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. 2004 Apr;2(2):67–87. doi: 10.1080/15401420490464295

The Use of Streptolysin O for the Treatment of Scars, Adhesions and Fibrosis: Initial Investigations Using Murine Models of Scleroderma

Stephen W Mamber 1, Vit Long 1, Ryan G Rhodes 1, Sunthorn Pond-Tor 1, Lyn R Wheeler 1, Kellie Fredericks 1, Brian VanScoy 1, Jean-Frederic Sauniere 1, Remy Steinschneider 2, Jean-Claude Laurent 2, John McMichael 3
PMCID: PMC2655705  PMID: 19330124

Abstract

Diseases and conditions involving the deposition of excessive amounts of collagen include scleroderma, fibrosis, and scar and surgical adhesion formation. Diseases such as scleroderma may result from acute and chronic inflammation, disturbances in the normal parenchymal area, and activation of fibroblasts. ML-05, a modified form of the hemolytic and cytotoxic bacterial toxin, streptolysin O, is being developed for the treatment of such collagen-related disorders. At sublytic concentrations in vitro, ML-05 was shown to activate CD44 expression. This may modulate production of collagen, hyaluronate, and their associated enzymes to allow a restoration of normal extracellular matrices within tissues. More importantly, ML-05 appeared to decrease skin collagen levels in two in vivo models of collagen disorders, the tight skin mouse (Tsk) model of scleroderma, and the bleomycin-induced mouse skin fibrosis model. In the Tsk model, levels of hydroxyproline (a measure of total collagen) decreased by 25% in the Tsk+ML-05 treatment group relative to the Tsk+saline control group over a 3-month period. In the bleomycin-induced skin fibrosis study, hydroxyproline levels decreased from 15–22% over a 6-week period in a bleomycin-induced ML-05 treatment group (relative to levels in a bleomycin-induced, untreated control group). Hydroxyproline levels in samples from this treatment group were only slightly greater than levels in an uninduced control group at 8 weeks. Thus, ML-05 treatment appeared to reduce collagen levels in two separate mouse skin fibrosis models, one genetically based and the other chemically induced.

Keywords: scleroderma, streptolysin O, treatment, animal model, collagen

INTRODUCTION

Scleroderma (literally, “hard skin”) is a chronic autoimmune connective tissue disease that affects thousands of patients. Scleroderma often progresses as an autoimmune disease in which the body’s immune system attacks its own connective tissues (Haynes and Gershwin, 1982; Krieg and Meurer, 1988; LeRoy et al., 1988; Mayes, 1997; Sapadin et al., 2001; Haustein, 2002; Trojanowska, 2002). There are two types of scleroderma: systemic scleroderma, which affects the internal organs, and localized scleroderma, which affects the local area of the skin. The disease is characterized by excessive collagen deposition in the affected skin, as well as in various internal organs (lungs, heart, kidneys, esophagus, myocardium, and gastrointestinal tract), and vascular injury (Haynes and Gershwin, 1982; Krieg and Meurer, 1988; LeRoy et al., 1988; Tuffanelli, 1998; Hawk and English, 2001; Sapadin et al., 2001; Haustein, 2002; Mori et al., 2002; Sehgal et al., 2002). The causes of scleroderma are unknown (Artlett et al., 1999; Johnson et al., 2002; Sapadin and Fleischmajer, 2002). Presently, there is no cure for scleroderma, but a number of treatments are available for specific symptoms. Such treatments include (i) methotrexate, a drug commonly used for rheumatoid arthritis and other inflammatory forms of arthritis; (ii) collagen peptides administered orally; (iii) halofuginone, a drug that inhibits the synthesis of type I collagen, a primary component of connective tissue; (iv) ultraviolet light therapy for localized forms of cutaneous scleroderma; and (v) bone marrow–derived stem cell transfusions, for early diffuse systemic sclerosis (Dutz, 2000; Hawk and English, 2001; Steen, 2001; Fisher and Kang, 2002; Mori et al., 2002; Sapadin and Fleischmajer, 2002; Stummvoll, 2002). Because scleroderma is an autoimmune disease, some investigators have observed that scleroderma results in a Th2-type humoral immune response, as opposed to a Th1-type cell-mediated immune response (Mavalia et al., 1997; Ong et al., 1999; Oliver, 2000). Th1 and Th2 immune responses can be characterized based on the types of cytokines produced in response to a given immune stimulus or as the result of a given disease state (Seder and Paul, 1994; Mosmann and Sad, 1996; Oliver, 2000). Cytokines involved in Th1-type immune responses include interleukin-2 (IL-2) and interferon-gamma, whereas cytokines such as IL-4, IL-5, IL-6, IL-10, and IL-13 are predominant in Th2-type immune responses (Seder and Paul, 1994; Mosmann and Sad, 1996). Accordingly, one therapeutic approach has been the administration of immunomodulatory substances that can shift the immune response in scleroderma from a Th2-type response to a Th1-type response, including anti-IL-4 antibodies (Ong et al., 1999), IL-12 (Tsuji-Yamada et al., 2001) and thalidomide (Oliver, 2000; Oliver et al., 2000).

ML-05, a nonhemolytic form of streptolysin O (SLO), is undergoing pre-clinical development as a possible immunomodulatory treatment for diseases involving abnormal or excessive collagen deposition (McMichael, 1998). SLO is an exotoxin produced by Streptococcus species of groups A, C, and G. It is a single chain protein that, as a thiol-activated, cholesterol-binding agent, can form pores in cell membranes in its reduced state but cannot readily form them after oxidation. Because red blood cells are particularly susceptible to cytolysis, they are used to assess the cytolytic properties of SLO. From a structural standpoint, the primary gene product consists of 571 amino acids with several structural and property domains including a cholesterol-binding domain. Secreted SLO is 540 amino acids (~60–70 kDa), whereas truncated SLO is 494 amino acids (~53–61 kDa). Similar toxins are secreted by other species of Gram-positive bacteria (Johnson et al., 1980; Alouf et al., 1984; Bhakdi et al., 1985; Palmer et al., 1996; Harris et al., 1998; Palmer, 2001). SLO and related substances have been shown to generate Th1-type immune responses (Kraakman et al., 1995; Baba et al., 2002). The application of low levels of oxidized SLO as a treatment for diseases involving abnormal or excessive collagen deposition was based on similar observations with other immunomodulatory substances (McMichael, personal communication; McMichael, 1998). In addition to its potential immunomodulatory properties, ML-05 may directly or indirectly alter production of collagen to allow a restoration of normal extracellular matrices within tissues by other mechanisms. At sublytic concentrations, pneumolysin, another bacterial protein chemically related to SLO, was capable of inducing collagenase production in fibroblasts (Johnson et al., 1988). Moreover, it has been hypothesized that compounds such as SLO could increase or maintain the expression of cell surface receptors that are involved in extracellular matrix organization, notably hyaluronan receptor CD44 (Aruffo et al., 1990; Lesley et al., 1993; Cichy and Pure, 2003). Appropriate changes in CD44 levels may have beneficial effects in both scleroderma (Kaya et al., 2000; Komura et al., 2002) and lung fibrosis (Teder and Heldin, 1997).

This paper addresses the hypothesis that low levels of ML-05 can modulate or reduce the excessive production of collagen in diseases such as scleroderma. The in vitro studies characterized the pharmacological effects of oxidized SLO on keratinocytes. The in vivo studies characterized the therapeutic effects of low levels of SLO on skin collagen levels in two murine models of scleroderma, the genetically based tight skin (Tsk) mouse model (Green et al., 1976; Menton et al., 1978) and the bleomycin-induced scleroderma model (Yamamoto et al., 1999).

MATERIALS AND METHODS

Streptolysin O

SLO for all in vivo and some in vitro studies was purchased from Sigma Chemicals, St. Louis, MO (catalog number S5265; 25,000–50,000 units/vial). SLO from a second source (Capricorn Products, Scarborough, ME) was used for certain in vitro experiments. Oxidized SLO was prepared by bubbling air or oxygen into solutions of SLO for defined time periods. Evidence of oxidation was obtained from the lack of activity of a given preparation in a standard hemolysis assay provided by Sigma Chemicals, except that sheep red blood cells were employed instead of human red blood cells. Solutions of the final formulation (ML-05) for in vitro experiments were prepared in saline or deionized water, filtered through a 0.22-μm filter and stored at 4 or –20°C. Solutions of ML-05 for in vivo use, containing 10, 50, and 250 units/ml, were prepared using sterile saline and stored at 4°C. A separate solution of sterile saline was the control vehicle for the in vivo experiments.

Primary Culture of Keratinocytes

The in vitro studies using primary human keratinocytes were performed at Bio Expertise Technologies Laboratory, Marseille, France. The primary cell culture procedures employed were based on methods previously described (Rheinwald and Green, 1975; Wilke and Bandemir, 1989). Human keratinocyte cell suspensions were obtained from five different normal donors undergoing plastic surgery. Keratinocytes were isolated from skin samples by a standard trypsinization procedure and were grown in primary culture (passage 0) on mitomycin C–treated 3T3 feeder cells. For all procedures involving keratinocyte cell cultures, cells were incubated at 37°C in a humidified 5% CO2 incubator. Alternatively, cells were harvested from the primary culture and cryopreserved at –80°C.

Keratinocyte Cell Growth/Cytotoxicity Assays

For studying cytotoxic effects of ML-05 on keratinocyte growth, keratinocytes at subconfluence were trypsinized, enumerated, and cultured in triplicate (passage 1) with KBM medium (Cambrex Bio Science [formerly known as BioWhittaker], Walkersville, MD) supplemented with bovine pituitary extract (30 μg/ml), epidermal growth factor (0.1 μg/ml), insulin (0.5 μg/ml), hydrocortisone (0.5 μg/ml), epinephrine, and transferrin in flat-bottomed microtiter plates with either 2000 or 5000 keratinocytes per well. ML-05 was tested at three different concentrations (0.02, 0.2, and 2 units/ml). ML-05 samples were added to the medium 24 hr after the cell seeding (day 1, T0) and cultures were maintained for 11 days. The activity of ML-05 on cell growth was evaluated daily using the MTT test, a tetrazolium salt-based colorimetric assay for cellular growth and survival (Mosmann, 1983). Briefly, MTT solution was added to cells in media at different days of culture. Mitochondrial succinate dehydrogenase activity was evaluated after 2 hr of MTT incorporation by first discarding the cell media and lysing the cells so that MTT was recovered in the supernatant of the lysis solution. Optical density of each well was measured at 540 nm with a background reading at 605 nm. The results were evaluated by comparing levels of cell proliferation over time for the ML-05–treated samples with that of the control (cells in culture medium without ML-05).

Immunostaining Assays for Keratinocyte Cell Surface Marker CD44

Cell surface marker CD44 was selected as a relevant target molecule for the keratinocyte experiments based on both the literature (Teder and Heldin, 1997; Kaya et al., 2000; Komura et al., 2002) and the results of initial immunostaining experiments with several cell surface markers other than CD44 (data not shown). For evaluation of the effects of ML-05 on the incidence of cell surface marker CD44, keratinocytes were used at passage 2. Briefly, keratinocytes harvested and preserved from the primary culture (passage 0) were thawed and cultured for one passage (passage 1) in order to have homogeneous cultures. Next, keratinocytes were dissociated and seeded in 96-well microplates (passage 2) at 15,000 or 20,000 cells/well. For qualitative evaluations of CD44 expression, five individual keratinocyte cell cultures seeded at 20,000 cells/well were employed. Each culture was treated with three different ML-05 concentrations (0.02, 0.2, and 2 units/ml). Plates with ML-05 and controls were incubated at 37°C. At 1- and 26-hr time points, cells were fixed with ethanol:acetic acid (95:5) and rinsed extensively with phosphate buffered saline (PBS). Direct immunofluorescence staining of CD44 cell surface markers of keratinocytes then was performed. Briefly, after saturation with 5% goat serum in PBS, phycoerythrin-labeled CD44 antibody (Caltag Laboratories, Burlingame, CA) was diluted 1:500 in PBS +1% bovine serum albumin and incubated at room temperature for 1 hr. After extensive washings, cells were observed under a fluorescence microscope (mercury lamp excitation filter 540 nm). Photographs of each well were taken and evaluated with respect to the fluorescence intensity of stained cells in the wells.

Animal Models for In Vivo Experiments

The genetically based animal model of choice for these studies was tight skin mutant mouse strain Tsk/+, which exhibits abnormally tight skin due to a mutation in the Tsk gene (Green et al., 1976; Menton et al., 1978). The heterozygotes (Tsk/+) develop substantial hypertrophy of loose connective tissue, resulting in hardening, thickening, and tightening of the skin. This hypertrophy is similar to that deposited in scleroderma patients (Green et al., 1976; Menton et al., 1978; Osborn et al., 1983; Russell, 1983; Walker et al., 1985). The nongenetic model utilized was a murine model of bleomycin-induced scleroderma. Yamamoto et al. (1999) employed subcutaneous injections of bleomycin as a means of chemically inducing scleroderma in C3H/He mice. This animal model is purported to reflect human progressive systemic scleroderma (PSS), because the dermis is thickened and appears pathologically similar to PSS in human skin (Yamamoto et al., 1999, 2000; Yamamoto, 2002).

Animals

For the genetically based scleroderma studies, female Tsk/+ (B6.Cg-Fbn1〈Tsk〉 +/+ Pldn〈pa〉) mice (initial age, 3 weeks) were obtained from Jackson Laboratory, Bar Harbor, ME. For the bleomycin-induced scleroderma studies, female C3H/He mice (initial age, 6 weeks) were used. They also were obtained from Jackson Laboratory. Mice were housed in groups of two in plastic solid-bottom cages (Thoren Caging Systems, Hazleton, PA) containing laboratory animal bedding (Paperchip; Canbrands, Moncton, Canada). Food (LabDiet 5015 Rodent Diet; PMI Nutrition International, Brentwood, MO) and water were given ad libitum. The vivarium in which the mice were housed was kept on an appropriate (12:12 hr) light–dark cycle, and the ambient temperature was maintained at about 22°C. An Institutional Animal Care and Use Committee approved all in vivo experimental procedures described.

Tight Skin Mouse Model: Experimental Design

After a standard conditioning period of 3 days, animals were divided into two groups of 10 Tsk/+ mice per group. The first group of Tsk/+ mice was given a twice-daily subcutaneous injection of ML-05 (10 units/ml, 0.2 ml/mouse). The second group of Tsk/+ mice was given a twice-daily subcutaneous injection of saline. Beginning at 4 months after treatment was initiated, and continuing at 4-week intervals for an additional 4 months of treatment, two mice were euthanized from each group. Animals were euthanized by CO2 inhalation. Once euthanized, three to four samples of shaved skin were taken from the dorsal surface between the shoulder blades with a 6-mm biopsy punch. Removed samples were wrapped in aluminum foil paper and placed on ice for up to 1 hr, then stored at –80°C. Skin samples were used for assays of hydroxyproline (a measure of total collagen content).

Bleomycin-Induced Scleroderma: Experimental Design

After a standard acclimation period of 3 days, a total of 68 mice (subsequently divided into three treatment groups and a positive control group) were given a daily subcutaneous injection of bleomycin (1 mg/ml, 0.2 ml/mouse/injection) for 4 weeks to induce dermal sclerosis. A separate group of 20 mice (designated as the negative control group) did not receive any bleomycin. The animals were visually monitored daily and weighed weekly. A daily subjective analysis was recorded for each mouse, which measured whether the mouse was showing signs of lethargy, a hunched position, and/or a ruffled coat. ML-05 treatment was initiated on the day after the final injection of bleomycin. At that point, the 68 bleomycin-injected animals were randomly divided into three treatment groups of 16 mice per group and a positive control group of 20 mice. The three treatment groups of mice were given twice-daily subcutaneous injections of ML-05 (10, 50, or 250 units/ml, 0.2 ml/mouse/injection, for doses of 2, 10, and 50 units/injection). Both control groups (bleomycin-induced positive control and uninduced negative control) were given twice-daily subcutaneous injections of saline. Animals were treated for up to 8 weeks. On treatment day 0, four members of the two control groups were euthanized by CO2 inhalation. Four mice from each group (treatment as well as controls) then were euthanized at 2, 4, 6, and 8 weeks post-treatment initiation. Once euthanized, a samples (3–4) of shaved skin from a given mouse were taken from the dorsal surface between the shoulder blades with a 6-mm biopsy punch. Removed samples were wrapped in aluminum foil paper, placed on ice for up to 1 hr, and then stored at –80°C. Skin samples were used for assays of hydroxyproline.

Hydroxyproline Assay Rationale

Because the hallmark of scleroderma is the excessive deposit of collagen, the primary endpoint for evaluation in both animal models was a reduction in the level of total collagen in skin samples from ML-05–treated mice relative to that in untreated control animals. This was accomplished by performing assays for the hydroxyproline content of skin biopsy samples. Hydroxyproline concentration is directly related to the total collagen content (Neuman and Logan, 1950; Woessner, 1961; Stegemann and Stalder, 1967; Cheng, 1969; Edwards and O’Brien, 1980; Reddy and Enwemeka, 1996).

Hydroxyproline Assay Reagents

Colorimetric assays of hydroxyproline in murine skin samples were performed to determine the amount of all forms of collagen present in those samples. With respect to reagents, sodium acetate, citric acid, chloramine-T, p-dimethylaminobenzaldehyde, acid-soluble collagen, and l-hydroxyproline were purchased from Sigma Chemicals, St. Louis, MO. Perchloric acid, n-propanol, hydrochloric acid, and acetic acid were purchased from Fisher Scientific Co., Pittsburgh, PA. Protease inhibitor tablets were purchased from Roche Biochemicals, Indianapolis, IN. The hydroxyproline stock solution contained 1 mg/ml of l-hydroxyproline in distilled water. Acetate citrate buffer (pH 6.5) was prepared by dissolving 120 g of sodium acetate trihydrate, 46 g of citric acid, 12 ml of acetic acid, and 34 g of sodium hydroxide in distilled water, adjusting the pH to 6.5, and then adjusting the final volume to 1 l using distilled water. Chloramine-T reagent (0.056 M) contained 1.27 g of chloramine-T dissolved in 20 ml of 50%n-propanol), and was brought to 100 ml with acetate citrate buffer. Erlich’s aldehyde reagent (1 M) was prepared by dissolving 15 g of p -dimethylaminobenzaldehyde in n-propanol:perchloric acid (2:1 v/v), which was then adjusted to a total volume of 100 ml using distilled water.

Hydroxyproline Assay Procedure

Approximately 5 mg of each skin sample was accurately weighed using an analytical balance. The sample was added to a glass 1-ml Dounce homogenizer tube (Wheaton, Millville, NJ), followed by the addition of 6N HCl containing protease inhibitor (one tablet per 7 ml of 6N HCl), with 1 ml of acid added per 5 mg of tissue. The sample was homogenized using a type B pestle, and the homogenate was added to a v-bottom glass vial (5-ml capacity) with a temperature-resistant phenolic screw cap and 20-mm TFE-silicone septum (Wheaton, Millville, NJ). The tube and its contents were baked at 110°C for 8 hr in a heating oven (Blue M Electric, Williamsport, PA). Next, 50 μl of each baked skin sample was removed to separate glass tubes (in triplicate), and 450 μl of chloramine-T was added to each sample. Oxidation reactions were allowed to proceed for 25 min at room temperature. To initiate chromophore development, 500 μl of Ehrlich ’s aldehyde reagent was added to each sample, followed by tube incubation at 65°C for 20 min in the water bath. Sample aliquots of 200 μl were transferred to the wells of a polystyrene 96-well microdilution tray (Corning Incorporated, Corning, NY), and absorbance was read at 550 nm using a microtiter plate reader (Thermo Lab Systems [formerly Dynex Technologies], Chantilly, VA). A standard curve was prepared using various dilutions of a 1 mg/ml stock solution of hydroxyproline, with final concentrations ranging from 2 to 200 μg/ml (typically 2, 10, 25, 50, 100, and 200 μg/ml). Values then were determined using the standard curve and expressed as μg hydroxyproline/mg skin.

RESULTS

Oxidation of SLO

Because ML-05 is a nonhemolytic form of oxidized SLO, it was necessary to demonstrate that oxidation of the parent molecule (SLO) resulted in a loss of hemolytic activity. Typical results of an oxidation experiment are shown in Figure 1. Based on hemolysis assays, SLO solutions that were oxidized for 30–60 minutes possessed only 1–2% hemolytic activity relative to that of the starting material.

FIGURE 1.

FIGURE 1

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Hemolysis of sheep red blood cells by SLO solutions prepared by bubbling air or oxygen through the solutions for varying periods of time (5–60 min). Hemolysis assays were conducted in accordance with standard protocols, except that sheep red blood cells were substituted for human red blood cells.

Effects of ML-05 on Keratinocyte Cell Growth

To ensure that ML-05 was not cytotoxic, it was tested for inhibitory effects on keratinocyte proliferation. Figure 2 shows the response of a primary keratinocyte culture to three test concentrations of ML-05 (0.02, 0.2, and 2 units/ml). All growth curves of cells treated with ML-05 were comparable to that of the untreated control. Thus, ML-05 at these test concentrations had little or no effect on keratinocyte growth or viability.

FIGURE 2.

FIGURE 2

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Effects of varying concentrations of ML-05 on growth of human primary keratinocytes in vitro. The numbers of cells per ml were calculated based on MTT absorbance readings of known cell concentrations. Triangle = cells plus ML-05, 0.02 units/ml; square = cells plus ML-05, 0.2 units/ml; circle = cells plus ML-05, 2 units/ml; diamond = negative control (cells plus media only).

Effects of ML-05 on Keratinocyte CD44 Immunostaining

Direct immunofluorescence staining of keratinocyte CD44 cell surface markers was conducted to evaluate the effects of ML-05 on the incidence of that marker. Figures 3 and 4 are fluorescent micrographs showing CD44 immunostaining localized to keratinocytes. In untreated (negative) control wells, the staining of keratinocytes for CD44 was stronger at 1 hr of incubation than at 26 hr (Figures 3A and 4A). At 1 hr of incubation with ML-05 (Figures 3B–D), there were few apparent differences between staining after incubation with ML-05 and without ML-05. However, at 26 hr of ML-05 incubation (Figures 4B–D), CD44 immunostaining was present in cells treated with ML-05 but was absent from the negative control cells. Thus, ML-05 treatment appeared to upregulate or maintain expression of CD44 in keratinocytes after 26 hr of culture.

FIGURE 3.

FIGURE 3

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Effects of varying concentrations of SLO on cell surface marker CD44 of keratinocytes after 1 hr of SLO exposure (immuno fluorescence staining of CD44). A = negative control; B = SLO, 0.02 units/ml; C = SLO, 0.2 units/ml; and D = 2 units/ml of SLO. Expression of CD44 is similar for the control and SLO-treated samples, although somewhat reduced at 2 units/ml.

FIGURE 4.

FIGURE 4

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Effects of varying concentrations of SLO (0.02–2 units/ml) on cell surface marker CD44 of keratinocytes after 26 hr of SLO exposure (immunofluorescence staining of CD44). A = negative control; B = SLO, 0.02 units/ml; C = SLO, 0.2 units/ml; and D = 2 units/ml of SLO. Expression of CD44 is comparable in the SLO-treated samples, but absent in the negative control sample, suggesting upregulation by SLO at this timepoint.

Effects of ML-05 on Skin Hydroxyproline Levels in the Tight Skin Model of Scleroderma

The cumulative results of hydroxyproline assays for total collagen in skin samples from the tight skin mouse study are shown in Table 1 and Figure 5. Figure 6 shows the percent change in hydroxyproline levels in the Tsk+ML-05 treatment group relative to the Tsk+saline control group. Hydroxyproline levels averaged 43% higher in the Tsk+ML-05 group relative to the Tsk+saline group at 4 and 5 months post-treatment initiation. This was followed by a 25% decrease in hydroxyproline levels in the Tsk+ML-05 treatment group relative to the Tsk+saline group at 6–8 months. Based on the relative differences between the treatment and control groups, it was speculated that both a loss of fibrotic collagen structures and a concurrent synthesis of new collagen are modulated as the result of ML-05 treatment. In addition to the hydroxyproline measurements, the mice were observed subjectively with respect to treatment effects on the animals’ appearance and overall health. The mice in the Tsk+ML-05 group appeared to have looser skin relative to the mice in the Tsk+saline group, although this was not quantified.

TABLE 1.

Cumulative Hydroxyproline Assay Results for Tight Skin (Tsk) Mouse Study

Group Month Hydroxyproline concentration, ug/mg skin
Average* SD
Tsk+ML-05 4 13.03 1.42
Tsk+ML-05 5 8.11 0.88
Tsk+ML-05 6 14.08 5.24
Tsk+ML-05 7 14.24 1.99
Tsk+ML-05 8 10.69 0.56
Tsk+saline 4 8.26 0.90
Tsk+saline 5 6.33 0.69
Tsk+saline 6 18.96 0.62
Tsk+saline 7 18.87 2.13
Tsk+saline 8 14.55 6.28
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*Each average value was calculated from the results of two skin samples assayed in duplicate or in triplicate. SD = standard deviation.

FIGURE 5.

FIGURE 5

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Effects of SLO treatment on mouse skin hydroxyproline levels in the Tsk scleroderma model (cumulative results). Gray = Tsk+ML-05 treatment group; vertical lines = Tsk+saline control group. Hydroxyproline levels in the treatment group were higher at 4 and 5 months, then lower at 6–8 months post-treatment initiation relative to that of the control group. Also, hydroxyproline levels for the duration of the study ranged from 8 to 14 μg/mg protein and 6 to 19 μg/mg protein for the treatment and control groups, respectively. This suggests that ML-05 modulated collagen synthesis over time.

FIGURE 6.

FIGURE 6

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Effects of SLO treatment on mouse skin hydroxyproline levels in the Tsk scleroderma model based on percent change in hydroxyproline levels in the treatment (Tsk+ML-05) group relative to that of the control (Tsk+saline) group over time.

Effects of ML-05 on Skin Hydroxyproline Levels in the Bleomycin-Induced Scleroderma Model

The cumulative results of hydroxyproline assays for total collagen in skin samples from the bleomycin-induced scleroderma study are shown in Table 2 and Figure 7. The percent change in hydroxyproline levels in the ML-05 treatment groups relative to that of the bleomycin-induced saline control (positive control) group is shown in Figure 8. Hydroxyproline levels in the 2 and 50 unit/injection treatment groups were similar to that of the positive control group. However, there was a 10 –15% decrease in hydroxyproline levels in the 10 unit/injection treatment group relative to the positive control group at 4–8 weeks after treatment was initiated. By week 8, hydroxyproline levels in this treatment group approached that of the uninduced saline control (negative control) group.

TABLE 2.

Cumulative Hydroxyproline Assay Results for Bleomycin-Induced Scleroderma Study

Group Week Hydroxyproline concentration, ug/mg skin
Average† SD
2 U/injection 2 14.79 0.95
2 U/injection 4 20.74 2.38
2 U/injection 6 16.06 0.98
2 U/injection 8 18.33 3.58
10 U/injection 2 17.38 1.93
10 U/injection 4 16.66 0.89
10 U/injection 6 16.07 0.56
10 U/injection 8 12.90 0.67
50 U/injection 2 13.66 1.65
50 U/injection 4 20.09 0.70
50 U/injection 6 23.89 6.21
50 U/injection 8 16.07 3.74
Positive control 2 14.25 1.25
Positive control 4 20.31 6.83
Positive control 6 20.50 4.48
Positive control 8 15.19 2.55
Negative control 2 9.17 0.94
Negative control 4 11.42 2.65
Negative control 6 7.97 0.95
Negative control 8 10.41 2.94
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†Each average value was calculated from the results of four skin samples assayed in duplicate or in triplicate. SD = standard deviation.

FIGURE 7.

FIGURE 7

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Effects of SLO treatment on mouse skin hydroxyproline levels in the bleomycin-induced scleroderma model (cumulative results). Note the differences in hydroxyproline levels between the treatment and control groups over time. Vertical lines = 10 units/injection; gray solid = 50 units/injection; horizontal lines = 250 units/injection; right slant lines = positive control; cross hatches = negative control. The 10 unit/injection treatment group showed a marked reduction in hydroxyproline levels at 4–8 weeks post-treatment initiation relative to that of the positive control group.

FIGURE 8.

FIGURE 8

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Effects of SLO treatment on mouse skin hydroxyproline levels in the bleomycin-induced scleroderma model based on percent change in hydroxyproline levels in the three treatment groups relative to that of the positive control (bleomycin-induced, untreated) group over time. Vertical lines = 10 units/injection; gray solid = 50 units/injection; horizontal lines = 250 units/injection.

DISCUSSION

The in vivo results of this research indicate that ML-05 can reduce and/or modulate collagen deposition in skin. This is based on decreased or altered levels of hydroxyproline (a direct measure of total collagen) in skin samples obtained from ML-05-treated tight skin (Tsk) mice as well as in samples from at least one of three treatment groups of mice with bleomycin-induced scleroderma. The initial in vivo scleroderma investigation was the tight skin mouse study, which employed only one test dose of ML-05 and a relatively lengthy evaluation period (8 months). Both dose and evaluation period were selected based largely on prior immunomodulatory research with SLO (McMichael, personal communication; McMichael, 1998). The bleomycin-induced scleroderma model was employed for a variety of reasons. One reason was general interest in testing ML-05 in a nongenetic model of scleroderma. Another reason was specific interest in determining if ML-05 could alleviate bleomycin-induced fibrosis. This was influenced by the research of Nici and Calabresi (1999) and Nici et al. (1998), who used a rat model of bleomycin-induced fibrosis. Building on experiences with the tight skin mouse study, the bleomycin-induced scleroderma experiment was designed to permit the evaluation of multiple doses of ML-05, including the same dose employed in the tight skin study. It was believed that an 8-week treatment period would have been sufficient to determine ML-05 efficacy. In retrospect, it would have been useful to conduct the bleomycin-induced scleroderma study for 12 or even 16 weeks. At the end of the 8-week treatment period, hydroxyproline levels in samples from the 10 unit/injection group were comparable to that of the negative control, continuing a downward trend that began at 2 weeks post-treatment initiation. Based on this observation, it is possible that ML-05 treatment beyond 8 weeks would result in further reductions or stabilization of hydroxyproline levels over time. On the other hand, because hydroxyproline levels were reduced in samples from only one (10 unit/injection group) of the three treatment groups in the bleomycin-induced scleroderma model, the effective concentration range obtained in that model was relatively narrow. Because bleomycin is a known cytotoxic agent and ML-05 is derived from a cytolytic substance (SLO), the combination of these two agents to induce and to treat scleroderma (even at nontoxic doses by themselves) may mask the effectiveness of ML-05 in this model. The genetically based tight skin mouse model thus may be preferable for evaluating ML-05 effectiveness in the treatment of fibrosis because there are no concerns that additional chemical substances could produce synergistic or antagonistic effects in that model.

The mechanism(s) by which ML-05 can reduce or modulate collagen levels requires further investigation. The literature suggests that SLO can act as an immunomodulator, capable of generating Th1-type immune responses (Kraakman et al., 1995; Baba et al., 2002). As a potential immunomodulator, ML-05 could be affecting cytokine expression, epithelial cell migration/proliferation, and other specific immune responses to the scleroderma disease state (Kraakman et al., 1995; Mavalia et al., 1997; Ong et al., 1999; Oliver, 2000; Tsuji-Yamada et al., 2001; Baba et al., 2002). In addition to its potential immunomodulatory properties, ML-05 may, either directly or indirectly, alter the process of fibrosis by other mechanisms. One intriguing possibility, based on the in vitro experiments reported here, is that ML-05 can induce the expression of hyaluronan receptor CD44 in keratinocytes. Upregulation of keratinocyte CD44 could lead to the accumulation of CD44 in the extracellular matrix via proteolytic cleavage (Cichy et al., 2002; Cichy and Pure, 2003). This, in turn, could promote the assembly of hyaluronan-rich extracellular matrices through the formation of CD44–hyaluronic acid complexes (Cichy et al., 2002; Cichy and Pure, 2003). These complexes could modulate epithelial cell behavior, including increased mobility, altered adhesion, and/or altered proliferation of keratinocytes and fibroblasts. Such physiological changes in the keratinocytes and fibroblasts present in sclerotic regions may ultimately result in a restoration of normal extracellular matrix organization in those regions (Kaya et al., 2000; Cichy et al., 2002; Komura et al., 2002; Cichy and Pure, 2003).

In summary, ML-05 was not cytotoxic to keratinocytes and appeared to have upregulated hyaluronan receptor CD44 in vitro. Moreover, ML-05 appeared to reduce or modulate collagen levels in two mouse scleroderma (skin fibrosis) models. Currently, there are no satisfactory drugs for the treatment of the different forms of scleroderma in the human population. However, based on these studies, ML-05 may be able to relieve the clinical symptomology associated with such conditions. With respect to reductions in total collagen, the effective concentration range was relatively narrow. Still, these results serve as a promising foundation for subsequent in vitro and in vivo evaluations of ML-05 activity in alleviating diseases that involve excess collagen deposition.

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