Aloxistatin

Effect of a Thiol Proteinase Inhibitor, E-64-d, on Susceptibility to Infection with Staphylococcus aureus in Chediak-Higashi Syndrome (Beige) Mice

Abstract

We previously reported that abnormally down-regulated protein kinase C (PKC) activity is responsible for the impaired cellular function of natural killer (NK) cells and polymorphonuclear cells (PMNs), and the giant granule formation in fibroblasts in the beige mouse, an animal model of Chediak-Higashi syndrome (CHS). Here, we examine the effect of oral or intraperitoneal administration of E-64-d, which protects PKC from calpain-mediated proteolysis, on the impaired cellular function in PMNs from beige mice. We found that oral administration of E-64-d (12.5 mg/kg body weight per day) for three consecutive days significantly improved the abnormally increased concanavalin A (Con A) cap formation and the decreased lysosomal enzyme activity in beige PMNs. In addition, E-64-d significantly improved the delayed bactericidal activity against Staphylococcus aureus. In contrast, E-64-d at the same dose did not affect these cellular functions in PMNs from C57BL/6J mice. We confirmed that the abnormal down-regulation of PKC after Con A stimulation was eliminated in PMNs from E-64-d-treated beige mice. We then examined whether the administration of E-64-d to beige mice improved the susceptibility to experimental infection with S. aureus (2 × 10⁸/mouse). Both intraperitoneal and oral administration of E-64-d to beige mice resulted in a significant increase in survival, whereas E-64-d at the same dose did not alter the survival rate in normal mice. These results suggest that the administration of E-64-d may be effective against severe bacterial infection in Chediak-Higashi syndrome.

Keywords: Chediak-Higashi syndrome, beige mice, protein kinase C, calpain, bacterial infection

1. Introduction

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disorder characterized by partial oculocutaneous albinism, recurrent pyogenic infections, defective natural killer (NK) activity, and abnormal giant lysosomes. There is no specific treatment, and most patients succumb to frequent bacterial infections.

Susceptibility to infection in CHS patients is associated with granulocytopenia, abnormal chemotaxis, and a delay in the killing of phagocytized bacteria in polymorphonuclear cells (PMNs). It has been reported that ascorbic acid, a known stimulant of cyclic GMP levels, improves the chemotaxis and bactericidal activity against Staphylococcus aureus in PMNs from beige mice. However, leukocyte dysfunction is not improved by ascorbic acid in human CHS patients.

The beige mouse has been used as an animal model of CHS. Human CHS patients and beige mice have homologous disorders associated with CHS1 mutation. However, the precise function of the CHS1 protein has not been elucidated, although it is suggested that the CHS1 protein regulates lysosomal fission.

Protein kinase C (PKC) is a Ca²⁺, phospholipid-dependent serine/threonine protein kinase that plays an essential role in intracellular signal transduction. We previously reported that PKC activity is abnormally down-regulated after stimulation with phorbol ester or concanavalin A (Con A) in PMNs, NK cells, and fibroblasts from beige mice. This abnormal down-regulation of PKC in beige cells is eliminated by treatment of cells with potent inhibitors of calpain, a thiol proteinase that proteolyzes PKC to an inactive form. In addition, we showed that the formation of giant granules, increased Con A cap formation, defective NK activity, and decreased elastase and cathepsin G activity in beige mice are recovered by treatment of cells in vitro with thiol proteinase inhibitors.

In the present study, we examined whether susceptibility to infection with S. aureus and the associated cellular functions of PMNs are improved when E-64-d, a cell-permeable thiol proteinase inhibitor, is given orally or intraperitoneally to beige mice.

2. Materials and Methods
2.1. Mice

C57BL/6J (+/+) and C57BL/6J-beige (bg/bg) mice were originally obtained from CLEA Japan Inc. (Tokyo, Japan). Eight- to twelve-week-old mice of the same age and sex (weighing 20–25 g) were used in all experiments. All animal experiments were approved by the Animal Experiment Committee at the University of Yamanashi.

2.2. Reagents

E-64-d [ethyl (+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-oxiranecarboxylate] was kindly provided by Taisho Pharmaceutical Co. (Saitama, Japan). Hanks’ balanced salt solution (HBSS) and phosphate-buffered saline (PBS) were from Invitrogen Co. (Carlsbad, CA, USA). [γ-³²P]ATP and the PKC enzyme assay system were purchased from GE Healthcare Bio-Science Co. (Piscataway, NJ, USA). The substrate of elastase was from Peptide Institute Inc. (Osaka, Japan). The substrates of cathepsin G and β-galactosidase, FITC-Con A, lysostaphin, and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). SCD agar and heart infusion broth were obtained from Eiken Chemical Co. (Tokyo, Japan).

2.3. Bacteria

The coagulase-positive and methicillin-sensitive strains of S. aureus used in this study were clinical isolates from the University of Yamanashi Hospital. Bacteria were grown for 12 h in heart infusion broth at 37°C. Subsequently, the bacterial culture was kept at 4°C until further use. Serial 10-fold dilutions were made from this bacterial culture and plated on SCD agar plates. After overnight culture, the colonies were counted and the bacterial culture was diluted to the desired concentrations.

2.4. Administration of E-64-d

E-64-d was suspended in carboxymethylcellulose solution containing 0.1% Tween 80 as described by Tamai et al. E-64-d was given to mice orally by gastric intubation (0.1 ml) using a 22-gauge intubation tube for three consecutive days. Alternatively, E-64-d was dissolved in dimethylsulfoxide and diluted with PBS and given by intraperitoneal injection (0.5 ml).

2.5. Isolation of Murine PMNs

PMNs were obtained by lavage with 5 ml of HBSS 16–20 h after intraperitoneal injection of 1 ml of sterile thioglycolate broth. The cells obtained (6–10 × 10⁶/mouse) were 80–85% PMNs as determined by May-Giemsa staining; the remainder were macrophages and lymphocytes.

2.6. Con A Cap Formation

FITC-conjugated Con A-induced cap formation was determined as previously described. Cells (1 × 10⁶/ml) were incubated with FITC-Con A at a concentration of 20 μg/ml at 37°C. The cells were fixed with 2% paraformaldehyde for 10 min at 37°C. Wet mounts were prepared using 10 μl of each cell mixture on a glass slide with cover slips. The cells were observed with an epifluorescence microscope fitted with an FITC interference filter and a 20× or 40× objective. Scoring was carried out in two categories: random clusters or capped. Two hundred cells were counted, and the percentage of capped cells was calculated.

2.7. Assay for Lysosomal Enzyme Activity

Cells (5 × 10⁶) were resuspended in cold 0.2% Triton X-100, 0.2 M sucrose, 5 mM EDTA in 20 mM imidazole-HCl, pH 7.3. The cells were disrupted by sonication for 20 s three times at 4°C and centrifuged at 15,000 rpm for 15 min at 4°C. The supernatant was used for enzyme assays. Elastase activity was measured with the fluorogenic substrate methoxysuccinyl-Ala-Ala-Pro-Val-4-methylcoumarinyl-7-amide. Cathepsin G activity was assayed using Suc-Ala-Ala-Pro-Phe-pNA as the substrate. β-Galactosidase activity was assayed according to the method of Brandt et al.

2.8. Bactericidal Assays

Bactericidal assays were performed as described by Gallin et al. For these studies, 5 × 10⁶ PMNs in HBSS with 10% fresh normal mouse serum were preincubated at 37°C for 10 min, and then 5 × 10⁷ bacteria were added. The PMN suspension was tumbled for 20 min to allow phagocytic uptake. Lysostaphin (final concentration 10 U/ml) was then added to kill extracellular bacteria. Portions (0.1 ml) were removed at intervals (30, 45, and 120 min), washed, lysed, and plated for total viable bacterial counts.

2.9. Assay for PKC Activity

Cells (5 × 10⁶) were disrupted by sonication in 20 mM Tris-HCl (pH 7.5), 0.25 M sucrose, 2 mM EDTA, 5 mM EGTA, 2 mM PMSF, 0.01% leupeptin, and 50 mM 2-mercaptoethanol. Cytosolic and membrane fractions were prepared, and PKC activity was assayed using a commercial PKC enzyme assay system.

2.10. Susceptibility to Infection with S. aureus

Beige and normal mice were divided into two groups. One group was administered E-64-d (12.5 mg/kg per day) orally or intraperitoneally for three consecutive days. The other group received control solution. Five hours after the final administration, all mice were inoculated intraperitoneally with viable S. aureus (2 × 10⁸/mouse). Mice were observed at 12-h intervals, and the number of surviving mice was recorded. Deaths within the first 4 h were excluded as technical.

2.11. Statistics

Statistical analysis was performed with Student’s t-test. Mortality rates were compared using the Wilcoxon test.

3. Results
3.1. Effect of Administration of E-64-d on Con A Cap Formation

Con A-capped cells were significantly increased in beige mice compared with C57BL/6J mice. Intraperitoneal administration of E-64-d for three consecutive days resulted in a dose-dependent decrease in Con A cap formation in beige mice, with no significant effect in control mice. Oral administration of E-64-d (12.5 mg/kg per day) for 3 days produced similar results. The number of peritoneal PMNs was not significantly altered by E-64-d at these doses.

3.2. Effect of Administration of E-64-d on Lysosomal Enzyme Activity

Elastase and cathepsin G activity in beige PMNs were significantly lower than in normal PMNs, while β-galactosidase activity was not significantly different. Oral administration of E-64-d (12.5 mg/kg per day) for 3 days significantly increased elastase and cathepsin G activity in beige PMNs, but did not affect enzyme activity in normal PMNs. Intraperitoneal administration produced similar results.

3.3. Effect of Administration of E-64-d on Bactericidal Activity Against S. aureus

The killing rate of S. aureus in PMNs from beige mice was significantly lower than in PMNs from normal mice. The bactericidal activity of PMNs from beige mice significantly increased after oral administration of E-64-d (12.5 mg/kg per day) for 3 days at all time points tested (30, 45, and 120 min). E-64-d had no significant effect on bactericidal activity in PMNs from normal mice.

3.4. Effect of Administration of E-64-d on PKC Activity

In normal mice, membrane-bound PKC activity increased after 10 min of Con A stimulation, while cytosolic enzyme activity decreased. In beige PMNs, membrane-bound PKC activity drastically decreased after Con A stimulation. Oral administration of E-64-d (12.5 mg/kg per day) for 3 days markedly increased the down-regulated membrane-bound PKC activity in beige PMNs. PKC activity in normal mice was not significantly altered by E-64-d. Intraperitoneal administration produced similar results.

3.5. Effect of E-64-d on Susceptibility to Infection with S. aureus

Administration of E-64-d to beige mice improved bactericidal activity and lysosomal enzyme activity, both strongly linked to defense against bacterial infection. After intraperitoneal inoculation with S. aureus (2 × 10⁸/mouse), 75% of beige mice died within 5 days, while only 33.3% of normal mice died. Survival increased significantly in beige mice treated with either intraperitoneal or oral administration of E-64-d (12.5 mg/kg per day) for 3 days. E-64-d did not significantly affect survival in normal mice.

4. Discussion

This study demonstrates for the first time that oral or intraperitoneal administration of E-64-d to beige mice improves defective leukocyte function, including Con A cap formation, lysosomal enzyme activity, and bactericidal activity against S. aureus. Administration of E-64-d also decreases cumulative mortality from systemic infection with S. aureus in beige mice. E-64-d is a specific inhibitor of cysteine proteinases, including calpain, and is readily absorbed through the intestinal membrane due to its lipophilicity.

Previous studies showed that E-64-d prevents giant granule formation in beige fibroblasts and eliminates calpain-mediated proteolysis of PKC in beige fibroblasts, PMNs, NK cells, and human CHS cell lines. This abnormal down-regulation of PKC is suggested to cause the cellular defects in CHS.

Con A cap formation is associated with membrane-cytoskeleton interaction. PKC inhibitors enhance Con A cap formation in normal murine PMNs, and in beige PMNs, abnormal PKC down-regulation after Con A stimulation is corrected by E-64-d. Lysosomal elastase and cathepsin G activity are selectively reduced in beige PMNs, contributing to high susceptibility to infection. E-64-d almost corrects the enzyme activity in CHS cell lines and beige fibroblasts. The processing and intracellular transport of these enzymes are considered deficient in CHS, and PKC may be involved in generating their active forms.

Intracellular destruction of S. aureus is impaired in leukocytes from CHS patients. Ascorbic acid improves PMN chemotaxis and bactericidal activity in beige mice but not in human CHS patients. Ceramide, produced by sphingomyelinase activation, promotes calpain-mediated PKC proteolysis and increases Con A cap formation and reduces lysosomal enzyme activity, further linking PKC breakdown to CHS pathology.

The genetic defect in CHS and beige mice (CHS1) has been identified, but the relationship between abnormal PKC breakdown and CHS1 protein remains unclear.

In summary, E-64-d corrects cellular defects in CHS mice in vitro and improves susceptibility to infection with S. aureus in vivo. These findings suggest that E-64-d may be effective Aloxistatin for preventing severe bacterial infection in patients with CHS.