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Induction of inflammatory cytokines by cartilage extracts
International Immunopharmacology 7 (2007) 383 – 391 www.elsevier.com/locate/intimp
Induction of inflammatory cytokines by cartilage extracts Liza Merly a,b , Shabana Simjee b,1 , Sylvia L. Smith a,b,⁎ a b
Department of Biological Sciences, Florida International University, University Park, Miami, FL 33199, United States Comparative Immunology Institute, Florida International University, University Park, Miami, FL 33199, United States Received 14 June 2006; received in revised form 17 November 2006; accepted 28 November 2006
Abstract Shark cartilage extracts were examined for induction of cytokines and chemokines in human peripheral blood leukocytes. Primary leukocyte cultures were exposed to a variety of aqueous and organic extracts prepared from several commercial brands of shark cartilage. From all commercial sources of shark cartilage tested the acid extracts induced higher levels of TNFα than other extracts. Different commercial brands of shark cartilage varied significantly in cytokine-inducing activity. TNFα induction was seen as early as 4 h and IFNγ at detectable levels for up to four days. Shark cartilage extracts did not induce physiologically significant levels of IL-4. Results suggest that shark cartilage, preferentially, induces Th1 type inflammatory cytokines. When compared to bovine cartilage extract, collagen, and chondroitin sulfate, shark cartilage induced significantly higher levels of TNFα. Treatment with digestive proteases (trypsin and chymotrypsin) reduced the cytokine induction response by 80%, suggesting that the active component(s) in cartilage extracts is proteinaceous. The induction of Th1 type cytokine response in leukocytes is a significant finding since shark cartilage, taken as a dietary supplement for a variety of chronic degenerative diseases, would be contraindicated in cases where the underlying pathology of the chronic condition is caused by inflammation. © 2006 Elsevier B.V. All rights reserved. Keywords: Cytokine; Shark cartilage; Immunomodulation; CAM; Natural product
Abbreviations: AA, Alkaline extract; AE, Acid extract; BCA, Bicinchoninic acid; BSA, Bovine serum albumin; Con A, Concanavalin A; FCS, Fetal calf serum; EDTA, Ethylenediaminetetraacetic acid; ELISA, Enzyme-linked immunosorbant assay; HPBL, Human peripheral blood leukocytes; IFNγ, Interferon gamma; IL, Interleukin; LPS, Lipopolysaccharide; MWCO, Molecular weight cut off; OE, Organic extract; PBS, Phosphate buffered saline; PHA, Phytohemagglutin; SC, Shark cartilage; SS, Salt soluble extract; TC, Tissue culture; TNFα, Tumor necrosis factor alpha. ⁎ Corresponding author. Department of Biological Sciences, Florida International University, University Park, Miami, FL 33199, United States. Tel.: +1 305 348 3183; fax: +1 305 348 1083. E-mail address: [email protected] (S.L. Smith). 1 Current address: International Center for Chemical Studies, H.E.J. Research Institute for Chemistry, University of Karachi, Karachi75270, Pakistan. 1567-5769/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2006.11.011
1. Introduction Intake of natural dietary supplements for the treatment of various diseases or for general health improvement is a form of complementary and alternative medicine that is gaining in popularity [1–3]. The biological activity, however, of many of the commercially available supplements has not been thoroughly characterized, nor has the treatment efficacy of many natural products been determined [4,5]. This study was undertaken to assess the biological activity of extracts of commercial shark cartilage with respect to the effect on immuno-regulation. While the initial interest in shark cartilage related to its alleged anticancer properties [6–11], recently, the
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interest is on the effect of shark cartilage on oxidative stress [12,13], the expression of adhesion molecules and fibrinolytic enzymes [14,15], and on immune function [16–18]. For these studies, shark cartilage has been purified preparations rather than commercial preparations of shark cartilage that are taken as oral supplements by consumers for therapeutic and/or prophylactic purposes. The purpose of the research conducted in our laboratory was to evaluate the immuno-effectiveness and bioactivity of commercial shark cartilage that is available as a non-regulated dietary supplement. Cytokines are extracellular protein/peptide messengers that play a critical role in cell-to-cell communication. They are the primary mechanism by which communication occurs between leukocytes such as lymphocytes and macrophages, and between other immune and non-immune cells and, thus, contribute significantly to regulating and controlling immune responses [19]. The profile of cytokines produced at any one time during an immune response is largely governed by two subsets of T-helper cells designated Th1 and Th2. The differentiation of T-helper cells into Th1 and Th2 cells is tightly controlled by the cytokines present in the local environment and the type of infection and/or immune stimulus [20]. Polarized Th1 and Th2 responses can contribute to the pathogenesis of immune-mediated diseases. Consequently, natural products and other therapeutic agents that are able to cause shifts in the Th1/Th2 balance could significantly influence the overall immune response [21]. We report here on the cytokine profile induced in human peripheral blood leukocytes following stimulation with extracts of commercial shark cartilage. 2. Materials and methods Shark cartilage was obtained from several commercial companies: (1) Cartilade (Solgar Laboratory, Leonia, NJ), (2) Advanced Shark Cartilage Extract (Twin Laboratories Incorporation, Ronkonkoma, NY), and (3) Natural Shark Cartilage (FDC Wholesale Corporation, Miami, FL). Bovine cartilage was obtained from Sigma Chemical Company, St. Louis, MO. All chemicals, enzymes, and enzyme inhibitors were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise stated. Dialysis membranes (MWCO 6–8 kDa) were obtained from Fisher (Fisher Scientific International, Inc. Liberty Lane, Hampton, NH). Filtration was carried out using Whatman (Whatman Inc., Florham Park, NJ) filter paper, and syringe filters purchased from Fisher Scientific. RPMI medium was purchased from Gibco/BRL (Invitrogen Corporation, Carlsbad, CA). Protein determination was carried out using the BCA kit purchased from Pierce (Pierce Biotechnology Inc., Rockford, IL). ELISA and TC plates were purchased from Fisher Scientific
(Falcon, Becton Dickinson). The cytokine detection kits were purchased from Pierce Endogen (Pierce Biotechnology Inc., Rockford, IL). The E-toxate kit from Sigma Aldrich was used to detect endotoxin. HPBL were obtained from healthy donors according to IRB approved protocol. 2.1. Cartilage extracts The starting material for all extract preparations was 2 g of commercial shark cartilage or bovine cartilage. Since commercial shark products are heterogeneous in chemical composition, the level of bioactivity of extracts prepared from different commercial sources was related to mg extract protein. Except for studies where extracts from different commercial brands of shark cartilage were compared, the acid extract used for all assays was prepared from Solgar cartilage. 2.1.1. Acid extract (SCAE) Acid extracts were prepared from both shark and bovine cartilage. Two grams of cartilage were suspended in 50 ml of 0.5 M acetic acid with 0.03% Toluene, pH 4.2, and incubated with stirring overnight at 4 °C. The extract was centrifuged (500 ×g, 45 min) and the supernatant carefully decanted and filtered to remove undissolved material through a series of filters that included Whatman filter paper followed by a 0.45 μm and a 0.22 μm syringe filter. The extract, in 15 ml aliquots was dialyzed twice against 500 ml of RPMI-1640 medium supplemented with HEPES (25 mM) and glutamine (0.3 mg/ml) using a molecular porous membrane with a MWCO of 6–8 kDa. The dialyzed extract was then filtered through a 0.22 μm filter and stored at −20 °C until further use. All cartilage extracts were assayed for endotoxin before use in bioassays. Each extract was also tested to determine whether it inhibited or interfered with ELISA used to measure cytokine levels. 2.1.2. Salt soluble extract (SCSS) Shark and bovine cartilage were dissolved in 50 ml of 0.1 M Tris–HCl, 0.15 M NaCl, pH 7.5, and incubated overnight at 4 °C. Insoluble material from each extract was removed by centrifugation and clarified by filtration. Fifteen (15) ml aliquots of extract were dialyzed against culture medium, filtered, and stored as described above. 2.1.3. Phosphate buffered saline extract (SCPBS) Shark and bovine cartilage were suspended in 50 ml of 0.02 M PBS (0.5 mM NaH2PO4, 1.9 mM of NaHPO4, 17.9 mM NaCl, pH 7.3) and incubated at 4 °C for 2 h. The extract was centrifuged and the supernatant containing soluble protein was filtered as described above. Extract aliquots were dialyzed against culture medium, filtered, and stored. 2.1.4. Alkaline extract (SCAA) Shark cartilage was suspended in 50 ml of 5% sodium hydroxide and incubated for 45 min at room temperature (22– 25 °C). The mixture was centrifuged and the supernatant filtered, dialysed against culture medium, and stored in 15 ml aliquots as described above.
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2.1.5. Organic extract (SCOE) Shark cartilage was dissolved in 10 ml of cold ethanol and kept on ice during the drop wise addition of 40 ml of a 1:3 isopropanol-hexane solution with stirring. The mixture was incubated on ice for no more than 30 min. The mixture was then centrifuged and the supernatant cleared by filtration, dialysed against culture medium, and stored in 15 ml aliquots. 2.2. Protein determination The protein concentration of each extract preparation was determined by the BCA protein assay using BSA as the standard [22]. 2.3. Assay for endotoxin The E-toxate test (Limulus Amebocyte Lysate Assay) was used for the detection and semi-quantitation of endotoxin in all test samples according to the manufacturer's instructions [23]. Endotoxin-free water provided by the manufacturer was used as a negative control and samples were also tested for the presence of inhibitors for the E-toxate test. The E-toxate test was chosen because it has a threshold sensitivity level between 0.05 and 0.1 endotoxin units per ml, a level considered extremely low for physiological significance. 2.4. Isolation of peripheral blood leukocytes For this study blood was provided by seven individuals. A single donor bleed was used for each experiment. HPBL were separated from freshly drawn heparinized peripheral blood of healthy donors by separation on 3% dextran in physiological saline. The harvested heterogeneous mixture of leukocytes was washed with saline, centrifuged, and the cell pellet subjected to hypotonic lysis to remove contaminating erythrocytes. The leukocytes were resuspended in 7 ml of 0.2% NaCl solution (15–20 s), gently mixed by inverting the tube several times, then an equal volume of 1.6% NaCl solution containing 0.2% dextrose was added to the cell suspension to achieve a final NaCl concentration equivalent to normal physiological saline (0.15 M). The suspension was centrifuged at 250 ×g for 10 min and the supernatant containing erythrocyte stroma was discarded. The cell pellet was washed several times in PBS and suspended in culture medium (RPMI-1640 medium supplemented with 0.3 mg/ml glutamine and 25 mM HEPES) containing 100 μg/ml of streptomycin and 100 U/ml of penicillin to inhibit bacterial contamination, and 10% fetal bovine serum. The cell suspension was standardized to 2.0–2.5 ×105 cells/ml by hemacytometer. Cell viability was checked by the Trypan Blue exclusion test, using 0.4% Trypan Blue in physiological saline. Only cell suspensions with a viability of greater than 94% were used for cell cultures. 2.5. In vitro stimulation of human leukocytes Primary cultures of HPBL were set up in triplicate in 24well flat bottom tissue culture plates. Each well contained 50 μl
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of test stimulant or control stimulant, 100 μl of culture medium (supplemented with 10% FBS), and 200 μl of leukocyte suspension (2.0–2.5 × 105 cells/ml). Depending on the individual experiment, test stimulants consisted of shark or bovine cartilage extract alone or in combination with mitogen, collagen or chondroitin sulphate. LPS (E. coli, Difco laboratories), PHA, or Con A served as stimulants in the positive control cultures. Unstimulated or negative control cultures contained culture medium in lieu of stimulant. Cultures were set up in triplicate for varying periods and plates were incubated in a humid chamber at 37 °C in 5% CO2. 2.6. Assessment of biological activity in vitro 2.6.1. In vitro induction of cytokines and chemokine HPBL culture supernatants were harvested at varying time intervals by aspiration, centrifuged, and stored at −20 °C. Supernatants were assayed for TNFα, IFNγ, IL-1β, IL-4, and IL-8, using commercially available ELISA kits for each individual cytokine/chemokine. Assays were set up in triplicate according to the manufacturer's instructions and cytokines were quantified spectrophotometrically. A standard curve was constructed for each cytokine from standards provided in each kit. 2.6.1.1. TNFα induction. Extracts (acid, alkaline, salt soluble, PBS and organic) using different commercial brands of shark cartilage were prepared. To make reliable comparisons between acid extracts each was standardized to contain between 0.350 to 0.450 mg protein/ml. Culture supernatants were harvested at 0, 4, 8, 12, 20, 24, 30, and 48 h and assayed for released TNFα. To determine whether the cytokine-inducing property was unique to shark cartilage, acid, salt soluble and PBS extracts were also prepared from purified bovine cartilage, but the culture supernatants were harvested at 24 h which represented peak level of cytokine production noted for shark cartilage. Since cartilage is a complex composite of proteins that includes collagen and chondroitin sulfate, TNFα induction, following stimulation of leukocytes by collagen and chondroitin sulfate was compared to that induced by shark cartilage treatment. HPBL cultures were set up as described previously and stimulated with SCAE (0.360 mg protein/ml), collagen (300 mg/ml) and chondroitin sulfate (100 mg/ml). A 1:2 dilution of each stimulant was also tested. Cultures were incubated for 24 h at 37 °C, 5% CO2. Culture supernatants were harvested and assayed for TNFα. 2.6.1.2. Miscellaneous cytokine induction. Cytokines IFNγ, IL-1β, IL-4 and IL-8 were assayed in HPBL cultures stimulated with SCAE (0.365 mg/ml) at 37 °C, 5% CO2. Culture supernatants were harvested and assayed for cytokine at various time intervals ranging from 0 h up to 96 h. In case of IFNγ induction was followed up to six days. Positive control cultures were stimulated with appropriate mitogens: LPS (5 μg/ml) for IL-1β induction, PHA (5 μg/ml) for IFNγ, Con A (5 μg/ml), a T cell mitogen, for IL-4 and all three mitogens individually for IL8 induction. Furthermore, to determine whether cartilage extract
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could affect the response of HPBL to PHA, cultures were also stimulated with a combination of both PHA (5 μg/ml) and SCAE (0.350 mg/ml) and culture supernatants were harvested and assayed for IFNγ daily for 6 days. Unstimulated cell cultures consisted of medium alone. To rule out the possibility that the extracts contained material that influenced and was reactive in the cytokine assays, SCAE alone and in medium was assayed for cytokine. 2.6.2. Protease susceptibility Because shark cartilage is a dietary supplement, SC extracts were treated with digestive proteases. Acid extracts of shark and bovine cartilage, dialyzed against 0.01 M Tris–HCl buffer, pH 7.5, were subjected to proteolysis using a combination of trypsin and chymotrypsin. Enzymes (2 mg each of trypsin and chymotrypsin) were added directly to 10 ml of dialyzed extract and the mixture incubated for 3 h at 37 °C. Digestion was stopped by the addition of 4 mg of trypsin– chymotrypsin inhibitor directly to the mixture. The digested extracts were dialyzed against RPMI-1640 culture medium and filtered before use in bioassays. Each digest was tested for endotoxin. 2.7. Statistical analyses Each experiment was repeated at least once for each treatment. Cultures were set up in duplicate and cytokine assays on culture supernatants were performed in triplicate. Standard errors above the mean were calculated and reported for each experiment for each treatment. Statistical analysis was performed by the Student's t-test, comparing induction level of cytokine in the treated culture with that of cultured cells in medium alone, and each treatment was compared to the positive control for each experiment. A p-value of b0.05 was considered statistically significant.
3.2.1. Stimulation of TNFα production Fig. 1 shows that of all the various extracts prepared (AE, SS and PBS) from each of three brands of shark cartilage, the acid extract (SCAE) was the most effective inducer of TNFα, yielding high levels of TNFα. This was true for acid extracts prepared from all three commercial brands of shark cartilage. When acid extract of three commercial brands of shark cartilage were compared to each other for cytokine-inducing activity, acid extract of shark cartilage from Solgar Laboratories induced higher level of cytokine than that of Twin Laboratories, but not significantly different from FDC acid extract. The AE, SS, and PBS extracts prepared from Twin Laboratories cartilage induced the least amount of TNFα. Similarly, when the cytokine-inducing properties of AE, SS, and PBS extracts of shark (Solgar) and bovine cartilage were compared (Fig. 2), the AE extracts from both sources induced significant levels of TNFα. When the level of TNFα produced was correlated to mg extract protein, the shark extracts produced 3–5 fold higher levels of TNFα than bovine extracts. Comparing the three bovine extracts, the SS extract induced the highest level of TNFα. The alkaline (AA) extract of shark cartilage induced TNFα production albeit to a lesser degree than SCAE, and the organic extract (SCOE) did not induce detectable level of TNFα over 72 h (results not shown). When HPBL were cultured in the presence of shark cartilage acid extracts (Solgar SCAE) significant amounts of TNFα were produced in vitro comparable to that induced by LPS, a strong inducer of TNFα (Fig. 3). In serum a level of b20 pg/ml of TNFα is considered normal [24]. No cytokine was detected in unstimulated cultures containing medium alone. TNFα could be detected in culture supernatants as early as 4 h. By 48 h detectable levels were low. A time-course study of TNFα release revealed that peak levels of TNFα were observed at 8 and 24 h
3. Results 3.1. Test for detectable endotoxin All cartilage extracts were determined to be free of detectable endotoxin by the E-toxate test. Extracts were also tested and found to be free of inhibitors for the E-toxate test (results not shown). 3.2. Cytokine-inducing activity of cartilage extracts Results for all cytokine and chemokine assays are expressed for each treatment as the mean of three replicate assays of duplicate cultures for two experiments. The p-values for each assay are provided in the figure captions and ranged from p b 0.001 to p b 0.01. All experiments were carried out for multiple time intervals and where significant cytokine induction occurred is reported for each cytokine. Extracts used as stimulants were determined to be free of inhibitory/interfering material for the cytokine ELISAs (results not shown).
Fig. 1. Cytokine response of cultured human leukocytes stimulated with different extract preparations from three commercial sources of shark cartilage. The production of TNFα by HPBL cultures stimulated for 24 h with SCAE, SCSS and SCPBS extracts prepared from three different brands of commercial shark cartilage was measured. To make reliable comparisons between brands and preparations, the level of TNFα in culture supernatants was related to mg extract protein. Error bars represent standard error above the mean. The level of TNFα induced in cultures stimulated with extracts was significantly higher (⁎) than that induced in cultures treated with medium alone ( p b 0.005).
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Fig. 2. TNFα response of human leukocytes stimulated by three different extracts prepared from shark (Solgar) and bovine cartilage (BC). Cultures were stimulated with acid (AE), salt soluble (SS) and phosphate buffered saline (PBS) extracts for 24 h. Protein concentration of extracts were SCAE 0.360 mg protein/ml, BC-AE 0.930 mg protein/ml; SC-SS 0.400 mg protein/ml, BC-SS 0.960 mg protein/ml; and SC-PBS 0.520 mg protein/ ml, BC-PBS 1.16 mg protein/ml. Culture medium alone and LPS (5 μg/ ml) served as control stimulants. Error bars represent standard error above the mean. The level of TNFα induced in cultures treated with SC extracts was significantly higher (⁎) than that induced in cultures treated with BC extracts or medium alone ( p b 0.01).
(Fig. 3). A similar pattern of cytokine release was produced with LPS over a 48 h period. The cytokine-inducing ability of SCAE (Solgar) was also compared to that of collagen and chondroitin sulfate, two components of cartilage. No cytokine induction was noted in response to stimulation with chondroitin sulphate (Fig. 4). Collagen did induce cells to produce TNFα, however, the level of cytokine produced was significantly less than that induced by SCAE. The level of TNFα produced appeared to be related to the concentration of the stimulant, suggesting a dose-related response.
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Fig. 4. TNFα response of leukocytes to stimulation by SCAE, collagen and chondroitin sulfate. The level of TNFα produced was measured at 24 h following stimulation. Culture medium and LPS were control stimulants. Error bars represent standard error above the mean. SCAE and LPS induced significantly higher levels (⁎) of TNFα than did treatment with medium alone ( p b 0.005). Collagen and chondroitin sulfate did not induce significant levels of TNFα except for collagen at high concentration, 300 mg/ml ( p b 0.05).
to SCAE by the 3rd day of culture, whereas PHA (a potent mitogen) induced comparable level of IFNγ by the 2nd day. However, when SCAE and PHA were combined to stimulate HPBL cultures simultaneously, there was a significant increase in the level of IFNγ produced. Furthermore, this increased activity was reached by the second day. Results indicate that SCAE enhanced the PHA response by inducing a higher level of IFNγ. The normal serum range of IFNγ is b 20 pg/ml [25].
3.2.2. Stimulation of IFNγ production HPBL cultured with SCAE produced significant amounts of IFNγ in vitro (Fig. 5). Most IFNγ was produced in response
Fig. 3. Time-course of TNFα induction in leukocytes stimulated with shark cartilage (Solgar). HPBL cultures were stimulated with LPS (5 μg/ml), SCAE (0.450 mg protein/ml) or culture medium alone at 37 °C, 5% CO2. Error bars represent standard error above the mean. SCAE and LPS induced significantly high levels (⁎) of TNFα compared to treatment with medium alone ( p b 0.005).
Fig. 5. Induction of IFNγ in HPBL cultures stimulated with PHA, SCAE (Solgar), and a combination of both PHA and SCAE. Cultures were stimulated with PHA (5 μg/ml), SCAE (0.367 mg protein/ml) and a combination of PHA and SCAE. Unstimulated cultures were grown in culture medium alone. Error bars represent standard error above the mean. SCAE induced significantly higher level (⁎) of IFNγ at 72 h when compared to cultures treated with medium alone ( p b 0.008). PHA induced a significantly higher level of IFNγ at 48 h, while the combination of SCAE and PHA induced higher levels of IFNγ at bother 48 and 72 h when compared to cultures treated with medium alone ( pb 0.008).
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Fig. 6. IL-1β production by leukocytes stimulated with SCAE (Solgar). HPBL were stimulated with SCAE (0.396 mg protein/ml), LPS (5 μg/ml) or culture medium at 37 °C. Error bars represent standard error above the mean. The level of IL-1β induced by SCAE was significantly high (⁎) at 12 and 24 h, while LPS induced significant levels of cytokine over 96 h ( p b 0.001).
3.2.3. Stimulation of IL-1β production The production of IL-1β in response to cartilage extracts was examined (Fig. 6). Results showed that SCAE (Solgar) induced IL-1β early in culture, reaching peak level by 12 h and declining to insignificant level by 72 h. The IL-1β response to LPS, however, was maintained at a significantly high level through 72 h showing a significant decline only at 96 h.
Fig. 8. Induction of IL-8 in leukocytes. HPBL cultures were stimulated with SCAE (0.396 mg protein/ml), LPS (5 μg/ml), PHA (5 μg/ml), Con A (5 μg/ml) or culture medium. Error bars represent standard error above the mean. SCAE and LPS induced significantly higher levels (⁎) of IL-8 than medium alone at 24, 48 and 72 h ( p b 0.001).
3.2.5. Stimulation of IL-8 production The release of IL-8 in response to cartilage stimulation was compared to chemokine induction following stimulation of cultures by mitogens, such as LPS, PHA and Con A (Fig. 8). Significant levels of IL-8 were induced by SCAE within 24 h and levels were sustained over a period of 72 h. The production of IL-8 in response to LPS however, reached peak levels at 48 h and by 72 h showed a gradual decline. Physiologically significant level of IL-8 was not produced by cultures stimulated with culture medium alone, PHA, or Con A.
3.2.4. Stimulation of IL-4 production When HPBL were cultured in the presence of shark cartilage extract (Solgar), there was no physiologically significant production of IL-4 over 48 h (Fig. 7), a response similar to that obtained with unstimulated cultures (medium alone). Positive control cultures stimulated with Con A, secreted a significant level (40 pg/ml) of IL-4.
The effect of proteolytic digestion on the TNFα-inducing ability of shark and bovine cartilage was examined by using
Fig. 7. IL-4 response of leukocytes stimulated with SCAE (Solgar). HPBL cultures were stimulated with Con A (5 μg/ml), or SCAE (0.360 mg protein/ml) or culture medium. Error bars represent standard error above the mean. SCAE did not induce levels of IL-4 greater than that induced in cultures treated with medium alone, while Con A induced significantly higher levels (⁎) of IL-4 than unstimulated (medium alone) cultures ( p b 0.005).
Fig. 9. The effect of proteolytic digestion on TNFα induction. Shark (Solgar) and bovine cartilage extracts were treated with trypsin and chymotrypsin and used to stimulate HPBL cultures for 24 h. Error bars represent standard error above the mean. In HPBL cultures stimulated with protease-treated extracts, significantly less (⁎) TNFα was produced than in cultures stimulated with untreated extracts derived from both shark and bovine cartilage ( p b 0.005).
3.3. Susceptibility to proteolytic digestion
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extracts treated with trypsin and chymotrypsin (digestive enzymes) prior to stimulating leukocyte cultures (Fig. 9). Following treatment, SCAE (Solgar) lost 80% activity suggesting a protein moiety is associated with the cytokine-inducing activity. A similar effect was noted with bovine cartilage extract.
4. Discussion The results of this study provide evidence that cartilage extracts induce the production of several different cytokines and/or chemokines by human peripheral blood leukocytes. The most well-characterized cytokine response of HPBL to cartilage stimulation, thus far, is TNFα production. When different commercial brands of shark cartilage were tested for their ability to induce TNFα, there was a significant variation in the level of cytokine induced, although each brand induced a detectable level of TNFα. When extracts were prepared from the amount of commercial shark cartilage equivalent to the manufacturer's recommended daily dose (i.e., approximately a three-fold increase in starting material), a 20% increase in the level of TNFα was noted compared to the level induced with extract prepared from 2 g of SC (data not shown). For all brands of cartilage tested, the most cytokineinducing activity was associated with the acid extracts of cartilage. Acid extracts simulate the acidic environment of the stomach. For a dietary supplement to be biologically effective at the time of absorption it must be acid resistant. Considering that the acid extract of shark cartilage was the most effective inducer of a cytokine response, the acidity of the stomach may very well play a role in the in vivo release of the active component(s) from crude cartilage preparations taken orally. Manufacturers are reticent to provide information on the source of the original shark material such as geographic location, species identity, age/sex of animal, tissue from which material was derived, and/or the process used to obtain material. These unknown differences most likely result in the variation in cytokine response seen with different commercial sources of shark cartilage. While both shark cartilage and bovine cartilage extracts induced the production of TNFα, shark cartilage induced significantly higher levels of TNFα, with the acid extract inducing highest levels, of all extracts tested. For bovine cartilage, the salt-soluble extract induced a higher level of TNFα than the acid extract. Taken together, the results suggest that although both types of cartilage contain cytokine-inducing activity, the active component(s) present in each might not be similar. Alternatively, the difference might reflect the nature of the starting material used to prepare the extracts.
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Both shark and bovine cartilage extracts, when treated with proteases, trypsin and chymotrypsin, lost considerable ability to stimulate TNFα production. Results suggest that the active component(s) in cartilage is proteinaceous. Since not all activity was abolished following enzyme treatment, this leads us to speculate that the extracts contain an additional active component(s) which is resistant to the proteolytic activity of trypsin and chymotrypsin. Given the complexity of the gut environment, the effect of proteases may not be as profound as that seen in vitro, and residual activity may be sufficient to stimulate the epithelial lining of the gut to produce a variety of immune messengers which in turn could have a systemic effect given the rich blood supply to the gut. When speculating on in vivo conditions, the presence of microbial enzymes must also be considered which could be a factor in determining the potential effectiveness of shark cartilage as a dietary supplement. Cartilage is a complex material composed of several proteinaceous components, including collagen and chondroitin sulphate. Chondroitin sulfate is least likely to be responsible for the cytokine-inducing effect of cartilage. Collagen alone was able to induce a cytokine response but not at the level seen with SCAE. It is unlikely that collagen present in commercial preparations of shark cartilage contributes significantly to the level of in vitro stimulation noted considering the large amount of collagen required (0.36 mg/ml SCAE versus 300 mg/ml collagen). An important aspect of this investigation was the time-course study of the induction of cytokines in response to cartilage stimulation, specifically TNFα. The biphasic response to SCAE stimulation may reflect TNFα produced by two or more different cell populations given that the cytokine is a pleiotropic cytokine [26]. Macrophages might be the cell type to initially respond to direct stimulation by cartilage extract [27], while other cell types (e.g., lymphocytes) account for peak activity noted at 24 h, which may be an indirect effect in response to additional cytokine messengers released by the cell type initially responding to cartilage stimulation [28]. Alternatively, the decrease in level of TNFα detected in supernatants at 12 h may not represent a decline in secretion but rather a binding of the free cytokine by up-regulated cytokine receptors on activated cells or by soluble TNFα receptors released into culture supernatants by responding cells. Shark cartilage extract induced the production of IFNγ at levels comparable to that obtained in positive control cultures stimulated with PHA, a known mitogen. Shark cartilage extract appeared to behave like a mitogen in stimulating the production of IFNγ after 72 h.
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When cultures were stimulated with PHA alone, IFNγ level peaked at 48 h. However, when PHA and SCAE were combined as stimulants, the cytokine response was enhanced with a significantly higher level (almost 800 pg/ml) of IFNγ detected at 48 h. These results suggest that an active component(s) in shark cartilage behaves like a mitogen stimulating leukocytes to produce potent mediators, and its presence appears to enhance the activity of PHA. An alternative interpretation might be the reverse, that is, that PHA induces an earlier and enhanced response to SCAE. What is undetermined is whether the enhanced response is due to one or more cell type(s) responding. The induction of proinflammatory cytokines, TNFα, IFNγ, and IL-1β suggests that cartilage preferentially induces Th1 type cytokines. The production of IL-4 (b5 pg/ml) was considered to be physiologically insignificant (normal serum range b30 pg/ml). In contrast, the chemotactic chemokine, IL-8, was produced by stimulated leukocytes at higher (20,000 pg/ml) than normal levels encountered in serum (0–10 pg/ml). Establishing “normal ranges” for cytokine serum levels derived from immune assays of serum/plasma of healthy individuals can be misleading, since recognized variation exists in cytokine levels in normal individuals to the extent that cytokine levels can often be undetectable by ELISA. It is, therefore, essential to include normal controls in each individual experiment to take into account possible variation [29]. The cytokine induction profile for shark cartilage, therefore, includes three potent Th1 type cytokines, TNFα, IFNγ and IL-1β, two of which are characteristically involved in inflammatory responses and an inflammatory chemokine, IL-8. Shark cartilage did not induce a significant level of IL-4, thus it not only preferentially stimulates a Th1 type response but appears to indirectly inhibit the development of a Th2 response through the action of IFNγ which is an inhibitor of Th2 cell population expansion [30,31]. From the cytokine profile induced by shark cartilage one can conclude that activation, proliferation, and differentiation of B cells (i.e., a humoral immune response) is not an integral part of the immune response to SCAE stimulation since IL-4, a key cytokine in the activation of B cells, was not induced to physiologically significant levels by SCAE [32]. Based on the differences in peak production of cytokines over time we can speculate on the sequence of cellular events. An early event is most likely to be monocyte/macrophage activation by SCAE inducing the autocrine and paracrine effects of TNFα, followed by Th cell activation, preferably Th1, in response to TNFα and IL-1β co-stimulation with IFNγ inhibiting proliferation of Th2 cells.
We have shown that cartilage induces various inflammatory cytokines and a potent chemokine in immune cells. Thus, if through intestinal absorption the active component(s) in shark cartilage were to reach systemic circulation and/or target sites in the body, immune regulation could be significantly influenced. Consequently, the inflammatory cytokine profile induced by cartilage might be deleterious to the health of individuals suffering from chronic diseases where the underlying pathology is associated with inflammation and where up regulation of Th1 type response is undesirable. For such individuals shark cartilage, taken as an unregulated dietary supplement, is counter indicated. Alternatively, this type of immune response, in which IL-4 production is restricted, could be beneficial for hypersensitive individuals whose allergic state is often associated with production of IgE. In the absence of Th2 stimulation, the IgE response would be down regulated to the benefit of the individual. Acknowledgments We thank Barbara Anderson for performing phlebotomy, Betzabel Gonzalez for her technical contributions, and John Makemson and Charles Bigger for their comments on the manuscript. LM was supported by an MBRS student fellowship and this study was supported in part by a summer research award to LM under the Comparative Immunology Initiative (R25 GM061347). Facilities for the project were provided by FIU Comparative Immunology Institute and this is contribution FIU-CII-003 from the Institute. The authors received no funding from and have no association with the manufacturers and/or distributors of the commercial brands of shark cartilage used in this study. References [1] Eisenberg DM, Davis RB, Ettner SL, Appel S, Wilkey S, et al. Trends in alternative medicine use in the United States, 1990–1997 — results of a follow-up national survey. JAMA 1998;280(18): 1569–75. [2] Kennedy J. Herb and supplement use in the US adult population. Clin Ther 2005;27(11):1847–58. [3] Tindle HA, Davis RB, Phillips RS, Eisenberg DM. Trends in use of complementary and alternative medicine by US adults: 1997– 2002. Altern Ther Health Med 2005;11(1):42–9. [4] Oneschuk D, Fennell L, Hanson J, Bruera E. The use of complementary medications by cancer patients attending an outpatient pain and symptom clinic. J Palliat Care 1998;14(4):21–6. [5] Suarez-Almazor M, Kendall CJ, Dorgan M. Surfing the NetInformation on the World Wide Web for persons with arthritis: patient empowerment or patient deceit? J Rheumatol 2001;28 (1):185–91.
L. Merly et al. / International Immunopharmacology 7 (2007) 383–391 [6] Brem H, Folkman J. Inhibition of tumor angiogenesis mediated by cartilage. J Exp Med 1975;141:427–39. [7] Lee A, Langer R. Shark cartilage contains inhibitors of tumor angiogenesis. Science 1983;221:1185–7. [8] Oikawa T, Ashino-Fuse H, Shimamura M, Koide U, Iwaguchi T. A novel angiogenic inhibitor derived from Japanese shark cartilage (I). Extraction and estimation of inhibitory activities toward tumor and embryonic angiogenesis. Cancer Lett 1990;51(3):181–6. [9] Gingras D, Renaud A, Mousseau N, Beliveau R. Shark cartilage extracts as antiangiogenic agents: smart drinks or bitter pills? Cancer Metastasis Rev 2000;19:83–6. [10] Gonzalez RP, Leyva A, Moraes MO. Shark cartilage as source of antiangiogenic compounds: from basic to clinical research. Biol Pharm Bull 2001;24(10):1097–101. [11] Loprinzi CL, Levitt R, Barton DL, Sloan JA, Atherton PJ, et al. Evaluation of Shark Cartilage in patients with advanced cancer: A North Central Cancer Treatment Group Trial. Wiley Interscience; 2005. Published online. [12] Felzenszwalb I, Peleilo de Mattos JC, Bernardo-Filho M, Caldeira-de-Araujo A. Shark cartilage-containing preparation: protection against reactive oxygen species. Food Chem Toxicol 1998;36:1079–84. [13] Conde CMS, Albano F, Bouskela E, Felzenszwalb I, Svensjo E. Inhibition of ischemia/reperfusion induced plasma leakage by alpha-tocopherol, trolox, and a shark cartilage preparation with anti-oxidant properties. Nutr Res 2001;21:1363–71. [14] Chen JS, Chang CM, Wu JC, Wang SM. Shark cartilage extract interferes with cell adhesion and induces reorganization of focal adhesions in cultured endothelial cells. J Cell Biochem 2000;78: 417–28. [15] Ratel D, Glazier G, Provencal M, Boivin D, Beaulieu E, et al. Direct-acting fibrinolytic enzymes in shark cartilage extract: potential therapeutic role in vascular disorders. Thromb Res 2005;115(1–2):143–52. [16] Feyzi R, Hassan Z, Mostafaie A. Modulation of CD4+ and CD8+ tumor infiltrating lymphocytes by a fraction isolated from shark cartilage: shark cartilage modulates anti-tumor immunity. Int Immunopharmacol 2003;3:921–6. [17] Kralovec J, Guan Y, Metera K, Carr RI. Immunomodulating principles from shark cartilage Part I. Isolation and biological assessment in vitro. Int Immunopharmacol 2003;3:657–69. [18] Hassan Z, Feyzi R, Sheikhian A, Bargahi A, Mostafaie A, et al. Low molecular weight fraction of shark cartilage can modulate
[19] [20] [21]
[22]
[23]
[24]
[25]
[26] [27] [28]
[29]
[30]
[31]
[32]
391
immune responses and abolish angiogenesis. Int Immunopharmacol 2005;5(6):961–70. Romagnani S. Immunologic influences on allergy and the Th1/ Th2 balance. J Allergy Clin Immunol 2004;113(3):395–400. Muraille E, Leo O. Revisiting the Th1/Th2 paradigm. Scand J Immunol 1998;47(1):1–9. Kidd P. Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev 2003;8(3): 223–46. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76–85. Nandan R, Brown DR. An improved in vitro pyrogen test: to detect picograms of endotoxin contamination in intravenous fluids using limulus amoebocyte lysate. J Lab Clin Med 1977;89 (4):910–8. Aggarwal BB, Reddy S. Tumor necrosis factor. In: Nicola NA, editor. Guidebook to cytokines and their receptors. A Sambrook and Tooze Publication of Oxford University Press; 1994. p. 103–4. Gray PW. Interferon Gamma. In: Nicola NA, editor. Guidebook to cytokines and their receptors. A Sambrook and Tooze Publication of Oxford University Press; 1994. p. 118–9. Tracey KJ, Cerami A. Tumor necrosis factor: a pleiotropic cytokine and therapeutic target. Annu Rev Med 1994;45:491–503. Mosser DM. The many faces of macrophage activation. J Leukoc Biol 2003;73(2):209–12. Wang J, Wakeham J, Harkness R, Xing Z. Macrophages are a significant source of type 1 cytokines during mycobacterial infection. J Clin Invest 1999;103(7):1023–9. Whiteside TL. Cytokine measurements and interpretation of cytokine assays in human disease. J Clin Immunol 1994;14(6): 327–39. Billiau A, Heremaus H, Vermeire K, Matthys P. Immunomodulatory properties of interferon gamma: an update. Ann NY Acad Sci 1998;856:22–32. Maggi E, Parronchi P, Manetti R, Simonelli C, Piccinni MP, et al. Reciprocal regulatory effects of IFN-gamma and IL-4 on the in vitro development of human Th1 and Th2 clones. J Immunol 1992;148(7):2142–7. Lund FE, Garvey BA, Randall TD, Harris DP. Regulatory roles for cytokine-producing B cells in infection and autoimmune disease. Curr Dir Autoimmun 2005;8:25–54.
Induction of inflammatory cytokines by cartilage extracts Liza Merly a,b , Shabana Simjee b,1 , Sylvia L. Smith a,b,⁎ a b
Department of Biological Sciences, Florida International University, University Park, Miami, FL 33199, United States Comparative Immunology Institute, Florida International University, University Park, Miami, FL 33199, United States Received 14 June 2006; received in revised form 17 November 2006; accepted 28 November 2006
Abstract Shark cartilage extracts were examined for induction of cytokines and chemokines in human peripheral blood leukocytes. Primary leukocyte cultures were exposed to a variety of aqueous and organic extracts prepared from several commercial brands of shark cartilage. From all commercial sources of shark cartilage tested the acid extracts induced higher levels of TNFα than other extracts. Different commercial brands of shark cartilage varied significantly in cytokine-inducing activity. TNFα induction was seen as early as 4 h and IFNγ at detectable levels for up to four days. Shark cartilage extracts did not induce physiologically significant levels of IL-4. Results suggest that shark cartilage, preferentially, induces Th1 type inflammatory cytokines. When compared to bovine cartilage extract, collagen, and chondroitin sulfate, shark cartilage induced significantly higher levels of TNFα. Treatment with digestive proteases (trypsin and chymotrypsin) reduced the cytokine induction response by 80%, suggesting that the active component(s) in cartilage extracts is proteinaceous. The induction of Th1 type cytokine response in leukocytes is a significant finding since shark cartilage, taken as a dietary supplement for a variety of chronic degenerative diseases, would be contraindicated in cases where the underlying pathology of the chronic condition is caused by inflammation. © 2006 Elsevier B.V. All rights reserved. Keywords: Cytokine; Shark cartilage; Immunomodulation; CAM; Natural product
Abbreviations: AA, Alkaline extract; AE, Acid extract; BCA, Bicinchoninic acid; BSA, Bovine serum albumin; Con A, Concanavalin A; FCS, Fetal calf serum; EDTA, Ethylenediaminetetraacetic acid; ELISA, Enzyme-linked immunosorbant assay; HPBL, Human peripheral blood leukocytes; IFNγ, Interferon gamma; IL, Interleukin; LPS, Lipopolysaccharide; MWCO, Molecular weight cut off; OE, Organic extract; PBS, Phosphate buffered saline; PHA, Phytohemagglutin; SC, Shark cartilage; SS, Salt soluble extract; TC, Tissue culture; TNFα, Tumor necrosis factor alpha. ⁎ Corresponding author. Department of Biological Sciences, Florida International University, University Park, Miami, FL 33199, United States. Tel.: +1 305 348 3183; fax: +1 305 348 1083. E-mail address: [email protected] (S.L. Smith). 1 Current address: International Center for Chemical Studies, H.E.J. Research Institute for Chemistry, University of Karachi, Karachi75270, Pakistan. 1567-5769/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2006.11.011
1. Introduction Intake of natural dietary supplements for the treatment of various diseases or for general health improvement is a form of complementary and alternative medicine that is gaining in popularity [1–3]. The biological activity, however, of many of the commercially available supplements has not been thoroughly characterized, nor has the treatment efficacy of many natural products been determined [4,5]. This study was undertaken to assess the biological activity of extracts of commercial shark cartilage with respect to the effect on immuno-regulation. While the initial interest in shark cartilage related to its alleged anticancer properties [6–11], recently, the
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interest is on the effect of shark cartilage on oxidative stress [12,13], the expression of adhesion molecules and fibrinolytic enzymes [14,15], and on immune function [16–18]. For these studies, shark cartilage has been purified preparations rather than commercial preparations of shark cartilage that are taken as oral supplements by consumers for therapeutic and/or prophylactic purposes. The purpose of the research conducted in our laboratory was to evaluate the immuno-effectiveness and bioactivity of commercial shark cartilage that is available as a non-regulated dietary supplement. Cytokines are extracellular protein/peptide messengers that play a critical role in cell-to-cell communication. They are the primary mechanism by which communication occurs between leukocytes such as lymphocytes and macrophages, and between other immune and non-immune cells and, thus, contribute significantly to regulating and controlling immune responses [19]. The profile of cytokines produced at any one time during an immune response is largely governed by two subsets of T-helper cells designated Th1 and Th2. The differentiation of T-helper cells into Th1 and Th2 cells is tightly controlled by the cytokines present in the local environment and the type of infection and/or immune stimulus [20]. Polarized Th1 and Th2 responses can contribute to the pathogenesis of immune-mediated diseases. Consequently, natural products and other therapeutic agents that are able to cause shifts in the Th1/Th2 balance could significantly influence the overall immune response [21]. We report here on the cytokine profile induced in human peripheral blood leukocytes following stimulation with extracts of commercial shark cartilage. 2. Materials and methods Shark cartilage was obtained from several commercial companies: (1) Cartilade (Solgar Laboratory, Leonia, NJ), (2) Advanced Shark Cartilage Extract (Twin Laboratories Incorporation, Ronkonkoma, NY), and (3) Natural Shark Cartilage (FDC Wholesale Corporation, Miami, FL). Bovine cartilage was obtained from Sigma Chemical Company, St. Louis, MO. All chemicals, enzymes, and enzyme inhibitors were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise stated. Dialysis membranes (MWCO 6–8 kDa) were obtained from Fisher (Fisher Scientific International, Inc. Liberty Lane, Hampton, NH). Filtration was carried out using Whatman (Whatman Inc., Florham Park, NJ) filter paper, and syringe filters purchased from Fisher Scientific. RPMI medium was purchased from Gibco/BRL (Invitrogen Corporation, Carlsbad, CA). Protein determination was carried out using the BCA kit purchased from Pierce (Pierce Biotechnology Inc., Rockford, IL). ELISA and TC plates were purchased from Fisher Scientific
(Falcon, Becton Dickinson). The cytokine detection kits were purchased from Pierce Endogen (Pierce Biotechnology Inc., Rockford, IL). The E-toxate kit from Sigma Aldrich was used to detect endotoxin. HPBL were obtained from healthy donors according to IRB approved protocol. 2.1. Cartilage extracts The starting material for all extract preparations was 2 g of commercial shark cartilage or bovine cartilage. Since commercial shark products are heterogeneous in chemical composition, the level of bioactivity of extracts prepared from different commercial sources was related to mg extract protein. Except for studies where extracts from different commercial brands of shark cartilage were compared, the acid extract used for all assays was prepared from Solgar cartilage. 2.1.1. Acid extract (SCAE) Acid extracts were prepared from both shark and bovine cartilage. Two grams of cartilage were suspended in 50 ml of 0.5 M acetic acid with 0.03% Toluene, pH 4.2, and incubated with stirring overnight at 4 °C. The extract was centrifuged (500 ×g, 45 min) and the supernatant carefully decanted and filtered to remove undissolved material through a series of filters that included Whatman filter paper followed by a 0.45 μm and a 0.22 μm syringe filter. The extract, in 15 ml aliquots was dialyzed twice against 500 ml of RPMI-1640 medium supplemented with HEPES (25 mM) and glutamine (0.3 mg/ml) using a molecular porous membrane with a MWCO of 6–8 kDa. The dialyzed extract was then filtered through a 0.22 μm filter and stored at −20 °C until further use. All cartilage extracts were assayed for endotoxin before use in bioassays. Each extract was also tested to determine whether it inhibited or interfered with ELISA used to measure cytokine levels. 2.1.2. Salt soluble extract (SCSS) Shark and bovine cartilage were dissolved in 50 ml of 0.1 M Tris–HCl, 0.15 M NaCl, pH 7.5, and incubated overnight at 4 °C. Insoluble material from each extract was removed by centrifugation and clarified by filtration. Fifteen (15) ml aliquots of extract were dialyzed against culture medium, filtered, and stored as described above. 2.1.3. Phosphate buffered saline extract (SCPBS) Shark and bovine cartilage were suspended in 50 ml of 0.02 M PBS (0.5 mM NaH2PO4, 1.9 mM of NaHPO4, 17.9 mM NaCl, pH 7.3) and incubated at 4 °C for 2 h. The extract was centrifuged and the supernatant containing soluble protein was filtered as described above. Extract aliquots were dialyzed against culture medium, filtered, and stored. 2.1.4. Alkaline extract (SCAA) Shark cartilage was suspended in 50 ml of 5% sodium hydroxide and incubated for 45 min at room temperature (22– 25 °C). The mixture was centrifuged and the supernatant filtered, dialysed against culture medium, and stored in 15 ml aliquots as described above.
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2.1.5. Organic extract (SCOE) Shark cartilage was dissolved in 10 ml of cold ethanol and kept on ice during the drop wise addition of 40 ml of a 1:3 isopropanol-hexane solution with stirring. The mixture was incubated on ice for no more than 30 min. The mixture was then centrifuged and the supernatant cleared by filtration, dialysed against culture medium, and stored in 15 ml aliquots. 2.2. Protein determination The protein concentration of each extract preparation was determined by the BCA protein assay using BSA as the standard [22]. 2.3. Assay for endotoxin The E-toxate test (Limulus Amebocyte Lysate Assay) was used for the detection and semi-quantitation of endotoxin in all test samples according to the manufacturer's instructions [23]. Endotoxin-free water provided by the manufacturer was used as a negative control and samples were also tested for the presence of inhibitors for the E-toxate test. The E-toxate test was chosen because it has a threshold sensitivity level between 0.05 and 0.1 endotoxin units per ml, a level considered extremely low for physiological significance. 2.4. Isolation of peripheral blood leukocytes For this study blood was provided by seven individuals. A single donor bleed was used for each experiment. HPBL were separated from freshly drawn heparinized peripheral blood of healthy donors by separation on 3% dextran in physiological saline. The harvested heterogeneous mixture of leukocytes was washed with saline, centrifuged, and the cell pellet subjected to hypotonic lysis to remove contaminating erythrocytes. The leukocytes were resuspended in 7 ml of 0.2% NaCl solution (15–20 s), gently mixed by inverting the tube several times, then an equal volume of 1.6% NaCl solution containing 0.2% dextrose was added to the cell suspension to achieve a final NaCl concentration equivalent to normal physiological saline (0.15 M). The suspension was centrifuged at 250 ×g for 10 min and the supernatant containing erythrocyte stroma was discarded. The cell pellet was washed several times in PBS and suspended in culture medium (RPMI-1640 medium supplemented with 0.3 mg/ml glutamine and 25 mM HEPES) containing 100 μg/ml of streptomycin and 100 U/ml of penicillin to inhibit bacterial contamination, and 10% fetal bovine serum. The cell suspension was standardized to 2.0–2.5 ×105 cells/ml by hemacytometer. Cell viability was checked by the Trypan Blue exclusion test, using 0.4% Trypan Blue in physiological saline. Only cell suspensions with a viability of greater than 94% were used for cell cultures. 2.5. In vitro stimulation of human leukocytes Primary cultures of HPBL were set up in triplicate in 24well flat bottom tissue culture plates. Each well contained 50 μl
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of test stimulant or control stimulant, 100 μl of culture medium (supplemented with 10% FBS), and 200 μl of leukocyte suspension (2.0–2.5 × 105 cells/ml). Depending on the individual experiment, test stimulants consisted of shark or bovine cartilage extract alone or in combination with mitogen, collagen or chondroitin sulphate. LPS (E. coli, Difco laboratories), PHA, or Con A served as stimulants in the positive control cultures. Unstimulated or negative control cultures contained culture medium in lieu of stimulant. Cultures were set up in triplicate for varying periods and plates were incubated in a humid chamber at 37 °C in 5% CO2. 2.6. Assessment of biological activity in vitro 2.6.1. In vitro induction of cytokines and chemokine HPBL culture supernatants were harvested at varying time intervals by aspiration, centrifuged, and stored at −20 °C. Supernatants were assayed for TNFα, IFNγ, IL-1β, IL-4, and IL-8, using commercially available ELISA kits for each individual cytokine/chemokine. Assays were set up in triplicate according to the manufacturer's instructions and cytokines were quantified spectrophotometrically. A standard curve was constructed for each cytokine from standards provided in each kit. 2.6.1.1. TNFα induction. Extracts (acid, alkaline, salt soluble, PBS and organic) using different commercial brands of shark cartilage were prepared. To make reliable comparisons between acid extracts each was standardized to contain between 0.350 to 0.450 mg protein/ml. Culture supernatants were harvested at 0, 4, 8, 12, 20, 24, 30, and 48 h and assayed for released TNFα. To determine whether the cytokine-inducing property was unique to shark cartilage, acid, salt soluble and PBS extracts were also prepared from purified bovine cartilage, but the culture supernatants were harvested at 24 h which represented peak level of cytokine production noted for shark cartilage. Since cartilage is a complex composite of proteins that includes collagen and chondroitin sulfate, TNFα induction, following stimulation of leukocytes by collagen and chondroitin sulfate was compared to that induced by shark cartilage treatment. HPBL cultures were set up as described previously and stimulated with SCAE (0.360 mg protein/ml), collagen (300 mg/ml) and chondroitin sulfate (100 mg/ml). A 1:2 dilution of each stimulant was also tested. Cultures were incubated for 24 h at 37 °C, 5% CO2. Culture supernatants were harvested and assayed for TNFα. 2.6.1.2. Miscellaneous cytokine induction. Cytokines IFNγ, IL-1β, IL-4 and IL-8 were assayed in HPBL cultures stimulated with SCAE (0.365 mg/ml) at 37 °C, 5% CO2. Culture supernatants were harvested and assayed for cytokine at various time intervals ranging from 0 h up to 96 h. In case of IFNγ induction was followed up to six days. Positive control cultures were stimulated with appropriate mitogens: LPS (5 μg/ml) for IL-1β induction, PHA (5 μg/ml) for IFNγ, Con A (5 μg/ml), a T cell mitogen, for IL-4 and all three mitogens individually for IL8 induction. Furthermore, to determine whether cartilage extract
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could affect the response of HPBL to PHA, cultures were also stimulated with a combination of both PHA (5 μg/ml) and SCAE (0.350 mg/ml) and culture supernatants were harvested and assayed for IFNγ daily for 6 days. Unstimulated cell cultures consisted of medium alone. To rule out the possibility that the extracts contained material that influenced and was reactive in the cytokine assays, SCAE alone and in medium was assayed for cytokine. 2.6.2. Protease susceptibility Because shark cartilage is a dietary supplement, SC extracts were treated with digestive proteases. Acid extracts of shark and bovine cartilage, dialyzed against 0.01 M Tris–HCl buffer, pH 7.5, were subjected to proteolysis using a combination of trypsin and chymotrypsin. Enzymes (2 mg each of trypsin and chymotrypsin) were added directly to 10 ml of dialyzed extract and the mixture incubated for 3 h at 37 °C. Digestion was stopped by the addition of 4 mg of trypsin– chymotrypsin inhibitor directly to the mixture. The digested extracts were dialyzed against RPMI-1640 culture medium and filtered before use in bioassays. Each digest was tested for endotoxin. 2.7. Statistical analyses Each experiment was repeated at least once for each treatment. Cultures were set up in duplicate and cytokine assays on culture supernatants were performed in triplicate. Standard errors above the mean were calculated and reported for each experiment for each treatment. Statistical analysis was performed by the Student's t-test, comparing induction level of cytokine in the treated culture with that of cultured cells in medium alone, and each treatment was compared to the positive control for each experiment. A p-value of b0.05 was considered statistically significant.
3.2.1. Stimulation of TNFα production Fig. 1 shows that of all the various extracts prepared (AE, SS and PBS) from each of three brands of shark cartilage, the acid extract (SCAE) was the most effective inducer of TNFα, yielding high levels of TNFα. This was true for acid extracts prepared from all three commercial brands of shark cartilage. When acid extract of three commercial brands of shark cartilage were compared to each other for cytokine-inducing activity, acid extract of shark cartilage from Solgar Laboratories induced higher level of cytokine than that of Twin Laboratories, but not significantly different from FDC acid extract. The AE, SS, and PBS extracts prepared from Twin Laboratories cartilage induced the least amount of TNFα. Similarly, when the cytokine-inducing properties of AE, SS, and PBS extracts of shark (Solgar) and bovine cartilage were compared (Fig. 2), the AE extracts from both sources induced significant levels of TNFα. When the level of TNFα produced was correlated to mg extract protein, the shark extracts produced 3–5 fold higher levels of TNFα than bovine extracts. Comparing the three bovine extracts, the SS extract induced the highest level of TNFα. The alkaline (AA) extract of shark cartilage induced TNFα production albeit to a lesser degree than SCAE, and the organic extract (SCOE) did not induce detectable level of TNFα over 72 h (results not shown). When HPBL were cultured in the presence of shark cartilage acid extracts (Solgar SCAE) significant amounts of TNFα were produced in vitro comparable to that induced by LPS, a strong inducer of TNFα (Fig. 3). In serum a level of b20 pg/ml of TNFα is considered normal [24]. No cytokine was detected in unstimulated cultures containing medium alone. TNFα could be detected in culture supernatants as early as 4 h. By 48 h detectable levels were low. A time-course study of TNFα release revealed that peak levels of TNFα were observed at 8 and 24 h
3. Results 3.1. Test for detectable endotoxin All cartilage extracts were determined to be free of detectable endotoxin by the E-toxate test. Extracts were also tested and found to be free of inhibitors for the E-toxate test (results not shown). 3.2. Cytokine-inducing activity of cartilage extracts Results for all cytokine and chemokine assays are expressed for each treatment as the mean of three replicate assays of duplicate cultures for two experiments. The p-values for each assay are provided in the figure captions and ranged from p b 0.001 to p b 0.01. All experiments were carried out for multiple time intervals and where significant cytokine induction occurred is reported for each cytokine. Extracts used as stimulants were determined to be free of inhibitory/interfering material for the cytokine ELISAs (results not shown).
Fig. 1. Cytokine response of cultured human leukocytes stimulated with different extract preparations from three commercial sources of shark cartilage. The production of TNFα by HPBL cultures stimulated for 24 h with SCAE, SCSS and SCPBS extracts prepared from three different brands of commercial shark cartilage was measured. To make reliable comparisons between brands and preparations, the level of TNFα in culture supernatants was related to mg extract protein. Error bars represent standard error above the mean. The level of TNFα induced in cultures stimulated with extracts was significantly higher (⁎) than that induced in cultures treated with medium alone ( p b 0.005).
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Fig. 2. TNFα response of human leukocytes stimulated by three different extracts prepared from shark (Solgar) and bovine cartilage (BC). Cultures were stimulated with acid (AE), salt soluble (SS) and phosphate buffered saline (PBS) extracts for 24 h. Protein concentration of extracts were SCAE 0.360 mg protein/ml, BC-AE 0.930 mg protein/ml; SC-SS 0.400 mg protein/ml, BC-SS 0.960 mg protein/ml; and SC-PBS 0.520 mg protein/ ml, BC-PBS 1.16 mg protein/ml. Culture medium alone and LPS (5 μg/ ml) served as control stimulants. Error bars represent standard error above the mean. The level of TNFα induced in cultures treated with SC extracts was significantly higher (⁎) than that induced in cultures treated with BC extracts or medium alone ( p b 0.01).
(Fig. 3). A similar pattern of cytokine release was produced with LPS over a 48 h period. The cytokine-inducing ability of SCAE (Solgar) was also compared to that of collagen and chondroitin sulfate, two components of cartilage. No cytokine induction was noted in response to stimulation with chondroitin sulphate (Fig. 4). Collagen did induce cells to produce TNFα, however, the level of cytokine produced was significantly less than that induced by SCAE. The level of TNFα produced appeared to be related to the concentration of the stimulant, suggesting a dose-related response.
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Fig. 4. TNFα response of leukocytes to stimulation by SCAE, collagen and chondroitin sulfate. The level of TNFα produced was measured at 24 h following stimulation. Culture medium and LPS were control stimulants. Error bars represent standard error above the mean. SCAE and LPS induced significantly higher levels (⁎) of TNFα than did treatment with medium alone ( p b 0.005). Collagen and chondroitin sulfate did not induce significant levels of TNFα except for collagen at high concentration, 300 mg/ml ( p b 0.05).
to SCAE by the 3rd day of culture, whereas PHA (a potent mitogen) induced comparable level of IFNγ by the 2nd day. However, when SCAE and PHA were combined to stimulate HPBL cultures simultaneously, there was a significant increase in the level of IFNγ produced. Furthermore, this increased activity was reached by the second day. Results indicate that SCAE enhanced the PHA response by inducing a higher level of IFNγ. The normal serum range of IFNγ is b 20 pg/ml [25].
3.2.2. Stimulation of IFNγ production HPBL cultured with SCAE produced significant amounts of IFNγ in vitro (Fig. 5). Most IFNγ was produced in response
Fig. 3. Time-course of TNFα induction in leukocytes stimulated with shark cartilage (Solgar). HPBL cultures were stimulated with LPS (5 μg/ml), SCAE (0.450 mg protein/ml) or culture medium alone at 37 °C, 5% CO2. Error bars represent standard error above the mean. SCAE and LPS induced significantly high levels (⁎) of TNFα compared to treatment with medium alone ( p b 0.005).
Fig. 5. Induction of IFNγ in HPBL cultures stimulated with PHA, SCAE (Solgar), and a combination of both PHA and SCAE. Cultures were stimulated with PHA (5 μg/ml), SCAE (0.367 mg protein/ml) and a combination of PHA and SCAE. Unstimulated cultures were grown in culture medium alone. Error bars represent standard error above the mean. SCAE induced significantly higher level (⁎) of IFNγ at 72 h when compared to cultures treated with medium alone ( p b 0.008). PHA induced a significantly higher level of IFNγ at 48 h, while the combination of SCAE and PHA induced higher levels of IFNγ at bother 48 and 72 h when compared to cultures treated with medium alone ( pb 0.008).
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Fig. 6. IL-1β production by leukocytes stimulated with SCAE (Solgar). HPBL were stimulated with SCAE (0.396 mg protein/ml), LPS (5 μg/ml) or culture medium at 37 °C. Error bars represent standard error above the mean. The level of IL-1β induced by SCAE was significantly high (⁎) at 12 and 24 h, while LPS induced significant levels of cytokine over 96 h ( p b 0.001).
3.2.3. Stimulation of IL-1β production The production of IL-1β in response to cartilage extracts was examined (Fig. 6). Results showed that SCAE (Solgar) induced IL-1β early in culture, reaching peak level by 12 h and declining to insignificant level by 72 h. The IL-1β response to LPS, however, was maintained at a significantly high level through 72 h showing a significant decline only at 96 h.
Fig. 8. Induction of IL-8 in leukocytes. HPBL cultures were stimulated with SCAE (0.396 mg protein/ml), LPS (5 μg/ml), PHA (5 μg/ml), Con A (5 μg/ml) or culture medium. Error bars represent standard error above the mean. SCAE and LPS induced significantly higher levels (⁎) of IL-8 than medium alone at 24, 48 and 72 h ( p b 0.001).
3.2.5. Stimulation of IL-8 production The release of IL-8 in response to cartilage stimulation was compared to chemokine induction following stimulation of cultures by mitogens, such as LPS, PHA and Con A (Fig. 8). Significant levels of IL-8 were induced by SCAE within 24 h and levels were sustained over a period of 72 h. The production of IL-8 in response to LPS however, reached peak levels at 48 h and by 72 h showed a gradual decline. Physiologically significant level of IL-8 was not produced by cultures stimulated with culture medium alone, PHA, or Con A.
3.2.4. Stimulation of IL-4 production When HPBL were cultured in the presence of shark cartilage extract (Solgar), there was no physiologically significant production of IL-4 over 48 h (Fig. 7), a response similar to that obtained with unstimulated cultures (medium alone). Positive control cultures stimulated with Con A, secreted a significant level (40 pg/ml) of IL-4.
The effect of proteolytic digestion on the TNFα-inducing ability of shark and bovine cartilage was examined by using
Fig. 7. IL-4 response of leukocytes stimulated with SCAE (Solgar). HPBL cultures were stimulated with Con A (5 μg/ml), or SCAE (0.360 mg protein/ml) or culture medium. Error bars represent standard error above the mean. SCAE did not induce levels of IL-4 greater than that induced in cultures treated with medium alone, while Con A induced significantly higher levels (⁎) of IL-4 than unstimulated (medium alone) cultures ( p b 0.005).
Fig. 9. The effect of proteolytic digestion on TNFα induction. Shark (Solgar) and bovine cartilage extracts were treated with trypsin and chymotrypsin and used to stimulate HPBL cultures for 24 h. Error bars represent standard error above the mean. In HPBL cultures stimulated with protease-treated extracts, significantly less (⁎) TNFα was produced than in cultures stimulated with untreated extracts derived from both shark and bovine cartilage ( p b 0.005).
3.3. Susceptibility to proteolytic digestion
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extracts treated with trypsin and chymotrypsin (digestive enzymes) prior to stimulating leukocyte cultures (Fig. 9). Following treatment, SCAE (Solgar) lost 80% activity suggesting a protein moiety is associated with the cytokine-inducing activity. A similar effect was noted with bovine cartilage extract.
4. Discussion The results of this study provide evidence that cartilage extracts induce the production of several different cytokines and/or chemokines by human peripheral blood leukocytes. The most well-characterized cytokine response of HPBL to cartilage stimulation, thus far, is TNFα production. When different commercial brands of shark cartilage were tested for their ability to induce TNFα, there was a significant variation in the level of cytokine induced, although each brand induced a detectable level of TNFα. When extracts were prepared from the amount of commercial shark cartilage equivalent to the manufacturer's recommended daily dose (i.e., approximately a three-fold increase in starting material), a 20% increase in the level of TNFα was noted compared to the level induced with extract prepared from 2 g of SC (data not shown). For all brands of cartilage tested, the most cytokineinducing activity was associated with the acid extracts of cartilage. Acid extracts simulate the acidic environment of the stomach. For a dietary supplement to be biologically effective at the time of absorption it must be acid resistant. Considering that the acid extract of shark cartilage was the most effective inducer of a cytokine response, the acidity of the stomach may very well play a role in the in vivo release of the active component(s) from crude cartilage preparations taken orally. Manufacturers are reticent to provide information on the source of the original shark material such as geographic location, species identity, age/sex of animal, tissue from which material was derived, and/or the process used to obtain material. These unknown differences most likely result in the variation in cytokine response seen with different commercial sources of shark cartilage. While both shark cartilage and bovine cartilage extracts induced the production of TNFα, shark cartilage induced significantly higher levels of TNFα, with the acid extract inducing highest levels, of all extracts tested. For bovine cartilage, the salt-soluble extract induced a higher level of TNFα than the acid extract. Taken together, the results suggest that although both types of cartilage contain cytokine-inducing activity, the active component(s) present in each might not be similar. Alternatively, the difference might reflect the nature of the starting material used to prepare the extracts.
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Both shark and bovine cartilage extracts, when treated with proteases, trypsin and chymotrypsin, lost considerable ability to stimulate TNFα production. Results suggest that the active component(s) in cartilage is proteinaceous. Since not all activity was abolished following enzyme treatment, this leads us to speculate that the extracts contain an additional active component(s) which is resistant to the proteolytic activity of trypsin and chymotrypsin. Given the complexity of the gut environment, the effect of proteases may not be as profound as that seen in vitro, and residual activity may be sufficient to stimulate the epithelial lining of the gut to produce a variety of immune messengers which in turn could have a systemic effect given the rich blood supply to the gut. When speculating on in vivo conditions, the presence of microbial enzymes must also be considered which could be a factor in determining the potential effectiveness of shark cartilage as a dietary supplement. Cartilage is a complex material composed of several proteinaceous components, including collagen and chondroitin sulphate. Chondroitin sulfate is least likely to be responsible for the cytokine-inducing effect of cartilage. Collagen alone was able to induce a cytokine response but not at the level seen with SCAE. It is unlikely that collagen present in commercial preparations of shark cartilage contributes significantly to the level of in vitro stimulation noted considering the large amount of collagen required (0.36 mg/ml SCAE versus 300 mg/ml collagen). An important aspect of this investigation was the time-course study of the induction of cytokines in response to cartilage stimulation, specifically TNFα. The biphasic response to SCAE stimulation may reflect TNFα produced by two or more different cell populations given that the cytokine is a pleiotropic cytokine [26]. Macrophages might be the cell type to initially respond to direct stimulation by cartilage extract [27], while other cell types (e.g., lymphocytes) account for peak activity noted at 24 h, which may be an indirect effect in response to additional cytokine messengers released by the cell type initially responding to cartilage stimulation [28]. Alternatively, the decrease in level of TNFα detected in supernatants at 12 h may not represent a decline in secretion but rather a binding of the free cytokine by up-regulated cytokine receptors on activated cells or by soluble TNFα receptors released into culture supernatants by responding cells. Shark cartilage extract induced the production of IFNγ at levels comparable to that obtained in positive control cultures stimulated with PHA, a known mitogen. Shark cartilage extract appeared to behave like a mitogen in stimulating the production of IFNγ after 72 h.
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When cultures were stimulated with PHA alone, IFNγ level peaked at 48 h. However, when PHA and SCAE were combined as stimulants, the cytokine response was enhanced with a significantly higher level (almost 800 pg/ml) of IFNγ detected at 48 h. These results suggest that an active component(s) in shark cartilage behaves like a mitogen stimulating leukocytes to produce potent mediators, and its presence appears to enhance the activity of PHA. An alternative interpretation might be the reverse, that is, that PHA induces an earlier and enhanced response to SCAE. What is undetermined is whether the enhanced response is due to one or more cell type(s) responding. The induction of proinflammatory cytokines, TNFα, IFNγ, and IL-1β suggests that cartilage preferentially induces Th1 type cytokines. The production of IL-4 (b5 pg/ml) was considered to be physiologically insignificant (normal serum range b30 pg/ml). In contrast, the chemotactic chemokine, IL-8, was produced by stimulated leukocytes at higher (20,000 pg/ml) than normal levels encountered in serum (0–10 pg/ml). Establishing “normal ranges” for cytokine serum levels derived from immune assays of serum/plasma of healthy individuals can be misleading, since recognized variation exists in cytokine levels in normal individuals to the extent that cytokine levels can often be undetectable by ELISA. It is, therefore, essential to include normal controls in each individual experiment to take into account possible variation [29]. The cytokine induction profile for shark cartilage, therefore, includes three potent Th1 type cytokines, TNFα, IFNγ and IL-1β, two of which are characteristically involved in inflammatory responses and an inflammatory chemokine, IL-8. Shark cartilage did not induce a significant level of IL-4, thus it not only preferentially stimulates a Th1 type response but appears to indirectly inhibit the development of a Th2 response through the action of IFNγ which is an inhibitor of Th2 cell population expansion [30,31]. From the cytokine profile induced by shark cartilage one can conclude that activation, proliferation, and differentiation of B cells (i.e., a humoral immune response) is not an integral part of the immune response to SCAE stimulation since IL-4, a key cytokine in the activation of B cells, was not induced to physiologically significant levels by SCAE [32]. Based on the differences in peak production of cytokines over time we can speculate on the sequence of cellular events. An early event is most likely to be monocyte/macrophage activation by SCAE inducing the autocrine and paracrine effects of TNFα, followed by Th cell activation, preferably Th1, in response to TNFα and IL-1β co-stimulation with IFNγ inhibiting proliferation of Th2 cells.
We have shown that cartilage induces various inflammatory cytokines and a potent chemokine in immune cells. Thus, if through intestinal absorption the active component(s) in shark cartilage were to reach systemic circulation and/or target sites in the body, immune regulation could be significantly influenced. Consequently, the inflammatory cytokine profile induced by cartilage might be deleterious to the health of individuals suffering from chronic diseases where the underlying pathology is associated with inflammation and where up regulation of Th1 type response is undesirable. For such individuals shark cartilage, taken as an unregulated dietary supplement, is counter indicated. Alternatively, this type of immune response, in which IL-4 production is restricted, could be beneficial for hypersensitive individuals whose allergic state is often associated with production of IgE. In the absence of Th2 stimulation, the IgE response would be down regulated to the benefit of the individual. Acknowledgments We thank Barbara Anderson for performing phlebotomy, Betzabel Gonzalez for her technical contributions, and John Makemson and Charles Bigger for their comments on the manuscript. LM was supported by an MBRS student fellowship and this study was supported in part by a summer research award to LM under the Comparative Immunology Initiative (R25 GM061347). Facilities for the project were provided by FIU Comparative Immunology Institute and this is contribution FIU-CII-003 from the Institute. The authors received no funding from and have no association with the manufacturers and/or distributors of the commercial brands of shark cartilage used in this study. References [1] Eisenberg DM, Davis RB, Ettner SL, Appel S, Wilkey S, et al. Trends in alternative medicine use in the United States, 1990–1997 — results of a follow-up national survey. JAMA 1998;280(18): 1569–75. [2] Kennedy J. Herb and supplement use in the US adult population. Clin Ther 2005;27(11):1847–58. [3] Tindle HA, Davis RB, Phillips RS, Eisenberg DM. Trends in use of complementary and alternative medicine by US adults: 1997– 2002. Altern Ther Health Med 2005;11(1):42–9. [4] Oneschuk D, Fennell L, Hanson J, Bruera E. The use of complementary medications by cancer patients attending an outpatient pain and symptom clinic. J Palliat Care 1998;14(4):21–6. [5] Suarez-Almazor M, Kendall CJ, Dorgan M. Surfing the NetInformation on the World Wide Web for persons with arthritis: patient empowerment or patient deceit? J Rheumatol 2001;28 (1):185–91.
L. Merly et al. / International Immunopharmacology 7 (2007) 383–391 [6] Brem H, Folkman J. Inhibition of tumor angiogenesis mediated by cartilage. J Exp Med 1975;141:427–39. [7] Lee A, Langer R. Shark cartilage contains inhibitors of tumor angiogenesis. Science 1983;221:1185–7. [8] Oikawa T, Ashino-Fuse H, Shimamura M, Koide U, Iwaguchi T. A novel angiogenic inhibitor derived from Japanese shark cartilage (I). Extraction and estimation of inhibitory activities toward tumor and embryonic angiogenesis. Cancer Lett 1990;51(3):181–6. [9] Gingras D, Renaud A, Mousseau N, Beliveau R. Shark cartilage extracts as antiangiogenic agents: smart drinks or bitter pills? Cancer Metastasis Rev 2000;19:83–6. [10] Gonzalez RP, Leyva A, Moraes MO. Shark cartilage as source of antiangiogenic compounds: from basic to clinical research. Biol Pharm Bull 2001;24(10):1097–101. [11] Loprinzi CL, Levitt R, Barton DL, Sloan JA, Atherton PJ, et al. Evaluation of Shark Cartilage in patients with advanced cancer: A North Central Cancer Treatment Group Trial. Wiley Interscience; 2005. Published online. [12] Felzenszwalb I, Peleilo de Mattos JC, Bernardo-Filho M, Caldeira-de-Araujo A. Shark cartilage-containing preparation: protection against reactive oxygen species. Food Chem Toxicol 1998;36:1079–84. [13] Conde CMS, Albano F, Bouskela E, Felzenszwalb I, Svensjo E. Inhibition of ischemia/reperfusion induced plasma leakage by alpha-tocopherol, trolox, and a shark cartilage preparation with anti-oxidant properties. Nutr Res 2001;21:1363–71. [14] Chen JS, Chang CM, Wu JC, Wang SM. Shark cartilage extract interferes with cell adhesion and induces reorganization of focal adhesions in cultured endothelial cells. J Cell Biochem 2000;78: 417–28. [15] Ratel D, Glazier G, Provencal M, Boivin D, Beaulieu E, et al. Direct-acting fibrinolytic enzymes in shark cartilage extract: potential therapeutic role in vascular disorders. Thromb Res 2005;115(1–2):143–52. [16] Feyzi R, Hassan Z, Mostafaie A. Modulation of CD4+ and CD8+ tumor infiltrating lymphocytes by a fraction isolated from shark cartilage: shark cartilage modulates anti-tumor immunity. Int Immunopharmacol 2003;3:921–6. [17] Kralovec J, Guan Y, Metera K, Carr RI. Immunomodulating principles from shark cartilage Part I. Isolation and biological assessment in vitro. Int Immunopharmacol 2003;3:657–69. [18] Hassan Z, Feyzi R, Sheikhian A, Bargahi A, Mostafaie A, et al. Low molecular weight fraction of shark cartilage can modulate
[19] [20] [21]
[22]
[23]
[24]
[25]
[26] [27] [28]
[29]
[30]
[31]
[32]
391
immune responses and abolish angiogenesis. Int Immunopharmacol 2005;5(6):961–70. Romagnani S. Immunologic influences on allergy and the Th1/ Th2 balance. J Allergy Clin Immunol 2004;113(3):395–400. Muraille E, Leo O. Revisiting the Th1/Th2 paradigm. Scand J Immunol 1998;47(1):1–9. Kidd P. Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev 2003;8(3): 223–46. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76–85. Nandan R, Brown DR. An improved in vitro pyrogen test: to detect picograms of endotoxin contamination in intravenous fluids using limulus amoebocyte lysate. J Lab Clin Med 1977;89 (4):910–8. Aggarwal BB, Reddy S. Tumor necrosis factor. In: Nicola NA, editor. Guidebook to cytokines and their receptors. A Sambrook and Tooze Publication of Oxford University Press; 1994. p. 103–4. Gray PW. Interferon Gamma. In: Nicola NA, editor. Guidebook to cytokines and their receptors. A Sambrook and Tooze Publication of Oxford University Press; 1994. p. 118–9. Tracey KJ, Cerami A. Tumor necrosis factor: a pleiotropic cytokine and therapeutic target. Annu Rev Med 1994;45:491–503. Mosser DM. The many faces of macrophage activation. J Leukoc Biol 2003;73(2):209–12. Wang J, Wakeham J, Harkness R, Xing Z. Macrophages are a significant source of type 1 cytokines during mycobacterial infection. J Clin Invest 1999;103(7):1023–9. Whiteside TL. Cytokine measurements and interpretation of cytokine assays in human disease. J Clin Immunol 1994;14(6): 327–39. Billiau A, Heremaus H, Vermeire K, Matthys P. Immunomodulatory properties of interferon gamma: an update. Ann NY Acad Sci 1998;856:22–32. Maggi E, Parronchi P, Manetti R, Simonelli C, Piccinni MP, et al. Reciprocal regulatory effects of IFN-gamma and IL-4 on the in vitro development of human Th1 and Th2 clones. J Immunol 1992;148(7):2142–7. Lund FE, Garvey BA, Randall TD, Harris DP. Regulatory roles for cytokine-producing B cells in infection and autoimmune disease. Curr Dir Autoimmun 2005;8:25–54.