisms leading to polarized Th1 cell responses
during infection, our studies clearly and unambiguously demonstrate
that IFN- is important in determining host resistance. This finding
contradicts one proposed role for IFN- in infection with African
trypanosomes. Trypanosomes have been reported to produce a factor known
as TLTF (31, 32, 62, 63), which stimulates naive
CD8+ cells to release IFN- in an Ag nonspecific manner.
It has been proposed that IFN- produced by CD8+ cells as
a result of TLTF stimulation is used by the parasite as a requisite
growth factor (31, 34). In support of this hypothesis, studies have
demonstrated that neutralization of IFN- in vivo was found to
enhance the survival of both susceptible C3H/HeJ mice and resistant
C57BL/6 mice (64). Furthermore, it was determined that IFN- added to
cultures of T. brucei stimulated the proliferation and
survival of the parasite (34). However, our cumulative findings with
T. brucei rhodesiense are inconsistent with TLTF-induced
IFN- serving as a growth factor for trypanosomes. First, we have
shown previously that the primary source of IFN- produced during
infection in response to parasite Ags is CD4+ T cells
expressing the TCR-ß and recognizing antigenic peptides in an
APC-dependent and MHC II-restricted manner (28). Second, we have also
demonstrated in earlier studies a correlation between high IFN-
levels in serum, low parasitemias, and host resistance; in contrast,
susceptible mice had no detectable serum IFN- and exhibited high and
uncontrolled parasitemias (57). Third, we have shown in the present
study that susceptible C3HeB/FeJ mice produce less (or no) IFN- in
response to parasite Ag, whereas resistant B10.BR mice produce high
levels of IFN- in response to Ag. And, fourth, we have demonstrated
in this study that mice genetically incapable of IFN- production
(IFN- KO mice) are as susceptible to infection as mice without
functional T and B cells (scid mice) and that although both
wild-type and IFN- KO mice controlled parasitemia with an Ab
response to VSG, the overall parasitemias in the IFN- KO mice were
significantly greater. Thus, our cumulative studies have demonstrated
an inverse relationship between IFN- production and parasite growth.
One possible explanation for these discrepancies may lie with the
parasite itself. While the growth-enhancing activity of IFN- was
characterized with several different species and strains of
trypanosomes, including strains of T. brucei rhodesiense
(33, 34), we repeatedly have been unable to confirm that trypanosomes
of the LouTar serodeme of T. brucei rhodesiense are growth
responsive to IFN- in vitro (unpublished observations), and similar
results with other trypanosomes have been obtained previously (65). It
may be that some trypanosomes are more susceptible to IFN--induced
host resistance mechanisms (see below) than strains of trypanosomes
deemed to be growth stimulated by IFN-; thus, the result of exposure
to IFN- in vivo may be that any potential growth stimulatory effects
of IFN- are negated.
Overall, the present work clearly shows that IFN- is essential for
host resistance to T. brucei rhodesiense LouTat 1 in animals
capable of controlling parasitemia with Ab responses; however, it is
not yet clear how IFN- affords protection to the host. One potential
mechanism might involve the nature of parasite-specific Abs. IFN-
promotes a Cµ to C2a Ig isotype switch in VSG-specific Abs (30),
and it may be that this isotype switch event is critically important
for effective parasite control. However, when immune sera from
wild-type or IFN- KO mice containing Abs to the infecting
trypanosome VSG as well as to the VSG of subsequently arising variant
types were passively transferred to infected IFN- KO recipients,
there was no detectable effect on host survival. In support of this
finding is an earlier body of work demonstrating that parasite
clearance is effectively mediated by the VSG-specific IgM response
alone and that Ab isotype switching events occur primarily after
elimination of the trypanosomes from blood (30, 50, 66).
It is more likely that IFN- exerts its protective effect via the
macrophage cell. For example, IFN- serves as a potent macrophage
activation agent, and we have found that when trypanosomes are cultured
in the presence of macrophages and IFN-, the parasites are rapidly
destroyed; this cytotoxicity was reversible by adding arginine
analogues or Ab to IFN- to the culture medium (J. M. Mansfield,
unpublished observations). We suspect that this cytotoxicity is due to
nitric oxide (NO) production by IFN--activated macrophages. In this
light, we have previously demonstrated that macrophages from
trypanosome-infected mice exhibit up-regulated inducible NO synthase
transcript levels and spontaneously release NO (46). Furthermore,
others have shown that NO exerts adverse effects on trypanosomes
(67, 68, 69), and products of activated macrophages such as TNF- have
also been demonstrated to exert cytotoxic effects on trypanosomes
(70, 71, 72). Furthermore, we recently completed studies showing that
resistant mice, including C57BL/6 mice, exhibit earlier and
quantitatively greater macrophage activation responses than susceptible
mice, including IFN- KO
mice.6 We suggest,
therefore, that quantitative or qualitative differences in
IFN--induced macrophage activation may dramatically affect the
course of infection.
Since we have shown that IFN- is critical for host resistance to
T. brucei rhodesiense, and activated macrophages may serve
as the basis for this resistance, we formally propose that the Th1 cell
cytokine response is linked to resistance through the action of IFN-
on host macrophages. It is likely that this IFN-- and
macrophage-dependent component of host immunity is most effective
within the extravascular tissue sites, where it complements
parasite-specific Ab responses that are most effective within the
vascular compartment. Together, therefore, both Th and B cell responses
provide the resistance mechanisms necessary to control parasite numbers
in all tissues of the trypanosome-infected host.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John M. Mansfield, Department of Bacteriology, FMT Bldg., 1925 Willow Dr., University of Wisconsin, Madison, WI 53706. E-mail address:
3 Abbreviations used in this paper: VSG, variant surface glycoprotein; SPC, spleen cells; PC, peritoneal cells; KO, knockout; QC-PCR, quantitative competitive PCR; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; MST, mean survival time; TLTF, T lymphocyte triggering factor; NO, nitric oxide.
4 C. J. Hertz, and J. M. Mansfield. 1998. IFN--dependent nitric oxide production is not linked to resistance in experimental African trypanosomiasis. Submitted.
5 J. M. Mansfield, C. J. Hertz, H. Filutowicz, L. R. Schopf, and J. Sypeck. 1998. IFN-independent IL-12 production during trypanosome infection directs the outgrowth of highly polarized Th1 cell responses. Submitted.
6 M. Imboden, H. Filutowicz, M. A. Lokuta, D. M. Paulnock, and J. M. Mansfield. 1998. Tissue-specific patterns of macrophage activation in experimental African trypanosomiasis. Submitted.
Received for publication May 11, 1998. Accepted for publication August 20, 1998.
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