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Infection and Immunity, February 2000, p. 809-814, Vol. 68, No. 2
Department of Tropical Public Health, Harvard School of
Public Health,1 and Department of Biological
Chemistry and Molecular Pharmacology, Harvard Medical
School,3 Boston, Massachusetts 02115;
Department of Molecular Microbiology, Washington University
Medical School, St. Louis, Missouri 631102; and
Department of Pathology, School of Veterinary Medicine and
Biological Sciences, Colorado State University, Fort Collins, Colorado
805234
Received 20 September 1999/Returned for modification 22 October
1999/Accepted 10 November 1999
To determine whether an ongoing response to Leishmania
major would affect the response to a non-cross-reacting,
non-leishmanial antigen, susceptible BALB/c mice and resistant C3H mice
were infected with L. major parasites expressing
Escherichia coli The two major subsets of the CD4
T-cell compartment of mice, Th1 and Th2, produce distinct repertoires
of cytokines. For example, Th1 cells secrete gamma interferon (IFN- These observations point to an interesting question regarding
immunoregulation. How might a heavily skewed Th1 or Th2
parasite-specific response influence the concomitant response of T
cells to a protein that was antigenically unrelated to
Leishmania?
To address this question, we utilized techniques developed for
introducing and expressing foreign genes in L. major
(7). Using this approach, we expressed a bacterial antigen,
namely Escherichia coli Parasites.
L. major promastigotes (LV39/Neal/P strain,
clone 5 [22]) were maintained in M199 or NNN medium as
previously described (8, 22). For experiments, promastigotes
were harvested from stationary phase cultures which contained the
infective (metacyclic) form of the parasite (26). To produce
amastigotes, promastigotes were injected subcutaneously (s.c.) into the
shaved rumps of BALB/c mice or BALB/c nu/nu mice, and amastigotes were
isolated according to published techniques (11).
Molecular constructs and transfection.
To express Determining Reagents.
Infection and lymphocyte stimulation assay.
Mice were
infected s.c. in one hind footpad with 2.5 × 106
L. major-NEO or L. major- Generating antigen-specific T-cell blasts and flow
cytometry.
The LNC (5 × 106/ml) draining lesions
on infected mice were cultured in 24-well flat-bottom plates (Costar)
and stimulated with L. major (5 × 105/ml)
promastigotes or
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Priming of a
-Galactosidase (-GAL)-Specific Type 1 Response
in BALB/c Mice Infected with -GAL-Transfected Leishmania
major
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-galactosidase (-GAL); this parasite
was designated L. major-GAL. BALB/c and C3H mice
responded to infection with L. major-GAL by mounting a
CD4 T-cell response to both parasite antigens and to the reporter antigen, -GAL. The phenotypes of these T cells were characterized after generating T-cell lines from infected mice. As expected, BALB/c
mice responded to infection with L. major-GAL by
producing interleukin 4 in response to the parasite and C3H mice
produced gamma interferon (IFN-
) in response to the parasite and
-GAL. Interestingly, however, BALB/c mice produced IFN- in
response to -GAL. Taken together, these results demonstrate that
priming of IFN--producing cells can occur in BALB/c mice despite the fact the animals are simultaneously mounting a potent Th2 response to
L. major.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
)
and interleukin 2 (IL-2) while Th2 cells secrete IL-4 and IL-5
(23). The functions of Th1 and Th2 cells are modulated by
the reciprocal cross-regulatory properties of these cytokines.
Moreover, other cells of the immune system, such as macrophages
(M
s), produce cytokines (e.g., IL-10, IL-12, and transforming growth
factor ) that can influence the activities of T cells. Infection of
mice with Leishmania major is perhaps the best-studied
example of a disease in which selective activation of Th cells leads to
opposite outcomes of infection. Most mouse strains (e.g., C57BL/6 and
C3H) mount a Th1 response to the parasite and cure the infection,
whereas susceptible mice (e.g., BALB/c) mount a Th2 response and
succumb to infection (4, 20, 24, 25, 32).
-galactosidase (-GAL), in
L. major and then infected mice with this transfected
parasite, which was designated L. major--GAL. Since the
expressed -GAL would be targeted to the same phagolysosome as
L. major and be exposed to the same set of phagolysosomal
degradative enzymes, it could serve as a "reporter antigen" to
determine how a Th1 or Th2 response to L. major influences
the immune response to -GAL. Therefore, we followed the T-cell
response to both L. major and -GAL in mice infected with
L. major-GAL. We anticipated that the response to -GAL would be type 1 in mice mounting a Th1 response to L. major,
whereas it would be type 2 in mice mounting a Th2 response to the parasite.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-GAL
within L. major, we used the vector pXG (strain B1288
[12]). The E. coli lacZ coding region was
introduced into the pXG expression site, yielding the plasmid
pXG-GAL (strain B1007; L. Borges and S. M. Beverley,
unpublished data). Parasites freshly recovered from infected mice were
transfected with pXG-NEO or pXG-GAL by electroporation, and clonal
lines were obtained by plating on semisolid media as previously
described (15). Transfected promastigotes were maintained in
medium containing 10 µg of Geneticin (Sigma, St. Louis, Mo.) per ml.
Several transfectants were inoculated into mice to confirm that they
remained infective, and one each bearing one or the other plasmid and
showing wild-type infectivity was identified and used in this work.
Infective L. major promastigotes containing pXG-NEO were
designated L. major-NEO while those containing pXG-GAL
were designated L. major-GAL.
-GAL activity.
Pellets containing 2.5 × 107 parasites (promastigotes or amastigotes) were snap
frozen in liquid nitrogen and then resuspended in 100 µl of TPI
buffer (19) containing 0.01% sodium dodecyl sulfate (SDS).
-GAL activity was determined as previously described (19), taking care to ensure that the amount of extract and
the time of the assay were in the linear range of the assay. Extract aliquots were diluted to 80 µl with TPI and added to 320 µl of reaction buffer containing 0.3 mM 4-methyl-umbelliferyl
-D-galactoside (Sigma). Aliquots of 40 µl were taken
at 10-min intervals and added to 200 µl of stop buffer. The
fluorescence of the 4-methylumbelliferone (4-MU) product was measured
in a Bio-Rad Fluoromark microplate fluorometer and compared to that of
known concentrations of 4-MU (Sigma). In these assays, 2,700 fluorescence units corresponds to 1 nmol of 4-MU product. Assays of
purified E. coli -GAL (Sigma) showed a specific activity
of 1.5 × 108 fluorescence units/min/mg with the
4-MU--GAL substrate. All assay time points and samples were done in duplicate.
-GAL was purchased from Sigma (G 6008) and
dialyzed extensively against sterile double-distilled water before use.
The protein content in the dialyzed preparation was determined by the
micro bicinchoninic acid assay (Pierce, Rockford, Ill.), and then the preparation was aliquoted and stored at 
70°C until use.
Methyl[3H]thymidine (5 Ci/mmol) was purchased from
Amersham (Arlington Heights, Ill.).
GAL amastigotes, in a
final volume of 50 µl. At intervals thereafter, the draining
popliteal and inguinal lymph node cells (LNC) were restimulated
(6) in vitro (4 × 105/well) with L. major promastigotes (106/ml) or -GAL (100 µg/ml)
in Dulbecco's modified Eagle medium (DMEM) containing 0.5% normal
mouse serum in 96-well flat-bottom plates (Costar, Cambridge, Mass.).
After 5 days of culture (optimal time), LNC proliferation was measured
by pulsing the plates with 1 µCi of [3H]thymidine per
well, harvesting 24 h later on an automated sample harvester, and
assaying the incorporated radioactivity by scintillation counting.
Triplicate cultures were used in all experiments. In addition,
supernatants were harvested at 48 h and assayed by capture enzyme-linked immunosorbent assays for their content of IFN- and
IL-4 by published techniques (5, 6, 30).
-GAL (100 µg/ml). The blast cells were isolated
on Percoll gradients (29).
Activating -GAL-specific T-cell hybridoma 1E3.03.H4 to produce
IL-2.
The T-cell hybridoma 1E3.03.H4 (kindly provided by J. Langhorne, Imperial College, London, England, and I. Muller, University of Notre Dame, Notre Dame, Ind.) is I-Ad restricted and
-GAL specific. As a result, it produces IL-2 when activated by
BALB/c Ms presenting -GAL (10). 1E3.03.H4 was
maintained in DMEM containing 5% fetal calf serum.
s [5]) were cultured for an additional 4 h
with either medium alone or 100 U of IFN- (13) plus 10 ng
of lipopolysaccharide (LPS)/ml (E. coli 055:B55W; Difco, Detroit, Mich.) (10 ng of LPS/ml is a subactivating dose
[28]). Cultures were rinsed, and Ms were cultured
for another 4 h at 34°C with either -GAL (50 µg/ml) or
various forms of L. major (see text for details). Finally,
the cultures were rinsed, 1E3.03.H4 cells were added (106
cells/well), and the cultures were incubated for 18 h. The
supernatants of the cultures were collected and analyzed for their
content of IL-2 by capture enzyme-linked immunosorbent assay using
published techniques (5, 6, 30) as an indicator of the
degree of activation of 1E3.03.H4.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Generating L. major expressing high levels of -GAL
throughout the infectious cycle.
We chose -GAL as our reporter
antigen since, when emulsified in complete Freund's adjuvant and
injected into mice, -GAL elicits a potent type 1 response (data not shown).
-GAL in L. major and Leishmania
mexicana (18, 21). However, subsequent data suggested
that -GAL expression was down-regulated in lesion amastigotes
(21; L. Borges and S. M. Beverley, unpublished
data). In this work, we used a related expression vector, pXG-NEO,
which yields consistently higher expression in the amastigote stage (L. Borges and S. M. Beverley, unpublished data).
We introduced pXG-NEO and pXG-GAL into L. major
promastigotes and identified infective clonal transfectants bearing a
plasmid. Those bearing pXG-NEO were designated L. major-NEO
while those containing pXG-GAL were designated L. major-GAL. L. major-GAL promastigotes expressed high
levels of -GAL, 46 ng/106 cells (about 2% of the total
cell protein). The -GAL in these L. major-GAL
parasites was exclusively intracellular, as revealed by the absence of
staining of colonies by the sensitive chromogenic substrate X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside; data
not shown).
To confirm that the production of -GAL remained high in the
amastigote form of L. major, L. major-GAL
amastigotes were purified from cutaneous lesions of BALB/c mice 3 weeks
after infection. These amastigotes showed high levels of -GAL, 10 ng/106 cells, or about 22% of the level present in
promastigotes when they were injected into mice. Preliminary studies
suggest that the majority of the decrease relative to the levels in
promastigotes arose from a decline in the episomal pXG-GAL plasmid
copy number during the period of growth in vivo, rather than specific
developmental regulation of pXG-GAL expression (data not shown).
Thus, L. major-GAL showed a sustained ability to express
-GAL throughout the parasite infectious cycle in mice infected with
the parasite. As a result, L. major-GAL was an ideal
candidate for the experiments presented here.
L. major-NEO induces only a leishmanial-specific
response, but L. major-GAL induces both a leishmanial
and -GAL-specific response in BALB/c and C3H mice.
We chose to
use BALB/c or C3H mice as our experimental model since they represent
mice that are highly susceptible or highly resistant to infection with
L. major, respectively (2, 27). These mice were
infected with either 2.5 × 106 amastigotes of control
parasites (L. major-NEO) or L. major-GAL s.c.
in one hind footpad. At various times thereafter, the draining LNC were
removed and restimulated in vitro with either L. major or
-GAL. As determined in vitro, BALB/c and C3H mice responded to
infection with L. major-NEO by generating a vigorous
parasite-specific response, but little or no response to -GAL (Fig.
1). In contrast, BALB/c and C3H mice
responded to both L. major and -GAL in vitro following
infection with L. major-GAL. At 3 weeks of infection, the
-GAL-specific response was approximately 40% of the response to the
parasite (Fig. 2).
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-GAL released from lysing
L. major-GAL promastigotes at the inception of the
infection might have influenced our results. However, to directly test
this possibility, 2.5 × 106 L. major-NEO
control parasites were purposely contaminated with soluble -GAL (100 µg), and these were coinjected s.c. into the footpads of BALB/c and
C3H mice. At varying times thereafter, the draining LNC were removed
and restimulated in vitro with either L. major or -GAL.
Importantly, this approach did not prime BALB/c mice to -GAL and
produced only a short-lived response to -GAL in C3H mice (Fig.
3).
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-GAL induces IFN- production in BALB/c mice.
Next, we
determined the cytokines secreted by LNC harvested from BALB/c mice
infected with L. major-GAL. Figure
4 shows that LNC from BALB/c mice
responded to parasite antigens in the anticipated fashion; namely, they
produced a substantial amount of IL-4 and little IFN-. In contrast,
these LNC produced predominantly IFN- and little IL-4 when
restimulated with -GAL in vitro. Therefore, (i) -GAL-specific
cells were primed in BALB/c mice infected with L. major-GAL, (ii) these cells proliferated to -GAL stimulation in vitro, and (iii) these cells made IFN- when stimulated with -GAL.
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GAL, IFN- was
produced in response to both leishmanial antigens and to -GAL,
whereas little or no IL-4 was produced in response to either antigen
(Fig. 4).
L. major-GAL primes CD4 L. major- and
-GAL-specific T cells.
Since the production of IFN- in
response to -GAL was not the result expected from the BALB/c mice,
we wished to characterize this response further. The principal cell
that responds to infection with L. major in mice is a CD4 T
cell (4, 20, 24, 25, 32). Therefore, we determined whether
the cells responding in our system were CD4 T cells. LNC draining the
site of infection with L. major-GAL were stimulated for 5 days in vitro with either L. major or -GAL, and the
responding T-cell blasts were isolated. Fluorescence-activated cell
sorting analysis (Table 1) revealed that
the surface phenotype of the cells that responded to either L. major or -GAL was largely that of CD4 T cells. However, some cells were CD8 (12 to 15%). Thus, it is likely that CD4 cells, and
perhaps CD8 cells, were the source of the IFN- shown in Fig. 4.
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L. major-GAL-infected Ms do not activate
-GAL-specific T cells unless the Ms are activated with IFN-
plus LPS.
Taken as a whole, the data presented show that when
L. major is transfected with the Th1 antigen -GAL, the
transfected parasite (L. major-GAL) induces a T-cell
response to itself and to -GAL in BALB/c mice (Fig. 2). Moreover,
this -GAL priming appears to occur only after L. major-GAL is internalized by phagocytic cells (e.g., Ms)
which then present -GAL to responding T cells. The latter conclusion
is supported by the data of Fig. 2 and 3, wherein it is shown (i) that
L. major purposely contaminated with soluble -GAL does
not induce a -GAL response in BALB/c mice (therefore, possible early
extracellular lysis of injected L. major-GAL parasites
with the release of -GAL was not responsible for the -GAL
response observed [Fig. 3]) and (ii) that the response to -GAL in
L. major-GAL-infected BALB/c mice was greatest at 3 weeks
of infection (Fig. 2)
a time by which L. major-GAL has presumably long since been internalized by phagocytes in the mice.
GAL-infected BALB/c
Ms could present -GAL to -GAL-specific T cells, we infected
BALB/c Ms in vitro with the parasite and tested whether these Ms
could activate the I-Ad-restricted, IL-2-producing
-GAL-specific T cell hybridoma, 1E3.03.H4. Following incubation with
soluble -GAL, starch-elicited peritoneal BALB/c Ms induced a
marked IL-2 response from 1E3.03.H4 (Fig. 5). In contrast, following infection with
L. major-GAL, BALB/c Ms were unable to present -GAL
unless the cells were activated with IFN- plus LPS (Fig. 5). This
was a dose-dependent response, in that the greater the degree of
infection with L. major-GAL, the greater was the IL-2
signal secreted by 1E3.03.H4 (Fig. 5). Finally, this IL-2 response was
specific for -GAL since no IL-2 was secreted when BALB/c Ms were
infected with L. major-NEO and activated with IFN- plus
LPS (Fig. 5).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We used an L. major parasite that expresses a
non-leishmanial reporter Th1 antigen (-GAL) to determine how the
response to this reporter antigen is affected by a simultaneous
response to L. major. L. major is known to induce strongly
polarized Th1 or Th2 responses in resistant or susceptible mouse
strains, respectively (reviewed in references 4, 20, 24, 25,
32). Our results suggest that (i) -GAL-specific T cells
were primed in susceptible BALB/c mice following infection with
L. major-GAL (Fig. 2), (ii) these cells were not primed
by soluble -GAL released from L. major-GAL that were
lysed when injected into mice (Fig. 3) (although this does not exclude
the possibility that later in infection, soluble -GAL would be
released from phagocytic cells degrading L. major-GAL),
and (iii) -GAL-specific T cells were capable of secreting large
quantities of IFN- when restimulated with -GAL in vitro (Fig. 4).
It is interesting that BALB/c Ms were able to present -GAL to
responding T cells in vitro following infection with L. major-GAL and activation with IFN- plus LPS (Fig. 5). This
suggests that Ms in BALB/c mice also present -GAL in vivo when
the cells become infected with L. major-GAL. The data of
Fig. 5 also suggest that efficient presentation of -GAL does not
occur unless BALB/c Ms are activated. The reason activation is
required is unknown. Since the location of -GAL in L. major-GAL parasites is exclusively intracellular, some
destruction of L. major-GAL by BALB/c Ms (by, for
example, activation with IFN-) may be required before the cells can
effectively present -GAL. Taken as a whole, these observations
suggest that priming of -GAL-specific T cells would not occur in
BALB/c mice infected with L. major-GAL unless
antigen-presenting cells in the animals are activated to kill L. major-GAL, thus releasing -GAL for antigen processing and
presentation. The literature supports this contention. Although there
is rapid multiplication of L. major in the first week of
infection in both resistant and susceptible mice, the rate of
multiplication of the parasite in BALB/c mice slows considerably beyond
the second week of infection (34), and the second week of
infection is the time by which -GAL-specific T cells could be
recovered from BALB/c mice (Fig. 2 and 4). Moreover, treating BALB/c
mice with a neutralizing anti-IFN- antibody worsens an infection
with L. major and results in rapid dissemination of the
parasite in BALB/c mice, which suggests that IFN- activates cells
infected with L. major to kill the parasite in vivo
(3). Finally, work by Wolfram et al. (35)
directly demonstrated that Ms were unable to present amastigote
cysteine proteinase antigens of L. mexicana to T cells
unless the Ms were activated to kill the parasite intracellularly.
Other investigators have examined whether an ongoing Th2-skewed immune response can influence the immune response to an unrelated antigen. For example, Kullberg et al. (17) showed that the response to sperm whale myoglobin was more Th2-like in mice infected with Schistosoma mansoni as compared to that in uninfected control mice. In addition, Barral-Netto et al. (1) and Doherty et al. (9) showed that an ongoing Th2-biased response in mice exacerbated infection with either Leishmania amazonensis or L. major, respectively. Curiously, Doherty et al. (9) showed that even though infection with L. major was exacerbated, the mice still produced Th1 cytokines when lymphoid tissue was restimulated with leishmanial antigens in vitro. This result is in agreement with the results presented here (Fig. 4).
Genetically engineered Leishmania organisms have been used
by others to study antigen processing in Ms infected with L. major (16) and to study the importance of cytotoxic T
lymphocytes in the resistance of mice to infection with L. mexicana (21). In this study, we used these
transfection systems to express -GAL in L. major. We then
used the resulting parasite (L. major-GAL) to study
immunoregulation in mice infected with L. major-GAL. The
advantage of this approach is that it allows one to study the effect of
L. major infection on an unrelated antigen (-GAL) without
having to coinfect mice with another pathogen. Compared to the L. major-GAL system, coinfecting mice with two pathogens introduces unnecessary complications which might confound the interpretation of results. A second advantage of the L. major-GAL system is that since -GAL is exclusively
intracellular in L. major-GAL, L. major and
its reporter antigen, -GAL, should be targeted to the same
phagolysosome of phagocytic cells in mice infected with L. major-GAL. Therefore, the ability to genetically manipulate
Leishmania offers a powerful tool to address matters as
diverse as mechanisms of immunoregulation in mice infected with the
parasite to the development of auxotrophic gene knockout parasites that
can be used as a platform for the development of safe live vaccines for
leishmaniasis (33).
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ACKNOWLEDGMENTS |
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This work was supported by NIH grants AI 29955 (R.G.T.) and 29646 (S.M.B.).
We thank Lucia Borges for providing the pXG-NEO- and
pXG-GAL-transfected L. major organisms and Monica Estay
for excellent technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathology, CVMBS, Colorado State University, Fort Collins, CO 80523. Phone: (970) 491-4964. Fax: (970) 491-0603. E-mail: rtitus{at}cvmbs.colostate.edu.
Present address: Department of Internal Medicine, James H. Quillen
College of Medicine, East Tennessee State University, Johnson City, TN 37614.
Present address: Department of International Health, Johns Hopkins
University School of Public Health and Hygiene, Baltimore, MD 21205.
Editor: W. A. Petri Jr.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, February 2000, p. 809-814, Vol. 68, No. 2
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