Switch of CD4⁺ T Cell Differentiation from Th2 to Th1 by Treatment with Cathepsin B Inhibitor in Experimental Leishmaniasis¹

Tcell activation is initiated by recognition of antigenic epitopes with Ag-specific receptors in the context of major histocompatibility Ags on the surface of APCs. Thus, before CD4⁺ T cell activation, Ags must be processed with lysosomal proteases after endocytosis, then presented to CD4⁺ T cells in association with class II molecules of the MHC (1, 2). However, the proteases responsible for this Ag processing have not well been defined. Cathepsin B is a lysosomal cysteine protease involved in Ag processing (3, 4, 5). We developed a specific inhibitor (CA074) of cathepsin B, a lysosomal cysteine protease, that selectively inhibits the activity of cathepsin B in vivo as well as in vitro (6, 7). Matsunaga et al. first reported that CA074 suppresses immune responses, especially the production of Ag-specific IgG1 or IgE (3, 8). These observations suggest that cysteine proteases in lysosomes play an important role in the functional differentiation of MHC class II-restricted CD4⁺ T cells that promote the production of IgG1 and IgE Abs.

Mice resistant to infection with Leishmania major, such as the DBA/2, C3H, CBA, or C57BL/6 strains, preferentially develop CD4⁺ Th1 cells specific for L. major Ags (9, 10). Th1 cells generate IL-2 and IFN-γ and support the production of Ag-specific IgG2a-type Ab (11, 12). On the other hand, susceptible BALB/c mice exclusively develop Th2 CD4⁺ T cells, which generate IL-4 and IL-5 and support production of specific Abs of the IgE and IgG1 classes. Furthermore, it is well established that Th1 and Th2 cells regulate each other in mice infected with L. major (13, 14). However, the mechanisms involved in the preferential development of Ag-specific Th1 or Th2 type CD4⁺ T cells remain to be defined.

In the present study, we have examined the effect of treatment with CA074 on murine leishmaniasis in susceptible BALB/c mice, in an effort to elucidate the participation of lysosomal cysteine proteases in the functional differentiation of Th1 and Th2 cells. We show here that modulation in Ag processing by a cathepsin B inhibitor suppresses the functional differentiation of Th2 type CD4⁺ T cells and in turn augments Th1-type immune responses. These findings indicate that the cathepsin B inhibitor modulates the polarity of Th differentiation by the alteration in Ag processing.

Female BALB/c CrSlc (BALB/c) and DBA/2 CrSlc (DBA/2) mice were purchased from Japan Shizuoka Laboratory Animal Center (Hamamatsu, Japan), and CB-17 scid/scid (SCID) mice were obtained from the Central Institute for Experimental Animals (Kawasaki, Japan). The animals were 8 to 10 wk of age at the beginning of the experiment.

Cultures of L. major (MHOM/SU/73/5ASKH) were provided by Dr. Furuya, Kochi Medical College, Japan. The parasites were propagated in Schneider medium containing 20% FBS (Life Technologies, Grand Island, NY). Promastigotes were harvested from stationary phase cultures by centrifugation and washed at least three times in PBS. Parasites were passed at intervals in BALB/c female mice to ensure that virulence was maintained.

Ags were prepared using stationary phase promastigotes of L. major. Soluble leishmanial Ag (SLA)³ was prepared as described (15) and the protein concentration determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL). Cathepsin B inhibitor, CA074, was synthesized in our laboratory and the specificities confirmed as described (6, 7). CA074 dissolved in PBS was administered at doses of 0.5 mg/mouse/day. The inhibitor was given 2 h before inoculation of the parasites, and administration of the inhibitor was continued every 12 h for the indicated periods. For the measurement of mouse IL-5, mAb to mouse IL-5 (Genzyme, Cambridge, MA) and biotin-conjugated mAb (TRFK4) against IL-5 (PharMingen, San Diego, CA) were used as detecting and coating Abs, respectively. Anti-mouse CD3 Ab (2C11) was a gift from Dr. Nomoto (Kyushu University, Fukuoka, Japan). Recombinant murine IL-2 was a gift from Shionogi (Osaka, Japan), and recombinant murine IL-4 and IL-12 were purchased from Intergen (Purchase, NY) and Genzyme, respectively.

Liver, spleen, lymph nodes (LN), and peritoneal macrophages were used to assay for cathepsin B activity. To obtain macrophages, BALB/c mice were injected i.p. with 2 ml of 4.05% (w/v) thioglycollate medium (Wako, Osaka, Japan). Four days after injection, peritoneal cells were collected and plated on plastic plate. After adherence for 1 h, monolayer cells were used as the peritoneal macrophages. Lysosomes were isolated for the assay by gently homogenizing samples using a Teflon pestle in 0.25 M cold sucrose. The suspension was centrifuged at 3,500 × g for 10 min at 4°C. The supernatant was centrifuged at 25,000 × g for 20 min at 4°C. The resulting pellet was resuspended with 50 mM acetate buffer (pH 5.0). The suspension fluid was frozen and thawed three times to disrupt lysosomal membranes. After three cycles of freezing and thawing, the fluid was centrifuged and the supernatant was used as a mitochondria and lysosome fraction (ML fraction). Cathepsin B activity in ML fraction was assayed with Z-Arg-Arg-methyl coumarylamide (Peptide Institute, Osaka, Japan) as substrate at pH 5.0 by the method of Barrett and Kirschke (16). The reaction was initiated by addition of 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 in a fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Emission at 460 nm was measured with excitation at 370 nm.

Mice were inoculated with L. major s.c. in the footpads. For primary infection, 5 × 10⁶ promastigotes were injected into the left hind footpad, and for rechallenge, the same number of promastigotes were injected into the right hind footpad 60 days after the first infection. The progression of infection was assessed by monitoring lesion development by measuring the footpad thickness with a vernier caliper.

Promastigotes of L. major were cultured in Schneider medium containing 20% FBS with or without CA074 for 72 h at 25°C. The proliferation of L. major was evaluated by counting the parasites every 24 h.

SLA was covalently coated onto 96-well CovaLink NH plates (Nunc, Roskilde, Denmark). After blocking with 0.1% BSA in PBS (blocking buffer), the SLA was incubated with serum samples serially diluted in the blocking buffer for 6 h at 4°C. The results were developed using alkaline phosphatase-conjugated murine isotype-specific mAb, anti-mouse IgG (Sigma, St. Louis, MO), anti-mouse IgG1 or IgG2a (Organon Teknika, West Chester, PA), or anti-mouse IgE (Southern Biotechnology, Birmingham, AL) followed by p-nitrophenyl phosphate (Sigma). After terminating the reaction with 3N, NaOH, OD at 415 nm was determined on a microplate reader (Corona Electric, Katsuta, Japan).

SLA was separated by SDS-PAGE, then the gel was electroblotted onto a polyvinylidine difluoride membrane (Millipore, Bedford, MA). Sera from L. major-infected BALB/c mice or L. major-infected BALB/c mice treated with CA074 were used as the first Abs. Peroxidase-conjugated goat anti-mouse IgG (Zymed, San Francisco, CA) was the second Ab. Binding Abs were detected by means of the enhanced chemiluminescence method (Amersham, Buckinghamshire, U.K.), based on a combination of enhanced chemiluminescence detection. Fluorescence emitted from luminol in the presence of peroxidase could be detected by autoradiography film (Fuji Film, Tokyo, Japan).

Cells from popliteal LNs draining the footpad lesions were cultured with SLA (20 μg/ml) for 72 h. IFN-γ and IL-4 production in the supernatants was measured using ELISA EM-IFNG and EM-IL4 (Endogen, Cambridge, MA) kits.

Splenocytes (2 × 10⁵/well) from BALB/c mice were stimulated with anti-CD3 Ab (2C11) or Con A (Sigma) for 72 h. For the proliferation assay, [³H]TdR (1 μCi/well) was added to each well 6 h before harvesting. The cytokines in the supernatants were measured by ELISA.

T cells of splenocytes from naive female BALB/c mice were purified using a nylon fiber column (Wako). This procedure was performed twice, and the purity of T cells was >95% as judged by flow cytometry analysis.

SLA was digested at pH5.0, 37°C, for 2 h with lysosomal enzymes in an ML fraction of splenocytes from naive or CA074-treated BALB/c mice. After digestion, reaction samples were separated by SDS-PAGE. The digested products of SLA were detected by immunoblotting method using Abs in serum from L. major-infected BALB/c mice.

In this study, we first investigated the inhibitory effect of CA074 on cathepsin B activity in vivo. After administration of CA074, the kinetics of cathepsin B activity was examined. As shown in Figure 1,A, the activity in liver cells was reduced to ∼20 to 25% at 2 h after administration of 0.25 mg of CA074 per mouse, with 50% inhibition at 12 h. Next, we used the thioglycollate-induced peritoneal adherent cells to evaluate in vivo inhibition of cathepsin B activity. Two hours before collection, 0.25 mg of CA074 was injected into the peritoneal cavity. In macrophages, ∼75 to 80% inhibition of cathepsin B activity was observed (Fig. 1 B). These findings suggested that the dose of cathepsin B inhibitor, CA074, used in this study was sufficient to inactivate the cathepsin B activity.

We examined the effect of CA074 on murine leishmaniasis. In comparison with PBS-treated BALB/c mice that showed progressive footpad swelling, BALB/c mice treated with CA074 (0.5 mg/day (0.25 mg/12 h)) became resistant to L. major infection (Fig. 2,A). Next, we examined whether this inhibitor modulated the resistance of DBA/2 mice. DBA/2 strain is compatible with H-2 in BALB/c mice but resistant to L. major. As shown in Figure 2 B, CA074 did not influence the disease progression in resistant DBA/2 mice, suggesting that cathepsin B inhibitor did not suppress the genetically strong Th1 response in this strain. E64d, a more broad inhibitor for cysteine proteases, induced a similar inhibition of L. major induced footpad swelling like CA074 (data not shown).

To exclude the possibility that CA074 exerts a direct cytotoxic effect on L. major, we examined the influence of CA074 on the in vitro proliferation of L. major promastigotes. As shown in Figure 3 A, L. major proliferated comparably regardless of presence or absence of CA074.

We then investigated whether or not this inhibitor influenced the in vivo growth of L. major in SCID mice, which are deficient in functional T and B cells. As shown in Figure 3 B, no difference in lesion footpad thickness was observed between PBS- and CA074-treated SCID mice. Moreover, the speed of disease progression and pattern of lesions were similar between protease inhibitor-treated or untreated nude mice (data not shown). Thus, cathepsin B-specific inhibitor did not directly influence the infectivity of L. major, suggesting that the resistance in CA074-treated BALB/c mice was acquired through an immunologic mechanism lacking in the immunodeficient SCID or nude mice.

We then investigated the mechanisms of resistance in BALB/c mice treated with cathepsin B-specific inhibitor. Infected BALB/c mice treated with CA074 or PBS were later reinfected into the contralateral footpad 60 days after the primary infection. Swelling in the reinfected footpad prominently progressed in naive and PBS-treated BALB/c mice, whereas CA074-treated BALB/c mice were completely resistant whether or not additional CA074 had been given (Fig. 4). Thus, infected BALB/c mice treated with CA074 acquired protective immunity.

We examined the proportion of each subclass of L. major-specific Ab in the serum to evaluate the immunologic status of infected mice (Fig. 5 A). No difference in the amount of L. major-specific IgG was observed between PBS- and CA074-treated BALB/c mice. The amount of IgG2a was slightly higher in CA074-treated mice than in PBS-treated mice. On the other hand, the production of IgG1 and IgE Abs promoted by Th2 cells was markedly suppressed in the CA074-treated mice as compared with that in PBS-treated mice, indicating possible inhibition of Th2 function by the cathepsin B-specific inhibitor.

We then examined whether the Ags recognized by specific Abs in serum were different each other (Fig. 5 B). In contrast to sera from PBS-treated BALB/c mice, those from CA074-treated mice strongly recognized 20- and 50-kDa Ags in SLA but did not bind 30-kDa Ags, suggesting that the Abs specific for 20- and 50-kDa Ags were increased, but those specific for 30-kDa Ags were decreased in serum from CA074-treated mice. That is, the production of Abs specific for L. major Ags may be changed by treatment with CA074 in isotype, and Abs of mice treated with CA074 may recognize different molecules in L. major Ags from those of PBS-treated mice.

We examined the functional phenotype of CD4⁺ T cells in BALB/c mice that acquired resistance against L. major infection after treatment with CA074. Cells from LNs draining the footpad lesions of infected mice were stimulated with SLA, then IL-4 or IFN-γ, indicating Th2 or Th1 responses, respectively, in the culture supernatants were assessed by ELISA (Fig. 6). High levels of IL-4, but not IFN-γ, were produced in PBS-treated mice. In contrast, treatment with CA074 increased IFN-γ production and decreased production of IL-4. Thus, the pattern of cytokines produced in PBS-treated mice was of the Th2 type and of Th1 type in CA074-treated mice. These results suggest that Th2 function had been suppressed in BALB/c mice by treatment with CA074 so that Th1 function could be sufficiently activated to resolve infection with L. major. Thus, inactivation of cathepsin B appears to inhibit the development of Th2-type immune responses that ordinarily develop in BALB/c mice infected with L. major and, as a result, favors the development of Th1 cells.

We then examined the cathepsin B activity in peripheral lymphoid organs. In popliteal LNs draining the footpad lesions of infected mice, cathepsin B activity remained ∼70 to 80% 2 h after i.p. treatment with CA074 (Fig. 7). Although cathepsin B activity was inhibited ∼70 to 80% in liver and APCs (Fig. 1), the activity in peripheral LNs was inhibited only ∼20 to 30% via i.p. administration. However, local treatment with CA074 could inhibit the activity in draining LNs (data not shown), suggesting that lymphoid organs are not resistant to this compound. At present, we do not know why peritoneal treatment did not inhibit the activity in LNs as well as in liver. These results suggested that only an ∼20 to 30% reduction of cathepsin B activity is sufficient to induce the inhibition of the development of Th2-type immune responses and the promotion of the development of Th1 cells.

Naive splenocytes from BALB/c mice were stimulated with Con A or plate-fixed anti-CD3 Ab. To evaluate the influence of CA074, we added this inhibitor to the culture. As shown in Figure 8, CA074 did not affect either the proliferative response or the production of IFN-γ or IL-4 (Fig. 8, B and C). Next, we examined the effect of CA074 on Th differentiation initiated with cytokines such as IL-4 or IL-12. The combination of some cytokines induces the Th1 or Th2 from naive T cells (17, 18, 19). When naive T cells were stimulated with IL-2 plus IL-4, they differentiated into IL-4-producing cells; however, the addition of CA074 in this culture did not affect the production of IL-4, IL-5, or IFN-γ (Fig. 9,A). It is well known that combined treatment with IL-2 and IL-12 causes naive T cells to differentiate into IFN-γ-producing cells. The presence of CA074 in the culture did not change the production pattern of cytokines (Fig. 9 B). Thus, the cathepsin B inhibitor did not directly influence the T cell responses, and the switch from Th2 to Th1 is not due to the direct effect on T cell responses.

We supposed that the shift of Th differentiation from Th2 to Th1 in CA074-treated mice was induced by the modulation in Ag processing owing to the inhibition of lysosomal cathepsin B activity. Therefore, we first examined in vitro the change in the digestion of SLA with splenic lysosomal fraction by adding CA074 to the reaction mixture. As shown in Figure 10,A, the amount of 28-kDa Ag in SLA (indicated with a closed triangle) was decreased according to the digestion with lysosomal enzymes, whereas 16-kDa protein (indicated with a open triangle) was increased compared with the reaction mixture without lysosomal fraction. However, when CA074 was added to the reaction mixture, 28-kDa Ag was not decreased, but rather, 16-kDa-digested product was decreased. Next, we compared the digestion pattern of SLA by lysosomal enzymes prepared from splenic macrophages of uninfected BALB/c mice treated with or without CA074 (Fig. 10 B). Similar to the in vitro effect of CA074, the 16-kDa-digested product almost disappeared in the reaction mixture with the lysosomal fraction from CA074-treated mice. Thus, CA074 appears to influence Ag processing in vivo as well as in vitro.

When susceptible BALB/c mice infected with L. major were treated with a specific inhibitor for cathepsin B (CA074), they completely controlled the infection. This acquired resistance was not attributed to a direct cytotoxic effect of this inhibitor on the L. major, but to the switch of CD4⁺ Th cell-mediated immune responses from disease-promoting Th2 to protective Th1 type, as evaluated by the pattern of cytokine production and the subclass of specific Ab. Thus, CA074 appears to block the development of the Th2-type immune response that ordinarily develops in BALB/c mice infected with L. major, and, in turn, to promote the development of Th1 cells.

Many factors, especially cytokines, regulate the functional differentiation of CD4⁺ T cells. Alteration in the cytokine environment changes the Th1/Th2 balance as has been shown by other studies with murine leishmaniasis (9, 10). For example, IFN-γ and IL-4 generated by Th1 and Th2 cells, respectively, reciprocally counterregulate the differentiation of Th1 and Th2 cells from their precursor cells (13, 14). That is, IFN-γ enhances the development of Th1 but inhibits development of Th2 cells (20, 21), and the in vivo administration of monoclonal anti-IFN-γ Abs attenuates Th1 responses (22). On the other hand, IL-4 promotes the opposite effect to IFN-γ in Leishmania-infected mice (23). Thus, CA074 may have a potential to induce the production of IFN-γ or IL-4 by T cells through a direct effect to them. This possibility, however, was negated (Fig. 8). Moreover, CA074 did not exert the direct influence on Th differentiation that was induced with cytokines such as IL-4 or IL-12 (Fig. 9).

IL-12 (24, 25, 26) and IFN-γ-inducing factor (IGIF or IL-18) (27) secreted by macrophages play key roles in the induction of Th1 responses, and NK1.1⁺ T cells promote the development of Th2 cells by secreting IL-4 (28, 29). However, CA074 exerts neither an in vivo inhibitory effect on IL-4 production by NK1.1⁺ T cells nor an in vitro promoting effect on the production of IL-12 and IGIF by macrophages (data not shown). CD80 and CD86 expressed on activated APCs reportedly transfer signals that promote the differentiation of CD4⁺ T cells into Th1 and Th2 T cells, respectively (30, 31, 32). Accordingly, we investigated whether CA074 directly influences the expression of these molecules, employing in vitro experiments that used LPS-activated B cells. However, this possibility was also excluded (data not shown).

Before CD4⁺ T cell activation, Ags must be processed with lysosomal proteases after endocytosis. Although lysosomal proteases responsible for Ag processing have not yet been well defined, cathepsin B (3, 4, 5), D (4, 33), and E (34) have been shown to play major roles. Thus far, there is no report suggesting that some proteases preferentially create motifs that select either Th1- or Th2-type Ag-specific CD4⁺ T cells. As shown here, the Th2 response was suppressed in BALB/c mice infected with L. major and treated with CA074, while the Th1 response, essential to protective immunity against this infection, became dominant. A similar effect with CA074 was observed when so-called IgE-inducible Ags such as OVA (8) or Nippostrongylus brasiliensis (data not shown) were used as Ags. Thus, inactivation of cathepsin B with a specific inhibitor is expected to influence immune responses, especially the functional differentiation of CD4⁺ T cells. At present, the detailed molecular mechanisms of how the inhibitor of cathepsin B induces the switching of polarity from Th2 type to Th1 type have not been clarified, although this inhibitor was confirmed to modulate Ag processing with lysosomal enzymes in vivo as well as in vitro.

It is important to elucidate in molecular detail the switching mechanisms responsible for T cell development from Th2- to Th1-type immune responses in mice treated with cathepsin B inhibitors. This approach may allow an understanding of the mechanism(s) underlying CD4⁺ T cell differentiation and the immune deviation toward IgE production that is often seen clinically.

We thank Drs. Nirbhay Kumar and Barbara S. Polla for helpful suggestions and a critical reading. We also thank Ms. Tazim Verjee for manuscript preparation and editing.