International Immunology, Vol. 13, No. 8, 975-982,
August 2001
© 2001 Japanese Society for Immunology
Treatment with cathepsin L inhibitor potentiates Th2-type immune response in Leishmania major-infected BALB/c mice
Department of Parasitology and Immunology, The University of Tokushima School of Medicine, 3 Kuramoto-cho, Tokushima 770-8503, Japan
1 Chemistry Laboratory, Taiho Pharmaceutical Co., Misugidai 1-27, Hanno 357, Japan
2 Institute for Health Sciences, Tokushima Bunri University, Tokushima 770–8514, Japan
Correspondence to: Y. Maekawa
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Abstract |
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Prior to the activation of CD4 + T cells, exogenous proteins must be digested by endo/lysosomal enzymes in antigen-presenting cells (APC) to produce antigenic peptides that are able to be presented on class II molecules of the MHC. Studies described here inspect the functional significance of cathepsin L inhibition for antigen processing and T h 1/T h 2 differentiation in experimental leishmaniasis. We first demonstrated using in vitro systems that cathepsin L is one of the candidate endo/lysosomal enzymes in processing of soluble Leishmania antigen (SLA) and that its specific inhibitor, CLIK148, modulated the processing of SLA. BALB/c mice are known to be susceptible to infection with Leishmania major . Interestingly, treatment of BALB/c mice with CLIK148 exacerbated the infection by enhancing the development of SLA-specific T h 2-type response such as production of IL-4 and generation of T h 2-dependent specific IgE/IgG1 antibodies. Moreover, addition of CLIK148 in incubation of a SLA-specific CD4 + T cell line with APC up-regulated the production of IL-4. However, CLIK148 did not exert any direct influence on the function of T cells themselves. Taken together, these findings suggest that treatment of host mice with CLIK148 affects the processing of SLA in APC, resulting in the potentiation of T h 2-type immune responses and thus leading to exacerbation of the infection. Furthermore, endo/lysosomal cathepsin L was found to be functionally distinct from previously described cathepsins B and D.
Keywords: antigen processing, lysosomal proteases, protozoan parasites, T h 1, T h 2
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Infection in experimental animals with Leishmania major , an obligate intracellular protozoan parasite, induces polarized T h 1 and T h 2 responses. BALB/c mice (H-2 d ) are susceptible to infection with L. major and readily develop a disease-promoting T h 2-type response, which produces IL-4, -5 and -10, resulting in the production of IgE and IgG1 antibodies. In contrast, DBA/2 (H-2 d ), C3H/j (H-2 k ) and C57BL/6 (H-2 b ) mice are resistant, and show a protective T h 1-type response ( 1 – 3 ). T h 1-type CD4 + T cells are known to generate IL-2 and IFN-
, and to support the production of antigen-specific IgG2a antibody (
4
). Before the activation of CD4 + T cells, exogenous antigens must be processed by endo/lysosomal enzymes within antigen-presenting cells (APC) to create antigenic peptides and then be presented by MHC class II molecules ( 5 , 6 ). Enzymes such as aspartate proteases (e.g. cathepsin D) and cysteine proteases (e.g. cathepsins B, S and L) are thought to be involved in this process. Cathepsins B and D are mainly located in APC, and have been reported to be involved in the processing of various antigens and also in the degradation of the invariant chain (Ii) of MHC class II molecules ( 7 – 10 ). It has also been reported that cathepsins L and S are essential for maturation of MHC class II molecules through degradation of Ii and for antigen presentation in the thymus and in the spleen respectively ( 11 – 14 ). Cathepsin L, which exists in virtually all mammalian tissues, plays an important role in degradation of both exogenous and endogenous proteins ( 15 ). It has recently been demonstrated that cathepsin L exists in APC of the spleen as a proform, procathepsin L, and that the maturation of this protease is induced when mice are immunized with foreign antigens such as ovalbumin and soluble Leishmania antigen (SLA) ( 16 ). The increase in mature cathepsin L in macrophages of mice immunized with foreign antigens should be important in enhancing the proteolytic process of the endocytosed antigens and in presenting these antigens to the immune system, since macrophages play special roles in phagocytosis of microbial pathogens and antigen–antibody complexes. It is not clear, however, whether endo/lysosomal cathepsin L is involved in creating antigenic epitopes and whether this protease regulates the development of functional subsets of CD4 + T cells.
We previously reported that CA074, a specific inhibitor of cathepsin B, modulates the immune responses of BALB/c mice infected with L. major ( 8 – 10 ). That is, CA074 suppressed the T h 2 response but augmented the T h 1 response, suggesting that cathepsin B functions as an antigen-processing protease and preferentially activates T h 2 response. We have established a specific inhibitor for cathepsin L, CLIK148 ( 17 ). In the present study, we found that treatment of L. major -infected BALB/c mice with CLIK148 exacerbates the infection by enhancing T h 2 response, in striking contrast to the results of treatment with CA074. Thus, cathepsin L may also function as an antigen-processing protease, but in a different way from that of cathepsin B.
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Methods |
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Animals
BALB/c Cr Slc (BALB/c) mice were purchased from Japan Shizuoka Laboratory Animal Center (Hamamatsu, Japan). Female mice aged 8–10 weeks were used in all experiments. The mice were bred in a specific pathogen-free environment at the Institute for Animal Experimentation, The University of Tokushima.
Parasites and infection
L. major
(MHOM/SU/73/5ASKH strain) was provided by Dr Furuya (Kochi Medical College, Japan) and maintained by serial passages of the promastigotes inoculated s.c. into the footpads of susceptible BALB/c mice. Mice were infected by inoculation of 5x10
6
promastigotes of
L. major
in a final volume of 50 µl into the hind footpad. Disease progression after infection was monitored weekly by measuring the swelling of footpads with a metric caliper. The lesion size was determined by comparing the thickness of the infected footpad with those of uninfected footpads. After 6 weeks, the popliteal lymph nodes (LN) were drained for analyses of proliferation, cytokine production and quantitation of viable parasites.
Preparation of SLA
SLA for immunoblotting, ELISA and proliferation assay of lymphocytes was prepared from stationary-phase promastigotes of
L. major
. Promastigotes were cultured in Schneider medium for 3 days, and then washed 3 times with cold PBS, resuspended in PBS, frozen and rapidly thawed 5 times, sonicated, and centrifuged at 10,000
g
for 20 min at 4°C. The supernatant containing SLA was harvested and the protein concentration was determined with bicinchoninic acid protein assay reagent (Pierce, Rockford, IL), as described previously with minor modifications (
8
).
Cathepsin inhibitors and assays of enzyme activities
CLIK148 and CA074 were synthesized in our laboratory, and were shown to selectively inhibit cathepsins L and B
in vitro
and
in vivo
respectively (
17
–
19
). CLIK148 (0.2 mg/mouse) or CA074 (0.25 mg/mouse) was given 2 h before and after infection, and their administrations were continued every 12 h for 6 weeks. The mitochondria/lysosome (ML) fraction was isolated according to the previously described method (
9
). We used the substrate Z-Phe-Arg-MCA (Peptide Institute, Osaka, Japan) for discrimination of cathepsins L and B, and CA074 was used as a specific inhibitor for cathepsin B. The reaction was initiated by addition of the substrate (10 µM final concentration) after preincubation with the test compound for 3 min at 37°C. The fluorescence of the liberated 7-amino-4-methylcoumarin was measured using a fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Emission at 460 nm was measured with excitation at 370 nm (
20
,
21
).
Detection of antigen-specific Ig in serum
Volumes of 100 µg/ml SLA were coated on Covalink 96-well microplates (InterMed Nunc. Kamstrup, Denmark) for 2 h using
N
-hydroxysulfosuccinimide (Pierce) and 1-ethyl-3-(3-dimethylaminoprophyl)carbodiimide (Sigma, St Louis, MO). After blocking overnight with 0.1% BSA (Gibco/BRL, Gaithersburg, MD) in PBS, serum samples serially diluted with blocking buffer were incubated for 6 h in microplates. Then those samples were examined with alkaline phosphatase-conjugated anti-mouse IgE, IgG1 (Southern Biotechnology, Birmingham, AL) or IgG2a (Cappel, Durham, NC) followed by
p
-nitrophenyl phosphate (Sigma). The reaction was stopped with 3N NaOH and the absorbance at 415 nm was read with a microplate reader (BioRad, Hercules, CA).
Generation of a short-term T cell line
Mice were immunized i.p. with10 µg of SLA adsorbed to 4 mg of alum for 2 weeks. In brief, spleen fragments were subjected to mild collagenase (Roche Diagnostics, Mannheim, Germany) digestion at 37°C for 2 h to release APC. Spleen cells (2x10
6
cells/ml) were incubated with SLA (25 µg/ml) for 1 week and then harvested, and the viable cells were collected by Ficoll gradients. Cells were resuspended in complete cell culture medium at 0.5x10
6
cells/ml and stimulated with SLA in the presence of 5x10
6
cells/ml mitomycin C (Sigma)-treated syngeneic spleen cells from normal BALB/c mice serving as APC. Subsequent rounds of stimulation followed the same procedure at 3-day intervals. The T cell line was expanded by 30 U/ml IL-2 (
22
). For subsets of the T cell line, cells stained with the panel of directly FITC-conjugated anti-CD8 (clone Ly-2) and phycoerythrin-conjugated anti-CD4 (clone L3T4) mAb (PharMingen, San Diego, CA) were analyzed by using a FACS (Becton Dickinson, Mountain View, CA).
Cytokine analyses
Lymphocytes (2x10
6
cells/well) from infected or SLA-immunized mice were cultured with SLA (25 µg/ml) or concanavalin A (Con A) (5 µg/ml) in 1 ml RPMI 1640 containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 5x10
–5
M 2-mercaptoethanol (2-ME) in 48-well flat-bottom microplates at 37°C in an atmosphere of 5% CO
2
for 72 h. The cell-free culture supernatants were used to quantitate the production of IFN- and IL-4 by ELISA. Subsequently, 2 µg/ml of the biotinylated detection antibody XMG1.2 (anti-IFN-) or BVD6-24G2 (anti-IL-4) was added and the plates were incubated overnight. The plate-bound secondary antibody was then visualized by adding streptavidin–alkaline phosphatase (Oncogene, Cambridge, MA) and
p
-nitrophenyl phosphate.
Protein-digestion assay
SLA was digested at pH 5.0 (the pH of endocytic vesicles) at 37°C for 1.5 h with endo/lysosomal enzymes, prepared from the ML fraction of splenocytes from ovalbumin-immunized mice, in the presence or absence of CLIK148 or CA074. After digestion, samples were denatured in sample buffer [0.025 M Tris–HCl, pH 6.8, 2% SDS, 10% (v/v) glycerol, 10% (v/v) 2-ME and 0.002% bromophenol blue] at 100°C for 5 min and then separated by SDS–PAGE. The gel was electroblotted onto a PVDF membrane (Millipore, Bedford, MA). The digested products were detected by immunoblotting using anti-serum from
L. major
-infected BALB/c mice in this study and peroxidase-conjugated goat anti-mouse IgG (Pierce) as the second antibody. Binding antibodies were detected with ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Little Chalfont, UK).
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Results |
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Endo/lysosomal cathepsin inhibitors modulate the processing pattern of SLA with proteases
First, we examined whether the SLA-digesting activities of endo/lysosomal proteases in the ML fraction from BALB/c mice are modulated when CLIK148 or CA074 is added to the reaction with SLA and ML fractions by a protein digestion assay as described in Methods. Immunoblot analysis using anti-serum of L. major -infected BALB/c mice in this study revealed that SLA was mainly composed of 26-, 28-, 65- and 75-kDa components of SLA ( Fig. 1a
, lane 1), and all of these components of SLA except for the 26-kDa component were almost digested by endo/lysosomal enzymes (
Fig.
1a
, lane 3). Notably, CLIK148 suppressed digestion of the 28-, 65- and 75-kDa components (
Fig.
1a
, lane 5). In contrast, CA074 mainly suppressed the digestion of the 28-kDa component (
Fig.
1a
, lane 4). These results indicate that endo/lysosomal cathepsin L digests the 28-, 65- and 75-kDa components, whereas cathepsin B mainly digests the 28-kDa component. This difference may result in a difference between the functions of CD4
+
T cells in CLIK148- and CA074-treated mice. Next, we confirmed the specificity of the cathepsin L inhibitor CLIK148. CLIK148 administered to BALB/c mice showed >50% inhibitory activity and was highly specific
in vivo
(
Fig.
1b
), suggesting that CLIK148 is capable of entering spleen cells to suppress the activity of endo/lysosomal cathepsin L.
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Treatment with CLIK148 enhances the course of infection with L. major
CLIK148- and CA074-treated mice were challenged by injection of promastigotes of L. major into their left hind footpads, and the course of infection was assessed by measuring the size of local swelling. Treatment of susceptible BALB/c mice with CLIK148 enhanced the progression of leishmaniasis, as evaluated by footpad swelling. On the other hand, treatment of BALB/c mice with CA074 suppressed progression of the disease ( Fig. 2a
). To confirm that footpad swelling was correlated with the tissue parasite burden, we microscopically counted the numbers of parasites recovered from draining LN of individual mice 6 weeks after infection. The number of viable parasites was increased by treatment with CLIK148, whereas CA074-treated mice had significantly lower numbers of viable parasites than did untreated mice (
Fig.
2b
). These findings correlated well with the results of footpad swelling. However, treatment with CLIK148 or CA074 did not influence the
in vitro
proliferation of the protozoan or its
in vivo
infectivity in SCID mice (data not shown), suggesting that cathepsin inhibitors affect the infection of
L. major
by modulating immune responses, not by directly affecting the parasite itself. These results demonstrate that treatment of BALB/c mice with CLIK148 exacerbated leishmaniasis, in striking contrast to the results of treatment with CA074.
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Treatment with CLIK148 enhances the development of T h 2 response in L. major-infected mice
We investigated whether T h differentiation was different in CLIK148- and CA074-treated mice during infection, by estimating the patterns of production of cytokines such as IL-4 and IFN-
. Popliteal LN cells drained from infected mice were re-stimulated with SLA to determine the pattern of antigen-specific cytokine secretion in culture supernatants. As shown in
Fig. 3(a)
, lymphocytes from untreated BALB/c mice showed a T
h
2 type of cytokine production pattern with a high level of IL-4 (>8-fold compared with that of
L. major
-infected resistant DBA/2 mice, data not shown) but not IFN-. Treatment of mice with CA074 caused a deviation of the immune response to the T
h
1 type from the ordinary T
h
2 type, producing a very low level of IL-4 and a high level of IFN- compared with those in untreated mice. Unlike CA074 treatment, CLIK148 treatment strongly enhanced the T
h
2 response in
L. major
-infected mice. That is, treatment of mice with CLIK148 produced a high level of IL-4 and a relatively low level of IFN- (
Fig.
3a
). These results suggest that treatment with CLIK148 promotes the T
h
2-type response, while treatment with CA074 suppresses the development of the T
h
2-type response but activates the T
h
1-type response. In addition, lymphocytes of CLIK148-treated mice as well as those of untreated mice proliferated in response to specific stimulation with SLA or non-specific stimulation with Con A (data not shown), suggesting that treatment with CLIK148 does not influence the proliferation of T cells.
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To confirm that Ig class switching to T h 2-mediated antibodies was enhanced in CLIK148-treated mice, sera were harvested to assess the production of SLA-specific IgG1, IgE and IgG2a by ELISA in the three experimental groups 6 weeks after infection with L. major . CLIK148-treated mice showed increased levels of antigen-specific IgG1 and IgE production, and a reduced level of IgG2a compared with those in untreated mice ( Fig. 3b
). On the other hand, CA074-treated mice showed low serum concentrations of SLA-specific IgG1 and IgE, whereas the level of IgG2a was increased. Based on these findings, we concluded that T
h
2-mediated antibodies were increased in CLIK148-treated mice, whereas T
h
1-mediated antibodies became dominant in CA074-treated mice, as was the pattern of cytokine production.
Treatment with CLIK148 induces the development of T
h
2 response in SLA-immunized mice
To further demonstrate that treatment with a cathepsin L inhibitor increases the production of IL-4, BALB/c mice treated with CLIK148 were immunized with SLA adsorbed to alum. The modulatory effects of the cathepsin L inhibitor on
in vivo
immune responses were confirmed by examining the production of cytokines and antigen-specific antibodies. Spleen cells from immunized mice were stimulated with SLA or Con A and then IL-4 or IFN- in the culture supernatants was assessed by ELISA. Treatment of mice with CLIK148 produced a higher level of antigen-specific IL-4 and a lower level of IFN- compared with those in untreated mice (
Fig.
4a
). Moreover, CLIK148-treated mice also showed high titers of SLA-specific IgG1 production and low titers of IgG2a compared with those in untreated mice (
Fig.
4b
), suggesting that a cathepsin L inhibitor is able to enhance the development of the T
h
2 response. In addition, the production of antigen-specific IgE was not measured since the amount of IgE was very low in SLA-immunized mice (data not shown). No significant differences were seen between untreated and CLIK148-treated mice in proliferation and cytokine production in response to non-specific stimulation with Con A (data not shown). These findings were clearly correlated with results in CLIK148-treated and
L. major
-infected mice. Thus, treatment with CLIK148 induces the development of a T
h
2-type response specific to SLA.
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The effect of treatment with cathepsin inhibitors on modulation of T h responses in vitro
We already demonstrated that treatment of L. major -infected and SLA-immunized mice with a cathepsin L inhibitor enhanced the T h 2 response. To further determine whether a modulated T h 2 response is observed even after in vitro culture with APC, SLA and CLIK148 or CA074, we generated a short-term T cell line derived from the spleens of SLA-immunized BALB/c mice. As shown in Fig. 5(a)
, 98% of the cells in the SLA-specific T cell line were CD4
+
. After culture for 3 days without a stimulator, an SLA-specific CD4
+
T cell line established from untreated mice was able to produce a high level of IL-4 and did not generate a detectable level of IFN-. However, production of IL-4 secreted by a CD4
+
T cell line established from CLIK148-treated mice was 2-fold larger than that of untreated mice (
Fig.
5b
), further indicating that SLA-specific CD4
+
T cells in CLIK148-treated mice were mainly T
h
2 type. Moreover, after culture for 3 days with SLA and mitomycin C-treated syngeneic spleen cells serving as APC, a CD4
+
T cell line established from untreated mice also showed a T
h
2-type cytokine production pattern with a high level of IL-4 and a low level of IFN- (
Fig.
5c
). On the other hand, when CLIK148 was added during incubation with T cells, SLA and APC, antigenic epitopes presented by APC potentiated the CD4
+
T cell line to secret more IL-4 and less IFN- than the control group, whereas treatment with CA074 induced the suppression of T
h
2 cytokine secretion. In addition, neither cathepsin inhibitors had any direct effects on the function of T cells themselves, e.g. cytokine production and proliferation, when the cells were stimulated with Con A or anti-CD3 mAb (
Fig.
6
). These results suggest that treatment with CLIK148 enhances the development of a T
h
2-type response via modulating antigen processing in APC, in striking contrast to the results of treatment with CA074.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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There are many reports on regulatory mechanisms of T h 1 and T h 2 responses such as the route of antigen entry, physical forms of the antigen, the type of adjuvant used and antigen doses ( 23 , 24 ). Antigen processing in the endo/lysosome is an essential step in the induction of MHC class II-restricted immune responses to various infectious microorganisms ( 25 ). We have proposed that some endo/lysosomal proteases play essential roles in the differentiation of functional CD4 + T cell subsets ( 8 – 10 ). Cathepsins in the endo/lysosomal compartments are known to be involved in the proteolytic process of endocytosed proteins generate antigenic peptides, which are presented by MHC class II molecules on APC ( 12 ). One candidate protease is cathepsin B, a cysteine protease, since its specific inhibitor, CA074, changes the digestion pattern by endo/lysosomal enzymes and modulates immune response from T h 2 type to T h 1 type in BALB/c mice infected with L. major , and then those genetically susceptible mice acquire resistance to the infection ( 8 ).
In this paper, we have suggested that cathepsin L, another cysteine protease, also functions as an antigen-processing protease, but in a different manner from that of cathepsin B. That is, treatment of mice with CLIK148, a specific inhibitor of cathepsin L, enhances the development of the disease-promoting T h 2 response, in contrast to CA074, which suppresses the T h 2 response. Thus, L. major antigens processed by cathepsin L may participate in the up-regulation of the T h 1 response and/or down-regulation of the T h 2 response, whereas those processed by endo/lysosomal cathepsin B may preferentially promote development of the T h 2 response and/or suppress the T h 1 response as previously reported.
Cathepsin L exists in most mammalian cells, including APC, and participates in protein degradation of both exogenous and endogenous proteins in the lysosome ( 15 ). We found that a proform of cathepsin L, procathepsin L, abundantly existing in the spleen cells readily matures to cathepsin L after immunization of mice with foreign antigens. Cathepsin L is first translated as preproforms, transported through the Golgi apparatus as proforms and localized as mature enzyme forms in the lysosomes via endosomes ( 26 ). Thus, immunization of mice with foreign antigens is one of the stimuli for the transportation and maturation of cathepsin L. The increase in mature cathepsin L in macrophages of mice immunized with foreign antigens is thought to be essential for enhancing the proteolysis of the endocytosed antigens and for presenting these antigens to the immune system, since macrophages play special roles in phagocytosis of microbial pathogens. Actually, we observed that the efficiency of antigen processing was increased in APC as cathepsin L matured ( 16 ). Nakagawa et al . reported that mice deficient in cathepsin L showed a profound defect in Ii degradation in thymic cortical epithelial cells and exhibited a dramatic impairment in thymic CD4 + T cell selection ( 11 ). It is therefore possible that CLIK148 administered to mice modulates immune responses through interfering with differentiation of thymocytes. Unlike cathepsin L-deficient mice, however, 40% of cathepsin L activity was still retained in lymphoid tissues of mice treated with CLIK148. Furthermore, the dose of this inhibitor used in the present study did not result in a significant decrease in cellularity of the thymus or spleen, and T lymphocytes appeared to be normal with typical proportions of CD4 + CD8 – and CD4 – CD8 + T cells in both thymus and peripheral lymphoid tissues (data not shown).
It is also possible that cathepsin L is involved in degradation of Ii molecules in peripheral lymphoid tissues. We examined the degradation of Ii in macrophages and B cells from CLIK148-treated mice using previously described methods ( 9 , 10 ). The results of this experiment suggested that treatment with CLIK148 did not influence the degradation of Ii in those cells (data not shown). Moreover, the results of our in vitro experiments demonstrated that APC treated with CLIK148 present antigenic epitopes to an SLA-specific CD4 + T cell line, resulting in the potentiation of production of T h 2 cytokines with a significantly increasing amount of IL-4 compared with the control group. It is noteworthy that in vitro treatment of APC with the cathepsin B inhibitor CA074, rather, suppresses the production of T h 2-type cytokines. Taken together, the results suggest that treatment of APC with cathepsin inhibitors influences immune responses to L. major via modulation of antigen processing. Cathepsin L thus appears to be involved in down-regulation of T h 2-type cytokines by processing antigens in the endo/lysosome of APC, whereas cathepsin B seems to play an essential role in the production of T h 2-type cytokines.
CD4 + T h cells in mice infected with L. major can be divided into two distinct subsets, i.e. protective T h 1 and disease-promoting T h 2 cells, and those cells regulate each other ( 27 , 28 ). Strangely, DBA/2 mice treated with CLIK148 did not become susceptible to L. major infection (data not shown). This phenomenon may be explained by the fact that 60% of cathepsin L activity was inhibited in BALB/c mice 6 h after treatment with CLIK148, whereas only 20% of cathepsin L activity was suppressed in the spleens of CLIK148-treated DBA/2 mice (data not shown). The dose of the inhibitor used in this study was not sufficient to suppress the activity of cathepsin L in DBA/2 mice. This may be the reason why treatment with CLIK148 did not change the resistance of DBA/2 mice to L. major infection.
Endo/lysosomal proteases other than cysteine proteases may also contribute to antigen processing and/or activation of CD4 + T cells as we reported previously ( 9 , 10 ). Another candidate protease is cathepsin D, a pepstatin A-sensitive aspartic protease, which plays important roles in the endosome to activate CD4 + T cells via either Ii degradation or via antigen processing. That is, we found that treatment of L. major -infected mice with pepstatin A, a specific inhibitor of aspartic proteases, interferes with the induction of T h 0, a prototype of T h 1 and T h 2, and then suppresses the appearance of both mature types of CD4 + T cell subsets.
The results of the experiments presented here show that treatment with CLIK148 potentiates the T h 2 response, in striking contrast to treatment with CA074, which enhances the T h 1 response in L. major- infected mice. In other words, antigen processing with cathepsin L may be one of the essential steps to evoke a T h 1 response, whereas that with cathepsin B appears to preferentially induce a T h 2 response. Taken together, the results suggest that various endo/lysosomal proteases in APC contribute to the development of MHC class II-restricted CD4 + T cells but in different manners. Further investigation is needed to determine the exact mechanism of this kind of immunoregulation in order to elucidate the process of T h 1/T h 2 differentiation.
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Acknowledgments |
|---|
This work was supported in part by grants-in-aid from the Japanese Ministry of Education, Science and Culture.
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Abbreviations |
|---|
| APC antigen-presenting cell |
| Con A concanavalin A |
| Ii invariant chain |
| LN lymph node |
| ML mitochondria and lysosome |
| SLA soluble Leishmania antigen |
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Notes |
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Transmitting editor: T. Watanabe
Received 28 February 2001, accepted 16 April 2001.
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