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The Cathepsin B Inhibitor, z-FA-FMK, Inhibits Human T Cell Proliferation In Vitro and Modulates Host Response to Pneumococcal Infection In Vivo¹

Clare P. Lawrence^*, Aras Kadioglu, Ai-Li Yang^*, William R. Coward^* and Sek C. Chow^2,*

^* Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom; and Department of Infection, Immunity, and Inflammation, University of Leicester, Leicester, United Kingdom

	Abstract

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The cathepsin B inhibitor, benzyloxycarbonyl-phenyl-alanyl-fluoromethylketone (z-FA-FMK) at nontoxic doses was found to be immunosuppressive and repressed human T cell proliferation induced by mitogens and IL-2 in vitro. We showed that z-FA-FMK suppresses the secretion of IL-2 and IFN- as well as the expression of IL-2R -chain (CD25) in activated T cells, whereas the expression of the early activated T cell marker, CD69, was unaffected. Furthermore, z-FA-FMK blocks NF-B activation, inhibits T cell blast formation, and prevents cells from entering and leaving the cell cycle. z-FA-FMK inhibits the processing of caspase-8 and caspase-3 to their respective subunits in resting T cells stimulated through the Ag receptor, but has no effect on the activation of these caspases during Fas-induced apoptosis in proliferating T cells. When administered in vivo, z-FA-FMK significantly increased pneumococcal growth in both lungs and blood, compared with controls, in a mouse model of intranasal pneumococcal infection. Because host response to bronchopneumonia in mice is T cell dependent, our collective results demonstrated that z-FA-FMK is immunosuppressive in vitro and in vivo.

	Introduction

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Peptidyl fluoromethylketones with amino acids phenylalanine and alanine in the P1 and P2 positions, respectively, have been shown to be irreversible inhibitors of some members of the cathepsin enzyme family (1, 2). In particular, benzyloxycarbonyl-Phe-Ala-fluoromethylketone (z-FA-FMK)³ was found to be a potent inactivator of the human cathepsin B and binds tightly to the active site of the enzyme. Once bound to the active site, the fluoromethylketone group alkylates the cysteine residue in the active site and forms a covalent bond, which irreversibly blocks its proteolytic activity.

Cathepsin B is a cysteine protease found mainly in the lysosomes (3). However, in rheumatoid arthritis (RA), cathepsin B is secreted into the synovial fluid and adjacent inflamed tissues by leukocytes, synoviocytes, and chondrocytes (4, 5). This enzyme activity was found to be increased in the synovial fluid and synovial lining in RA patients. In addition, cathepsin B level increases progressively during the course of experimental arthritis in animal joint tissues (6, 7, 8). These studies suggest that cathepsin B may be a good target for therapeutic intervention for the treatment of RA using z-FA-FMK. Indeed, a number of early in vivo studies have shown that z-FA-FMK is very efficient in preventing the destruction of articular cartilage and bone in chronic inflammatory arthritis induced by adjuvant in mice (2, 8, 9).

Recently, z-FA-FMK was found to inhibit LPS-induced cytokine production in macrophages by blocking the transactivation potential of NF-B (10). This suggests that the therapeutic action of z-FA-FMK in the treatment of RA may not be due to the inhibition of secreted cathepsin B alone, but may involve the inhibition of NF-B dependent gene expression. In addition to the inhibition of cathepsin B, z-FA-FMK also inhibits the caspases during apoptosis and downstream apoptotic events such as phosphatidylserine externalization and DNA fragmentation in T cells treated with synthetic retinoid-related molecules (11). Although caspases play a pivotal role in apoptosis, recent studies clearly showed that these cysteine proteases, particularly caspase-8, play an important role in T cell activation and proliferation (12, 13). Inhibition of caspases using the peptidyl fluoromethylketone caspase inhibitor z-VAD-FMK has been shown to block T cell proliferation (12, 14, 15). Furthermore, T cells in mice that have germline deficiency in caspase-8 proliferate poorly upon stimulation and defective caspase-8 in human T cells display lymphocyte activation defects along with the expected defects in lymphocyte apoptosis and homeostasis (13, 16). The nonspecific inhibition of caspases and the blocking of NF-B transactivation by z-FA-FMK suggest that this peptidyl fluoromethylketone may have potential immunosuppressive properties, which could have contributed to the remarkable efficacy of z-FA-FMK on RA in vivo. Moreover, immunosuppressive agents, such as steroids and cyclosporine, have been proven to be effective in the treatment of inflammatory diseases such as RA (17, 18, 19).

In the present study, we examined the potential immunosuppressive properties of z-FA-FMK on primary human T cells. Our results demonstrated that z-FA-FMK is immunosuppressive and blocks T cell proliferation induced by mitogens as well as IL-2 in vitro. The peptidyl fluoromethylketone also blocked NF-B activation and downstream cytokine production in T cells as well as blocking cell cycle progression. We showed that z-FA-FMK blocks caspase-8 and caspase-3 processing to their respective subunits during T cell activation but had no effect on caspase-mediated cell death. When administered in vivo, z-FA-FMK significantly exacerbated both pulmonary and systemic pneumococcal infection in mice. The host immune response to pneumococcal infection has recently been shown to be T cell dependent (20). The data presented in this paper are in keeping with a protective role for T cells during pneumococcal infection. Collectively, our results demonstrate that z-FA-FMK is immunosuppressive in vitro and in vivo.

	Materials and Methods

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Reagents

Benzyloxycarbonyl-valyl-alanyl-aspartic-acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) and benzyloxycarbonyl-phenyl-alanyl-acid-fluoromethylketone (z-FA-FMK) were purchased from ICN. mAb against CD3 (clone OKT3) was purified from hybridoma (American Type Culture Collection) culture supernatants and anti-CD28 mAb was purchased from R&D Systems. Rabbit anti-caspase-3 was a gift from Xiao-Ming Sun (Medical Research Council Toxicology Unit, University of Leicester, Leicester, U.K.). FITC-conjugated anti-CD25 and RPE-conjugated anti-CD69 were acquired from Transduction Laboratories and DakoCytomation, respectively. Recombinant Fas ligand (FasL), anti-Flag, and anti-poly(ADP-ribose) polymerase (PARP) were obtained from Alexis Biochemicals. Goat-anti caspase-8 and rabbit anti-NF-B p65 (C-20) were from Santa Cruz Biotechnology. Agonistic anti-Fas (IgM, Clone CH-11) was obtained from Medical and Biological Laboratories. [³H]Thymidine was obtained from Amersham Biosciences, and PHA, PMA, and ionomycin were purchased from Sigma-Aldrich. MACS columns and MACS beads conjugated with anti-CD4 and anti-CD8 were obtained from Miltenyi Biotec. Lymphoprep was from Axis-Shield PoCAS, and RPMI 1640 and FCS were from Invitrogen Life Technologies. Hoechst 33358 and CFSE were from Molecular Probes.

Cell isolation

Peripheral venous blood was obtained from normal healthy volunteers and collected into heparinized Vacutainers (BD Biosciences). PBMC were purified using density gradient centrifugation with lymphoprep. In brief, heparinized blood was layered onto lymphoprep (density gradient of 1.077), and centrifuged at 800 x g for 30 min. The mononuclear cells at the interface were collected, washed, and resuspended in RPMI 1640 medium containing 10% (v/v) FCS, 10 mM L-glutamine (Invitrogen Life Technologies), penicillin (100 U/ml), and streptomycin (100 µg/ml). The viability of the lymphocyte population routinely obtained was >95% as assessed by trypan blue exclusion. Purified CD4⁺ (97%) and CD8⁺ T (98%) cells were isolated from PBMCs using anti-CD4 and anti-CD8 mAb-conjugated MACS beads.

Cell cultures and treatments

PBMCs (5 x 10⁶ cells per ml) or purified CD4⁺ and CD8⁺ T cells (1 x 10⁶ cells per ml) in RPMI 1640 supplemented with 10% FCS were stimulated with either 5 µg/ml PHA, costimulated with plate-bound 5 µg/ml anti-CD3 (OKT3 mAb) and 2.5 µg/ml anti-CD28 or with 10 ng/ml PMA and 200 ng/ml ionomycin in the absence or presence of z-FA-FMK for various time periods in an atmosphere of 5% CO₂ in air at 37°C. To generate proliferating T cells, purified CD4⁺ and CD8⁺ T cells were coactivated with anti-CD3 and anti-CD28 for 24 h and then reseeded in medium containing rIL-2 (25 U/ml). The activated T cells were cultured for 7 days before use. The human leukemic T cell line, Jurkat, clone E6–1 (ATCC) were maintained in log-phase of growth in RPMI 1640 (Life Technologies) supplemented with 10% FCS and 2 mM L-glutamine in an atmosphere of 5% CO₂ in air at 37°C. To induce apoptosis in proliferating T cells, a total of 1 x 10⁶ cells per ml in complete medium was stimulated with recombinant Flag-tagged FasL (100 ng/ml), followed by cross-linking with anti-Flag (1 µg/ml) for 16 h. Apoptosis in Jurkat T cells was induced by incubating the cells (1 x 10⁶ cells per ml) with 100 ng/ml anti-Fas (IgM, clone CH-11) for 6 h. Apoptotic cells were determined using UV microscopy as described previously (21). Cell viability was determined by suspending treated cells in 500 µl of ice-cold PBS with 10 µl of 20 µg/ml propidium iodide (PI), and the uptake of PI was analysis using flow cytometry.

Cell proliferation and division assays

T cell proliferation following mitogen stimulation was determined using [³H]thymidine incorporation. In brief, PBMCs or purified T cells were seeded at 1 x 10⁶ cells per ml in a 96-well plate and stimulated with either PHA (5 µg/ml), costimulated with anti-CD3 mAb (5 µg/ml) and anti-CD28 mAb (2.5 µg/ml) or PMA plus ionomycin in the presence or absence of z-FA-FMK. The cells were cultured for 72 h with the last 16 h pulsed with [methyl-³H]thymidine (0.037 MBq). The cells were harvested onto glass fiber filter mats using a Tomtec automated multiwell harvester (PerkinElmer). Wallac Betaplate scintillation reagent (PerkinElmer) was added to the glass fiber filter mats, and the radioactivity was determined on a 1450 Microbeta liquid scintillation counter (PerkinElmer). T lymphocyte division following mitogen stimulation was determined using CFSE labeling of the cells (22). In brief, PBMCs were suspended in PBS at a density of 5 x 10⁷ per ml and incubated with 5 µM CFSE at 37°C for 10 min. Following incubation with CFSE, the labeled PBMCs were washed twice in RPMI 1640 medium to remove excess CFSE. The CFSE-labeled cells were treated with mitogens as described previously in the presence or absence of z-FA-FMK. As the T lymphocytes divide, CFSE is sequentially diluted, resulting in a decreased in fluorescence intensity in the cells, which can be followed by flow cytometry.

Determination of cell surface CD25 and CD69 expression using flow cytometry

Following treatments, PBMCs (5 x 10⁵ cells) were centrifuged down and the supernatants discarded. The cell pellets were resuspended in 50 µl of staining buffer (2% BSA in PBS). FITC-conjugated anti-CD25 (10 µl), RPE-conjugated anti-CD69 (10 µl), or the appropriate fluorochrome-conjugated mouse IgG (isotype control) were added to the cells and incubated on ice for 30 min in the dark. The cells were then washed twice in staining buffer before analyzed by flow cytometry.

Measurement of secreted IL-2 and IFN- in culture supernatants

Following treatments, the cells were removed by centrifugation and the supernatants collected and kept frozen. The secreted IL-2 and IFN- in the supernatants were detected using the DuoSet ELISA kits from R&D Systems, according to the manufacturer’s instructions.

Nuclear translocation of NF-B RelA, p65

This is essentially as described previously (23). Purified T cells (3 x 10⁶ cells) were costimulated with anti-CD3 and anti-CD28 for 2 h, washed with cold PBS, and fixed with 1 ml of paraformaldehyde (4%) for 20 min at room temperature. The cells were permeabilized with PBS containing 3% BSA and 0.2% Triton X-100 for 2 min in room temperature. The permeabilized cells were washed twice and resuspended in 100 µl of PBS with 3% BSA and rabbit anti-p65 Ab (1/50 dilution) for 45 min at room temperature. The cells were then washed and incubated with anti-rabbit Ab conjugated with AlexaFluor (1/2000 dilution) in a final volume of 200 µl for 30 min in the dark. Following this, the cells were washed twice and resuspended in 10 µl PBS: glycerol (50/50, v/v). The cells were mounted onto slides and viewed using confocal microscopy. Images were randomly acquired from each sample, and cells with NF-B p65 nuclear localization were counted. A minimum of 500 cells was analyzed for each sample.

Western blotting

Following treatments, a total of 2 x 10⁶ cells was washed in PBS and resuspended in 30 µl of lysis buffer (0.1 M NaCl, 1 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1% Triton-X-100, and 1 mM PMSF). The cells in lysis buffer were taken through 3x freeze/thaw cycles on dry ice. Protein concentration was measured using the Bradford assay (Bio-Rad). Protein (20 µg) from whole-cell lysates was diluted in loading buffer (2% SDS, 10% glycerol, 50 mM Tris-HCl (pH 6.8), 0.2% bromphenol blue, and 100 mM DTT) and resolved using 13% SDS-PAGE. The separated proteins were transfer onto Hybond C membrane (Amersham Biosciences) and probed with Abs to caspase-8, –3, PARP, and -actin. Detection was conducted using chemiluminescence (Amersham).

Bacteria

Streptococcus pneumoniae serotype 2, strain D39 was obtained from National Collection of Type Cultures (London, U.K.). Bacteria were identified as pneumococci before experiments by Gram stain, catalase test, -hemolysis on blood agar plates and by optochin sensitivity. To standardize virulence of pneumococci, bacteria were passage through mice as described previously (24) and subsequently recovered and stored at –70°C. When required, suspensions were thawed at room temperature and bacteria harvested by centrifugation before resuspension in sterile PBS.

Infection of mice with S. pneumoniae

Eight- to 10-wk-old female MF1 outbred mice weighing 30–35 g (Harlan Breeders) were anesthetized slightly with 2.5% (v/v) fluothane (AstraZeneca) over oxygen (1.5–2 L/min). As described previously (20), 50 µl of PBS containing 1 x 10⁶ CFU of S. pneumoniae was administered into the nostrils of each mouse. The inoculum was confirmed by plating on blood agar plates following infection. At preselected times following infection, groups of mice were deeply anesthetized with 5% fluothane (v/v), and blood was collected by cardiac puncture. Subsequently, the mice were sacrificed by cervical dislocation, and the lungs were removed into 10 ml of PBS and weighed before homogenized in a Stomacher-Lab blender (Seward Medical). Viable counts in lung homogenates and blood were determined by serial dilution in sterile PBS and plating out in blood agar plates (Oxoid) supplemented with 5% horse blood. For z-FA-FMK studies, mice were given the peptidyl fluoromethylketone (25 mg/kg weight in 100 µl) i.v. 24 h and 1 h before infection with S. pneumoniae. All animal care and experiments were conducted under guidelines of the United Kingdom Home Office for Laboratory Animal Care and Experimentation.

Statistical analysis of the data

The experimental data were analyzed using one-way ANOVA followed by Dunnet’s test and the Mann-Whitney U test was used for analysis of the pneumococcal virulence studies.

	Results

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z-FA-FMK inhibits T cell proliferation without causing cell death or the induction of apoptosis

To determine whether z-FA-FMK has any effect on T cell proliferation, PBMCs were stimulated with TCR mimetics, such as PHA, anti-CD3 plus anti-CD28, and PMA plus ionomycin in the presence or absence of this peptidyl fluoromethylketone. As illustrated in Fig. 1A, T cell proliferation induced by these mitogens was inhibited by z-FA-FMK in a dose dependent manner as determined by the incorporation of [³H]thymidine. Although a lower concentration of z-FA-FMK (25 µM) was less effective in blocking mitogen-induced T cell proliferation, near complete inhibition was observed with 100 µM. The concentrations of z-FA-FMK used were not toxic to resting T cells as shown by the lack of PI uptake in the cells after 24 h incubation with the peptidyl fluoromethylketone (Fig. 1B). With activated T cells, higher concentrations of z-FA-FMK (100 µM) induced a small increase in toxicity after 72 h (not significant). These results suggest that z-FA-FMK is not toxic at the concentrations used in our present studies. To further confirm that T cell proliferation was inhibited by z-FA-FMK, we examined the ability of activated T cells to divide in the presence of peptidyl fluoromethylketone using CFSE labeling (22). The sequential dilution of the dye in CFSE-labeled cells as they divide following mitogenic stimulation was monitored using flow cytometry. As shown in Fig. 1C, CFSE-labeled nonactivated cells retained a very high fluorescence, which remained unchanged in the cells after 72 h in culture. There was no change in the fluorescence intensity of these cells after 24 h activation with PHA or costimulated with anti-CD3 and anti-CD28. However, after 72 h of activation, there was a sequential decreased in the fluorescence of the activated CFSE-labeled cells, indicating that the T cells are dividing. In the presence of z-FA-FMK, the sequential dilution of the CFSE dye in the cells induced by PHA, and costimulation with anti-CD3 and anti-CD28 was suppressed. In addition, the effect of z-FA-FMK on T cell blast formation also was examined following T cell activation. As illustrated in Fig. 1D, resting PBMCs have very few blasts (12.6%); however, upon costimulation with anti-CD3 and anti-CD28, nearly 50% of the cells have transformed to blasts, indicating that the cells are activated. In the presence of 50 and 100 µM of z-FA-FMK, the percentage of T cell blasts formed was markedly reduced to 27 and 14%, respectively. DMSO (>0.1%), which is the carrier solvent for z-FA-FMK, was included in all the studies and was found to have no effect on T cell proliferation (results not shown).

View larger version (38K): [in this window] [in a new window]   FIGURE 1. z-FA-FMK inhibits T cell proliferation and blasts formation at nontoxic concentrations. T cell proliferation in PBMCs was stimulated with PHA, costimulated with anti-CD3 and anti-CD28, or with PMA and ionomycin in the presence or absence of various concentrations of z-FA-FMK. T cell proliferation was assayed using [3H]thymidine incorporation (A). B, The toxicity of z-FA-FMK was assessed after 24 h in resting T cells and after 72 h in activated T cells using PI uptake. C, Activated T cells undergoing cell division are measured using CFSE labeling, and T cell activation was determined by the formation of T cell blasts (D). The results are means ± SEM from five (A) or three (B and D) experiments. C, The results are one representative of three experiments. D, Region 1 (R1) consists of T cell blasts, whereas region 2 (R2) consists of mostly dead cells and debris. *, Significantly decreased (p < 0.05) from control with mitogens alone.

Following T cell activation, the cytokine IL-2, which is a T cell growth factor, acts in an autocrine and paracrine fashion to drive T cell proliferation (25). To understand the underlying mechanisms of z-FA-FMK-induced inhibition of T cell proliferation, we examine whether z-FA-FMK has any effect on the production of IL-2 in activated T cells. As illustrated in Fig. 2A, control unstimulated T cells do not produce any IL-2. However, when the T cells were costimulated with anti-CD3 and anti-CD28 for 24 h, there was a huge increased in the concentration of IL-2 in the culture supernatants as detected using ELISA. The presence of 50 µM of z-FA-FMK in the cell culture inhibited the production of IL-2 by 65%, and higher concentration of z-FA-FMK (100 µM) further reduced the amount of IL-2 secreted into the culture supernatants. DMSO has no effect on IL-2 secretion in activated T lymphocytes. The inhibition of IL-2 production by z-FA-FMK suggests that gene transcription dependent processes may be inhibited by z-FA-FMK. To this end, we examined whether z-FA-FMK has any effect on IFN- secretion following T cell activation. As shown in Fig. 2B, resting T cells produced low levels of IFN-, but upon stimulation for 24 h through the Ag receptor, the concentration of this cytokine in the culture supernatants was markedly increased. The presence of z-FA-FMK (50 and 100 µM) markedly inhibited the production of IFN- in these activated T cells.

View larger version (15K): [in this window] [in a new window]   FIGURE 2. z-FA-FMK inhibits the production of IL-2 and IFN- in activated purified T cells. Purified CD4+ and CD8+ T cells were cotreated with anti-CD3 and anti-CD28 for 24 h in the absence or presence of z-FA-FMK. The amount of IL-2 (A) and IFN- (B) secreted into the culture supernatants were determined using ELISA as outlined in Materials and Methods. Results are the means ± SEM from three experiments. *, Significantly reduced (p < 0.05) from control.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. The inhibition of T cell proliferation induced by z-FA-FMK is not reversed by exogenous rIL-2. T cell proliferation was induced by costimulation with anti-CD3 and anti-CD28 in the presence or absence of z-FA-FMK (50 µM) for 72 h. rIL-2 (25 U/ml) was added to the cell cultures where indicated at the beginning of the experiments. The incorporation of [3H]thymidine was determined as in Materials and Methods. The results are the means ± SEM from triplicate samples from one representative experiment of four.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. The effect of z-FA-FMK on CD25 and CD69 expression in T cells following activation. The T cells in PBMCs were costimulated with anti-CD3 and anti-CD28 in the presence or absence of z-FA-FMK for 24 h. The cells were incubated with FITC-conjugated anti-CD25 and PE-conjugated anti-CD69 before analysis using flow cytometry as outlined in Materials and Methods. The results (inset) are the means ± SEM from three experiments.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. z-FA-FMK inhibits IL-2-driven T cell proliferation. Preactivated T cells were cultured in medium supplemented with rIL-2 (25 U/ml) in the absence and presence of z-FA-FMK (50 µM) for 72 h. The incorporation of [3H]thymidine was determined as outlined in Materials and Methods. Results are means ± SEM from three experiments. *, Significantly decreased (p < 0.05), compared with IL-2 treatment alone.

To further characterize the antiproliferative property of z-FA-FMK, we examined whether the cell cycle progression in these cells was being blocked. The DNA content of human T lymphocytes costimulated with CD3 and CD28 Abs in the presence or absence of z-FA-FMK was labeled with PI and analyzed using flow cytometry. As illustrated in Fig. 6, after 72 h of costimulation with anti-CD3 and anti-CD28, a large number of the T cells have entered the cell cycle and were distributed in the S/G2/M phases. In the presence of 50 µM z-FA-FMK, the number of cells at G2/M was slightly increased, suggesting that cells are being prevented from leaving the cell cycle. At higher concentration of z-FA-FMK (100 µM), the number of T cells entering S/G2/M phase was markedly reduced. Our results suggest that z-FA-FMK at high concentration blocked the cells from entering the cell cycle, whereas lower concentration of z-FA-FMK may prevent cells from leaving the cell cycle.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Effect of z-FA-FMK on DNA content in T cells following activation. Resting T cells were coactivated with anti-CD3 and anti-CD28 in the presence and absence of z-FA-FMK for 72 h. The DNA content of the activated T cells was analyzed after staining with PI as outlined in the Materials and Methods. The results (inset) are the means ± SEM from three experiments.

z-FA-FMK inhibits the nuclear translocation of NF-B RelA (p65) in activated T lymphocytes

A previous study has shown that z-FA-FMK blocks LPS-induced cytokine production in monocytes by interfering with NF-B signaling (10). Because NF-B is required for IL-2, IFN-, and CD25 gene transcription, IL-2 signaling as well as T lymphocyte activation (27), we examined the effect of z-FA-FMK on the signaling of this transcription factor. The nuclear translocation of Rel A (p65) in T lymphocytes activated through the Ag receptor was examined as reported previously using immunohistochemistry to localize p65 (23). Using this approach, we showed that the translocation of Rel A into the nuclei was detected in 65% of the T lymphocytes when costimulated with anti-CD3 and anti-CD28 for 2 h (Fig. 7), indicating that NF-B signaling was activated (23). In the presence of z-FA-FMK (50 and 100 µM), there was a significant decreased in nuclear translocation of p65 in the stimulated T lymphocytes, suggesting that NF-B signaling was suppressed by this peptidyl fluromethylketone. Because NF-B plays a pivotal role in gene transcription and T cell activation, its inhibition by z-FA-FMK could account for the decreased in cytokine (IL-2 and IFN-) production, CD25 up-regulation in the activated T cells, and the inhibition of IL-2 signaling during T cell proliferation.

View larger version (17K): [in this window] [in a new window]   FIGURE 7. The effect of z-FA-FMK on NF-B signaling in activated T cells. Purified T cells were costimulated with anti-CD3 and anti-CD28 in the presence or absence of z-FA-FMK and the translocation of cellular p65 to the nucleus were determined as outlined in Materials and Methods. Results are the mean ± SEM from three separate experiments. *, Significantly different (p < 0.05) from cells coactivated with anti-CD3 and anti-CD28 only.

In recent years, a number of studies (12, 13, 14, 15, 16) have shown that various caspases were activated during T cell proliferation. However, to date, only caspase-8 has been shown to play an important role in T cell activation and proliferation (13, 16). Because z-FA-FMK has been shown previously to block caspases (11), we examined its effect on the activation of caspases during T cells activation through the Ag receptor. Primary T cells were costimulated with anti-CD3 and anti-CD28 in the presence of z-FA-FMK for 24 h. As illustrated in Fig. 8, both caspase-8 and caspase-3 were activated to their respective subunits, p43/41 and p20 in T cells following 24-h stimulation, which confirmed previous reports (14, 15). In the presence of z-FA-FMK (50 and 100 µM), the processing of caspase-8 and caspase-3 to their subunits in activated T cells was completely abolished, whereas the caspase inhibitor, z-VAD-FMK, at 50 and 100 µM has no effect (Fig. 8). This suggests that the inhibition of caspase-8 and caspase-3 processing by z-FA-FMK was not due to the blocking of caspase activity per se. To confirm that z-FA-FMK is not blocking caspase activity, we examined its effect on FasL-induced apoptosis in proliferating T cells, where caspases play a pivotal role in the apoptotic-signaling pathway (28). To this end, T cells that have been proliferating for 7 days were exposed to FasL, followed by cross-linking with anti-Flag. As shown in Fig. 9A, proliferating T cells readily undergo apoptosis when treated with FasL and was unaffected by z-FA-FMK (up to 100 µM). However, z-VAD-FMK at 50 µM effectively blocked FasL-induced apoptosis in these cells. Western blot analysis (Fig. 9B) revealed that some caspase-8 was processed in control proliferating T cells. However, as the cells undergo apoptosis following FasL treatment, there was a further increased in the activation and processing of caspase-8 as shown by the loss in the proenzyme and a concomitant increased in the cleaved p43/41 subunits above control levels. All these were abolished in the presence of z-VAD-FMK but not with z-FA-FMK. Interestingly, we could not detect any cleaved caspase-3 fragments in control proliferating T cells. This could be due to rapid degradation of the cleaved caspase-3 fragments in these proliferating cells because resting T cells activated for 24 h retained some of the cleaved caspase-3 fragments (Fig. 8). However, when the proliferating T cells were exposed to FasL, caspase-3 was readily processed to its p17 subunit. Similar to caspase-8, z-FA-FMK has no effect on the activation of caspase-3, although it did prevent the processing of the p20 fragments to the p17 subunits. In contrast, z-VAD-FMK completely blocked the processing of caspase-3 (Fig. 9B).

View larger version (45K): [in this window] [in a new window]   FIGURE 8. z-FA-FMK blocks caspase-8 and caspase-3 activation during T cell activation. Primary T cells were coactivated with anti-CD3 and anti-CD28 in the presence and absence of z-FA-FMK for 24 h. Following treatments, 20 µg of protein of whole cell lysates were resolved by SDS-PAGE, transferred onto nitrocellulose membrane, and then probed with Abs to caspase-8, caspase-3, and -actin as outlined in Materials and Methods. Results shown are representative of three separate experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 9. Effect of z-FA-FMK on caspase-8 and caspase-3 activation during FasL-induced apoptosis. Proliferating T cells were exposed to FasL and cross-linked with anti-Flag in the presence or absence of z-FA-FMK and z-VAD-FMK. Apoptosis (A) and Western blot analysis (B) were conducted as described in Materials and Methods. The immunoblots are one representative experiment from three, and the results in A are the means ± SEM from three separate experiments. *, Significantly decreased (p < 0.05) from cells treated with FasL alone.

View larger version (27K): [in this window] [in a new window]   FIGURE 10. Effect of z-FA-FMK on caspase-8 and caspase-3 activation in Jurkat T cells treated with agonistic anti-Fas. Cells were incubated with agonistic anti-Fas for 6 h. A and B, Apoptosis was determined using UV microscopy and the activation and processing of caspases and PARP were analyzed using Western blotting analysis. Results are the means ± SEM from three separate experiments in A and one representative of three for B. *, Significantly increased; **, decreased (p < 0.05) from cells treated with anti-Fas alone.

Our in vitro results demonstrated that z-FA-FMK have immunosuppressive effects on T cells. To determine whether this peptidyl fluoromethylketone has similar effects in vivo, we examined the effect of z-FA-FMK in a mouse model of pneumococcal disease. Previous studies had shown that CD4⁺T cells played an important early role in the protective host response to pneumococcal infection, and mice deficient in CD4⁺ T cells were more susceptible to pneumococcal bronchopneumonia and septicemia, compared with their wild-type parent mice (20). Therefore, we proposed that if z-FA-FMK inhibits T cell function in vivo, mice administered with this peptidyl fluoromethylketone would have less protection against pneumococcal infection, compared with control mice. Indeed, as illustrated in Fig. 11A, there was a significant increase in the growth of pneumococci at 24 and 48 h post infection in the lungs of mice given z-FA-FMK, compared with mice given solvent carrier (Fig. 11A), whereby there was a 2.3-log increase at 24 h, compared with time zero and a 3.3-log increase at 48 h, compared with time zero (p < 0.05 for both). In contrast, control mice exhibited a <1-log increase in pneumococcal numbers by 48 h, compared with time zero. In blood, there was no significant difference in pneumococcal numbers from mice given z-FA-FMK or carrier solvent at 24 h post infection. However, by 48 h post infection, there was a significant increase of 1.5-log growth of pneumococci in the blood of mice given z-FA-FMK, compared with control mice given solvent carrier. These results are in keeping with previous studies using mice deficient in CD4⁺ T cells and suggest that z-FA-FMK is suppressing the activation of T cells during S. pneumoniae infection.

View larger version (11K): [in this window] [in a new window]   FIGURE 11. z-FA-FMK treatment increases pneumococcal load in the blood and lungs of MFI mice. Changes in numbers of pneumococci in the lungs (A) and blood (B) of control and z-FA-FMK-treated mice infected intranasally over a 48-h period (n = 10 mice for each time point). *, Significantly increased (p < 0.05), compared with mice given S. pneumoniae alone.

	Discussion

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It is well established that the NF-B family of transcription factors play a central role in the coordination of a wide variety of genes that control immune responses. NF-B complexes are normally localized in the cytoplasm, where they are bound to inhibitory proteins known as IBs. In T lymphocytes the predominant form of NF-B complexes activated is a heterodimer of the p65 subunit associated with either p50 or p52 subunit, although c-Rel/p50 is also present (29, 30). Following T cell activation, the inhibitory IB proteins are rapidly phosphorylated and degraded via ubiquination through the proteosome pathway (31, 32). This releases the NF-B transcription factors and allows their translocation into the nuclei and, together with the activator protein 1, regulates the transcription of cytokine and immunoreceptor genes (30). We observed that a large number of primary T cells activated through the Ag receptor were stained positive for p65 in the nucleus, which is in good agreement with previous study (23). In the presence of z-FA-FMK the nuclear translocation of p65 in the activated T cells was markedly suppressed, suggesting that NF-B signaling induced by Ag receptor stimulation is blocked. This confirmed a previous finding where z-FA-FMK inhibits the activation of NF-B in LPS-stimulated macrophages (10). It also accounts for the inhibition of IL-2 and IFN- production and as well as the expression of CD25 because NF-B-regulated gene transcription is known to be required for these processes (29, 30). However, CD69 expression was unaffected by z-FA-FMK because it is constitutively stored in intracellular pools and can be expressed in the absence of gene expression (26). In addition, Mortellaro et al. (27)showed that IL-2 signaling also requires the activation of NF-B, which explains the lack of cell proliferation in activated T cells driven by supplemented rIL-2 in the presence of z-FA-FMK. Besides blocking NF-B signaling, z-FA-FMK also affect cell cycle progression by impeding cells from leaving and entering the cell cycle. This correlates with the CFSE studies where cell division was markedly inhibited by z-FA-FMK. It would be interesting to see whether z-FA-FMK have any effect on the cell cycle genes during T cell proliferation.

In recent years, a number of studies (13, 14, 15, 16) have shown that the caspases, besides promoting cell death via apoptosis also play an important role in T cell activation. A number of peptidyl fluoromethylketone caspase inhibitors, which efficiently blocked apoptosis through the inhibition of caspases have been shown to block T cell proliferation induced by mitogens (12, 14, 15). Although z-FA-FMK was often used as a control for fluoromethylketone caspase inhibitors in apoptosis studies (33, 34, 35), it was reported to block effector caspases during T cell apoptosis induced by retinoid related molecules (11). We confirmed that both caspase-8 and caspase-3 were processed following T cell activation via the Ag receptors in primary T cells and z-FA-FMK completely inhibited the processing of both caspases. Surprisingly, the caspase inhibitor, z-VAD-FMK has virtually no effect on the processing of caspase-8 and caspase-3 during T cell activation, suggesting that the activation of these two enzymes is not caspase dependent. This is in contrast to Fas-induced apoptosis where both caspase-8 and caspase-3 were effectively blocked by z-VAD-FMK. These results suggest that the inhibition of caspase-8 and caspase-3 processing by z-FA-FMK in activated T cells was not due to the inhibition of caspase activity per se. In addition, z-FA-FMK has no effect on caspase-8 and caspase-3 activation or their activity during Fas-induced apoptosis, which involves the sequential activation of these enzymes (36). These results suggest that the processing of caspase-8 and caspase-3 during T cell activation is regulated differently from Fas-induced apoptosis. It is possible that both caspase-8 and caspase-3 are cleaved by the same enzyme during T cell activation, because the processing of both caspases was inhibited by z-FA-FMK and not z-VAD FMK. Although granzyme B has been shown to cleave both caspase-8 and caspase-3 and initiate apoptosis in target cells during cytotoxic killing (37, 38), cell death triggered by granzyme B is not inhibited by z-FA-FMK (39, 40). Another protease that plays a role in the activation of caspases is cathepsin B from the lysosomes (41, 42), which has been shown to activate the proinflammatory caspase-11 in vitro (43). Although z-FA-FMK was originally designed to block cathepsin B, z-VAD-FMK also blocks this cysteine protease activity and yet has no effect on the processing of caspase-8 and caspase-3 during T cell activation (44). This effectively rules out the involvement of cathepsin B in cleaving the caspases.

Although z-FA-FMK has no effect on the activation of caspase-8 and caspase-3 during Fas-induced apoptosis, we consistently observed an accumulation of both cleaved caspase-3 p20 and p17 subunits in the Jurkat cells. The caspase-3 p20 fragment is an intermediate, formed during the activation of the proenzyme and undergoes autoprocessing to yield the p17 subunit (36). The accumulation of both fragments during Fas-induced apoptosis in the presence of z-FA-FMK, suggests that the autoprocessing of p20 subunits may be suppressed. However, this is unlikely because only p20 will accumulates when autoprocessing is inhibited. It is more likely that z-FA-FMK is blocking the degradation of both caspase-3 subunits (p20 and p17), presumably by inhibiting the ubiquitin-proteosome system. Similar accumulation of cleaved caspase-3 fragments have been reported previously with the proteosome inhibitor, lactacystin, where both caspase-3 activity and apoptosis were also increased (45). This is in good agreement with our results where PARP cleavage (caspase-3 activity) and apoptosis were increased in the presence of z-FA-FMK.

The immunosuppressive effect of z-FA-FMK was confirmed in vivo in an intranasal mouse model of pneumococcal infection. Previous studies have shown that mice, which are devoid of CD4⁺ T cells are significantly more susceptible to pneumococcal infection, with higher bacterial loads in both lungs and blood, compared with wild-type parents (20). In keeping with this, we have now shown that mice administered with z-FA-FMK exhibited 40- to 50-fold higher pneumococcal numbers in both lungs and blood, compared with control mice, suggesting that the host T cell response to pneumococcal infection was significantly compromised by the peptidyl fluoromethylketone. Indeed, previous in vitro studies have shown that up-regulated CD25 expression in CD4⁺ T cells is a consequence of interactions with pneumococci (20). The immunosuppressive effect of z-FA-FMK may account for the remarkable therapeutic effect in suppressing articular cartilage and bone destruction in chronic inflammatory arthritis in mice, where T cells play an indispensable role (2, 8, 9). In addition, z-FA-FMK also was found to increase the mortality and parasitemia in mice infected with Trypanosoma cruzi (46). This parasitic disease affects a wide variety of cell types and tissues and cell-mediated immunity, particularly the CD8⁺ T cells play an important role in the immune response to T. cruzi (47, 48). The increased in mortality induced by z-FA-FMK treatment in mice infected with T. cruzi is likely to be mediated by its immunosuppressive properties.

In summary, we have shown that the cathepsin B inhibitor z-FA-FMK is an immunosuppressive agent and inhibits pleiotropic processes involved in T cell activation and proliferation in vitro. When administered in vivo using a mouse model of pneumococcal disease where T cells play an important role in the protective host response to pneumococcal infection, z-FA-FMK significantly increases pneumococcal loads in both lungs and blood. Taken together, our results demonstrated that z-FA-FMK is immunosuppressive in vitro and in vivo.

	Acknowledgments

 
We thank Drs. Ayman Marei and Renata Walewska for their help in collecting blood samples from volunteers.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Medical Research Council, United Kingdom.

² Address correspondence and reprint requests to Dr. Sek C. Chow, Medical Research Council Toxicology Unit, Hodgkin Building, Lancaster Road, University of Leicester, Leicester LE1 9HN, U.K.

³ Abbreviations used in this paper: z-FA-FMK, benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone; z-VAD-FMK, benzyloxycarbonyl-valine-alanine-Asp (OMe) fluoromethylketone; RA, rheumatoid arthritis, PI, propidium iodide; PARP, poly(ADP-ribose) polymerase.

Received for publication May 4, 2006. Accepted for publication July 7, 2006.

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