http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, Early Online: 1–8 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2014.999160
RESEARCH ARTICLE
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Pro-inflammatory properties of shark cartilage supplement Liza Merly* and Sylvia L. Smith Department of Biological Sciences, Florida International University, Miami, FL, USA
Abstract
Keywords
The erosion and breakdown of cartilage is generally recognized to be an integral manifestation of arthritic disease, which is often accompanied by the development and progression of inflammation associated with it. Commercial shark cartilage (SC) is a popular dietary supplement taken for the prevention and/or control of chronic disease, including arthritis. The efficacy of SC in maintaining joint health remains questionable; there is a lack of sufficient reliable information on its effect on immunocompetent cells, and the potential health risks involved have not been adequately assessed. Our earlier in vitro studies showed that SC extracts induce a Th1-type inflammatory cytokine response in human leucocytes, and collagen type II alpha 1 protein was shown to be an active cytokine-inducing component in SC. In this study, we further define the cellular response to SC stimulation by classifying leucocytes into primary and secondary responders employing enriched leucocyte subpopulations. Inhibitors of specific signaling pathways were used to verify the functional effect of SC on specific pathway(s) utilized. Results indicate the monocyte/macrophage as the initially responding cell, followed by lymphocytes and the production of interferon-g. Chemokines, MCP-1 and RANTES, were produced at significant levels in stimulated leucocyte cultures. Initial cellular activation is likely followed by activation of Jun Kinase and p38 mitogen-activated protein kinase signal transduction pathways. This study presents evidence of significant immunological reactivity of components of commercial SC supplement, which could pose a potential health risk for consumers, particularly those with underlying inflammatory disease such as irritable bowel syndrome and arthritis.
Immunomodulation, innate immunity, leucocyte response, pro-inflammatory cytokines, shark cartilage
Introduction Cytokines are involved in the development and progression of inflammation associated with chronic arthritic disease1. The cause of the initial lesion and factors involved in the initiation of an inflammatory response remains a matter of discussion2. However, the erosion and breakdown of cartilage is generally recognized to be an integral manifestation. Cartilage is mostly composed of a single cell type, the chondrocyte, and an extracellular matrix of collagen, proteoglycans and water. Fragments of glucosaminoglycans of proteoglycans when released from the ECM following injury to bones and joints can act as innate immune ‘‘danger’’ signals indicative of tissue injury/trauma/distress and induce inflammatory responses3–5. Thus, it appears that components of cartilage ECM play a role in maintaining the integrity of healthy tissue through surveillance by initiating innate immune responses
*Present address: Department of Marine Biology and Ecology, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149, USA Address for correspondence: Sylvia L. Smith, PhD, Department of Biological Sciences, OE 243, Florida International University, 11200 SW 8th St, Miami, FL 33199, USA. Tel: 305-348-3183 or 305-858-5485. Fax: 305-858-2151. E-mail: [email protected]
History Received 28 August 2014 Revised 1 December 2014 Accepted 12 December 2014 Published online 20 January 2015
following tissue stress. Consequently, it is reasonable to consider that cartilage-derived factors might represent a group of molecules that are able to activate antigen-presenting cells, such as tissue macrophages, leading to the modulation of overall cellular activity in such microenvironments resulting in disruption of normal physiology at localized and/or systemic sites6,7. Recently, an immune-active component was identified in a commercial preparation of shark cartilage (SC) as collagen type II, alpha 1 protein, which was shown to stimulate human leukocytes to produce significant levels of tumor necrosis factor (TNF)-a in culture8. Collagen type II, alpha 1 protein, purified from fetal bovine articular cartilage, has been experimentally used to induce arthritis in a mouse rheumatoid arthritis (RA) model referred to as collageninduced arthritis9. Commercial, over-the-counter products, such as chondroitin sulfate, glucosamine and cartilage, from a variety of sources, are taken by members of the public as therapeutic and/or preventative measures, and their use in the practice of complementary alternative medicine has increased significantly over the past decade10,11. SC, a dietary supplement, is one such product. To date, in vivo and in vitro studies on the biological and pharmacological properties of SC mostly focus on its antiangiogenic properties and effectiveness as an anticancer drug12–18. Most studies investigating its role as a
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modulator of immune function use laboratory-purified SC, which does not represent the potential bioactivity of commercial SC, which is the focus of this study. Immunomodulators often act through the induction of specific patterns of growth factors, cytokines and chemokines which contribute significantly to regulating and controlling immune responses19,20. Cellular responses include a cascade of intracellular signaling events, often involving members of the kinase family of proteins that lead to the production of immune mediators21. Merly et al.22 showed that SC extracts induce, preferentially, a Th1-type inflammatory cytokine response in human leukocytes and suggested that the cytokine pattern was the cumulative effect of several successive cellular responses of different leucocyte populations involving inter-cell communication through cytokines and chemokines. The question remained as to the role played by different leucocyte subpopulations in the Th-1 cytokine response and which signal transduction networks might be involved. The aim in this study was to test the hypothesis that the Th-1 cytokine response, consisting of an upregulation of TNF-a, interleukin (IL)-1b, IL-6 and IL-8, was the functional effect of distinct leucocyte types responding in a differential manner to SC stimulation. The objective was to stimulate leucocyte subpopulations and, using the production of TNF-a as an indicator of cytokine induction, to determine which cell type(s) was initially responding to SC and which leucocyte subpopulation was a secondary responder that produced cytokine in response to mediators released by primary responders and not the result of direct stimulation by immunomodulating factor(s) in SC extract. A simple fourstep cell separation protocol was employed to obtain highly enriched leucocyte subpopulations from peripheral blood leukocytes. SC-stimulated leukocytes were also examined for production of MCP-1 and RANTES, which have been shown to play a role in the development of arthritic lesions23,24. To further our understanding of the signaling networks that are most likely activated following SC stimulation, specific inhibitors of signaling pathways were used to determine the effect of inhibition on subsequent cytokine response.
Methods Cartilage extracts An acid extract of shark cartilage (SCE; 0.575 mg protein/ml) was prepared from commercial SC capsules (Solgar Vitamin and Herb, Leonia, NJ) of a single lot (Lot #47580)22. SCE was further subjected to solid-phase extraction (SPE)8 on a prepacked SPE C18 column (Waters, Sep-Pak 6 g 1 cc, Miliford, MA). Bound material was eluted with 50% acetonitrile with 0.1% TFA, and acetonitrile was removed by vacufugation. The remaining solid extract (SC-SPE) was re-suspended in culture medium (CM) consisting of RPMI-1640 supplemented with 0.3 mg/ml glutamine and 25 mM HEPES, 100 mg/ml of streptomycin and 100 U/ml of penicillin. All extracts were assayed for endotoxin using the E-Toxate assay (Limulus polyphemus amoebocyte lysate assay, Sigma catalog #210-500, St. Louis, MO), and only endotoxin-free extracts were used to stimulate cells. The detection limit of the endotoxin assay was 0.015 EU/ml.
Immunopharmacol Immunotoxicol, Early Online: 1–8
Leucocyte subpopulations Employing a combination of dextran sedimentation, Ficoll Paque separation and adherence to plastic highly enriched subpopulations of leucocytes were isolated from peripheral blood. Peripheral blood leucocytes (PBL) were separated from heparinized blood from healthy donors by dextran sedimentation22. An aliquot of the same blood sample used to isolate PBL was used to prepare mononuclear cell-enriched leucocyte populations (peripheral blood mononuclear cell; PBMC) by density-gradient centrifugation. Blood was diluted 1:1 with sterile, physiological saline and layered onto MonoPoly Ficoll-Paque Resolving medium (Sigma, Cat#171440-02). The mixture was centrifuged immediately at 250 g for 30 min and the buffy layer containing the mononuclear cell-enriched fraction was aspirated, washed several times in phosphate-buffered saline and suspended in serum-free CM. Further separation of mononuclear leucocytes into sub-populations was achieved by adding 1 ml of PBMC suspension (1 106cells/ml) per well of a 24-well culture plate and incubated in complete culture medium (CCM) consisting of RPMI-1640 medium supplemented with 10% fetal bovine serum and incubated for 2 h at 37 C in 5% CO2 with high humidity. The monocyte/macrophage-enriched (MME) subpopulation (approximately 5 105 cells) adhered to the bottom of wells, whereas non-adherent lymphocyteenriched (LE) cell population was harvested and standardized to 2.5 105cells/ml. CCM was added to the remaining adherent monocyte/macrophage (MME) sub-population and cultures stimulated as described below. The LE were re-plated on 24-well plates by adding 200 ml of the standardized suspension to each well and stimulated. For each leucocyte population, cytospin monolayers were examined to confirm the homogeneity of leucocyte type(s) in each subpopulation. Final cell suspensions were prepared in serum-free culture medium and standardized to desired concentrations. Cell viability was maintained at 495% and was required for subsequent cell culture (Mosmann, 1983; Strober, 2001)25,26. A differential count was performed on PBL to determine the proportion of leucocyte types present. Induction of cytokine and chemokine response in leucocytes Concurrent leucocyte cultures of PBL, PBMC, MME and LE cells, similarly treated, were set up in triplicate in 24-well flat-bottom tissue culture plates. Each culture contained 50 ml of cartilage extract or control stimulant, 100 ml of CM and 200 ml of a standardized cell suspension (2.0–2.5 105 cells/ ml) except for MME cultures, which contained approximately 3–5 105 adherent cells per well. PBMC, MME and LE cultures were stimulated with SC-SPE (1.5 mg protein/ml), lipopolysaccharide (LPS; 5 mg/ml) or CM for 24 h. Previous studies had shown that collagen type II, alpha 1 protein is the component in SC-SPE that induces TNF-a in stimulated leucocytes (Merly and Smith, 2013). LPS (Escherichia coli, Difco Laboratories) served as the positive-control stimulant. Unstimulated cultures contained CM in lieu of stimulant. Cultures were incubated in a humid chamber at 37 C in 5% CO2. Culture supernatants were harvested at 24 h from the four sets of leucocyte subpopulations and assayed for TNF-a.
DOI: 10.3109/08923973.2014.999160
Table 1. Protein kinase inhibitors of specific signaling pathways. Inhibitory molecule
Signaling pathway
BI 78D3 Wortmannin Rottlerin SB202190
C-Jun N-terminal kinase (JNK) Phosphatidylinositol 3-kinase (PI-3) Protein kinase C (PKC) p38 Mitogen-activated protein kinase (p38 MAPK) Raf/MEK/ERK2 MAPK kinase
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GW 5074
To determine production of interferon (IFN)-g, LE cultures were stimulated with SC-SPE, LPS, PHA or medium and supernatants collected at 72 h and assayed for IFN-g. Release of RANTES and MCP-1 by PBMC was measured following SCE stimulation by assaying PBMC culture supernatants, collected at 4–24 h intervals for 96 h, for chemokines. Since it was possible that a component of cartilage other than collagen type II, alpha 1 protein (the cytokine-inducing component of SC-SPE) could induce leucocytes to produce RANTES and MCP-1, PBMC were stimulated with SCE in lieu of SC-SPE. Cultures stimulated with LPS or CM served as controls. Harvested supernatants representing 0 h were estimated to be samples collected within 5 min immediately following culture set-up. Analysis of intracellular signaling A series of inhibitors of specific signaling pathways were used to verify the functional effect of SC and to validate the utilization of a specific pathway (Table 1). For each pathway studied, two concentrations of each inhibitor were used in the pre-treatment of leucocytes (PBL) prior to stimulation. Triplicate cultures were established in a 24-well tissue culture plate by adding 200 ml of cell suspension (2.5 105 cells/ml) to each test well. An additional 100 ml of CM was added to each well. Cell cultures were pre-incubated with 50 ml of either a specific inhibitor or a DMSO/PBS vehicle control for 60 m. The two negative control cultures consisted of: preincubation with CM alone and pre-incubation with DMSO/ PBS vehicle with no inhibitor. Following pre-treatment, leucocytes were stimulated with 50 ml of SC-SPE, LPS (5 mg/ml) or medium alone and incubated for 24 h. All cultures were incubated in a humid chamber at 37 C in 5% CO2. Culture supernatants were harvested at 24 h and assayed for TNF-a to determine the effect of kinase inhibition on TNF-a production. The concentrations of each test kinase inhibitor used for specific signaling pathways are listed in Table 1. Statistical analysis Leucocyte cultures were set up in duplicate and cytokine/ chemokine assays performed in triplicate. Experiments for each treatment were repeated at least twice. The student t test and ANOVA was used to identify significant differences between untreated and treated cell cultures. A p value of less than 0.05 was considered statistically significant.
Results PBLs represent a heterogeneous population of cells that play a significant role in orchestrating and coordinating host
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immune response (Figure 1a). Using a combination of separation methods, highly enriched leucocyte sub-populations were isolated from PBLs: the mononuclear cell-enriched fraction (PBMC) composed of monocytes and lymphocytes (Figure 1b), which upon further fractionation yielded the MME subpopulation (Figure 1c) and the LE subpopulation (Figure 1d). Cytospin monolayers confirmed the relative homogeneity of leucocytes in each subpopulation where other leukocyte types were either absent or accounted for less than 3% of preparations (triplicate counts of 200 cells/cytospin performed). Cytokine and chemokine response Cultures of PBL, PBMC, MME and LE were concurrently stimulated with SCE or a solid-phase extract of SCE (SCSPE), LPS (positive control) or CM (unstimulated control). Supernatants were assayed for TNF-a, MCP-1 or RANTES. PBL and MME cultures produced significant levels of TNF-a in response to both LPS and SC-SPE stimulation (Figure 2a and b). The level of TNF-a was considerably higher in MME cultures than that in PBL cultures, which was not unexpected since the relative numbers of monocytes/macrophages contained in PBL cultures was proportionately less. The TNF-a response of LE cultures to SC-SPE was insignificant, whereas the response to LPS stimulation, although low, was significant (Figure 2c). Supernatants from SC-SPE-stimulated MME and LE cultures were also assayed for IFN-g. At 24 and 72 h, no significant level of IFN-g was detected in MME culture supernatants (results not shown). Furthermore, SC-SPEstimulated LE cultures did not produce significant levels of IFN-g when compared with the IFN-g response of control cultures stimulated with LPS or CM (Figure 3). However, the IFN-g response of LE cultures to PHA stimulation (positive control) was significant indicating that the IFN-g response is absent when lymphocytes are stimulated in the absence of monocytes and granulocytes, further suggesting that the release of IFN-g by PBLs is a response to a mediator produced by a primary responding cell, probably, the monocyte/macrophage, rather than a direct effect of SCE stimulation on LE subpopulation. Supernatants from SCE-stimulated PBMC cultures were assayed for MCP-1 and RANTES: MCP-1 was produced through 96 h, at levels significantly higher than that produced in response to LPS or by unstimulated cells (Figure 4a). The level of MCP-1 was significant as early as 8 h following stimulation and continued to rise through 96 h, unlike the response to LPS, which remained relatively constant between 24 and 96 h (p ¼ 0.0004). Monocyte activation is likely maintained in an autocrine fashion by MCP-1 following initial SC stimulation. In contrast, a threshold level of RANTES was present in PBMC cultures for the first 12 h in both unstimulated and stimulated cultures, which was not significantly different (Figure 4b). However, after 24 h, RANTES levels in unstimulated cultures decreased steadily through 96 h, while the level in stimulated cultures increased and remained elevated (p ¼ 0.003) albeit at significantly different levels. This could be a reflection of changes in lymphocyte function as a consequence of the culture environment, although cell viability remained relatively
L. Merly & S. L. Smith
Immunopharmacol Immunotoxicol, Early Online: 1–8
Figure 1. Cytospin monolayers of leucocyte subpopulations. (a) Heterogeneous mixture of several types of leucocytes (PBL), (b) peripheral blood mononuclear cells (PBMC), (c) monocyte/macrophage-enriched leucocyte sub-population (MME) and (d) lymphocyte-enriched leukocyte (LE) subpopulation. The relative homogeneity of leucocytes in each subpopulation was confirmed by either the absence of other leucocyte types or accounted for less than 3% of preparations (triplicate counts of 200 cells/cytospin performed). (M, monocyte/macrophage; L, lymphocyte; and N, neutrophil).
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Figure 2. TNF-a response of leucocytes stimulated with SC-SPE. Supernatants of leucocyte cultures stimulated with SC-SPE, LPS or CM over 24 h were assayed for TNF-a. (a) TNF-a response of PBL cultures, (b) response of MME cultures and (c) LE culture response. **Indicates statistical significance.
stable through 96 h (495%). Alternatively, there may be protection of RANTES expression patterns in leucocytes cultured in a stimulated environment that is lacking in leucocytes cultured in medium alone.
Signaling pathways Leucocyte cultures were pretreated with either low or high concentrations of a specific inhibitor (Table 1) for Jun Kinase
Pro-inflammatory properties of SC supplement
DOI: 10.3109/08923973.2014.999160
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Figure 3. Interferon-g produced by LE subpopulation. IFN-g production by LE cells was measured following stimulation by SC-SPE, LPS, PHA and medium alone. IFN-g was not detected in supernatants of cultures stimulated with SC-SPE at 72 h. Significant levels of IFN-g were produced by lymphocytes stimulated by PHA (positive control). **Indicates statistical significance.
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Figure 5. TNF-a production following inhibition of the PI-3 kinase and PKC pathway. PBLs were pre-treated with either Wortmannin or Rottlerin, CM or DMSO/PBS control for 1 h prior to stimulation with either SC-SPE, LPS or medium alone for 24 h. (a) At both high and low PI-3 kinase inhibitor concentration (2 mM and 0.2 mM, respectively), TNF-a production was not blocked and an increased cytokine response seen in SC-stimulated cultures (p50.002). (b) There was no significant inhibition of TNF-a production in response to SC-SPE by blocking PKC. TNF-a response was significantly inhibited following LPS stimulation (p50.02). **Indicates statistical significance.
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Figure 4. MCP-1 and RANTES response of PBMC to SCE stimulation. Supernatants of PBMC cultures stimulated with SCE, LPS or CM for 96 h were harvested at intervals and assayed for (a) MCP-1 or (b) RANTES. MCP-1 was induced in response to SCE unlike cultures stimulated with CM or LPS (p50.005). There was no significant difference in the level of RANTES through 12 h; after 12 h, it decreased in CM-stimulated cultures and increased in response to LPS. RANTES response to SCE was constant over 96 h and did not decrease over duration of the experiment (p50.004). **Indicates statistical significance.
(JNK), p38 mitogen-activated protein kinase (MAPK) and Raf/MEK/ERK members of the MAPK kinase family prior to SC or LPS (control) stimulation. Control cultures pre-treated with DMSO served as vehicle control. When phosphoinostidol-3 (PI-3) kinase was blocked, TNF-a production was not inhibited in SC-SPE-stimulated cultures. Interestingly, inhibition of the PI-3 kinase significantly up-regulated the cytokine
response to SC, while the LPS response remained unchanged (p50.002; Figure 5a). When the protein kinase C (PKC) signaling pathway was inhibited, the production of TNF-a in response to LPS was significantly reduced compared with pre-treated leucocyte controls (p ¼ 0.019), while its production in response to SC was not affected. Results indicate that while PKC is a likely mediator of the response to LPS, it is not essential for the cytokine response to SC-SPE (Figure 5b). When the JNK pathway was blocked, cytokine production was completely inhibited in SC-SPE-stimulated leucocytes, suggesting that JNK is an important intracellular signal transduction mediator for TNF-a induction by SC-SPE (p50.02; Figure 6a). In addition, the TNF-a response to both LPS and SC-SPE stimulation was reduced (p50.05) when the p38 MAPK kinase pathway was blocked suggesting that p38 MAPK is a critical mediator in TNF-a response to cartilage (Figure 6b). However, when another member of the MAPK family of kinases, the Raf/MEK/ERK2 pathway, was
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Immunopharmacol Immunotoxicol, Early Online: 1–8
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Figure 6. TNF-a production following inhibition of the MAPK pathways. PBLs were pre-treated with either BI 78D3, SB202190, GW 5074, CM or DMSO/PBS control for 1 h prior to stimulation with either SC-SPE, LPS or CM for 24 h. (a) When JNK was blocked, there was complete inhibition of TNF-a production (p50.02). (b) When p38 MAPK was inhibited at both high and low concentrations, TNF-a response to both LPS and SC-SPE was significantly reduced (p50.05). (c) When the Raf/MEK/ERK2 MAPK kinase pathway was blocked, TNF-a response to neither LPS nor SC-SPE was inhibited. **Indicates statistical significance.
blocked, no significant difference in the TNF-a-inducing activity of either LPS or SC-SPE was noted indicating that inhibiting the Raf/MEK/ERK2 pathway does not affect the leukocyte TNF-a response to cartilage stimulation (Figure 6c). Both JNK and p38 MAPK signaling appear to be important signaling molecules in TNF-a production in SC-stimulated leucocytes, whereas the inhibition of the Raf/ MEK/ERK pathway does not affect cytokine production in responding cells. Results show that there are several, likely interrelated, signaling pathways activated in response to SC and that at least two are members of the MAPK kinase family.
Discussion This study demonstrates that the cytokine response pattern expressed in cartilage-stimulated leucocytes is the cumulative result of successive leukocyte responses involving the monocyte/macrophages initially responding to SC with the release of TNF-a, followed by lymphocytes as secondary responders producing IFN-g. Although the contribution of neutrophils in the initial response cannot be ruled out, a significant amount of TNF-a was produced by monocytes alone (in MME cultures) when compared to TNF-a produced by heterogeneous hPBL cultures. The difference can be explained by the relative number and proportion of MM responding in hPBL
cultures. Lymphocytes do not appear to contribute to this initial response. They appear to be activated later, producing significant levels of IFN-g after 72 h. The lymphocyte IFN-g response is absent in the absence of other cell types (such as in LE cultures). This suggests that lymphocytes do not respond directly to SC stimulation, but instead produce cytokine in response to early mediators like TNF-a released by primary responding cells present in PBMC cultures. MCP-1 is a potent recruiter of monocytes and macrophages in inflamed tissues27. It is produced by chondrocytes in articular joints in response to factors present in cartilage and thought to play a critical role in the underlying pathology of arthritis by initiating monocyte infiltration. RANTES is a potent pro-inflammatory chemokine for monocytes and T lymphocytes24. The MCP-1 and RANTES response of leucocytes to SC or LPS stimulation is distinctly different; suggesting that stimulation by these two substances is via distinct cell receptors and/or employs different activation/ signaling pathways. This is particularly evident with the MCP-1 response where SC induces at least a 10-fold higher level of cytokine production when compared to LPS. Differences in activation pathways for SC and LPS are further borne out by results obtained using kinase inhibitors. When the PKC signaling pathway was inhibited, the production of TNF-a in response to LPS was significantly reduced,
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DOI: 10.3109/08923973.2014.999160
whereas its production in response to SC was not affected. This may be due at least, in part, to lipid signaling within the cell membrane associated with LPS stimulation that is absent when leucocytes are stimulated with SC28,29. The results indicate that PKC is not essential for SC-stimulated cytokine production. A slightly different phenomenon was observed for PI-3 kinase where inhibition significantly up-regulated the cytokine response to SC, while the LPS response remained relatively unchanged. PI-3 kinase is involved in endosomal trafficking, particularly with regards to the recycling of receptors to the cell surface30. It may be that blocking the PI-3 kinase pathway increases signaling in response to SC because the receptor remains on the cell surface, thereby increasing its initial activation. Alternatively, it might be that there is another regulatory protein that normally attenuates the cellular response to SC that is governed by PI-3 kinase. When PI-3 kinase is inhibited, this regulatory protein is absent, and the response to SC is up-regulated. MAPK signaling is involved in almost every aspect of innate immune responses and is responsible for signaling events that lead to cytokine gene expression, especially for the production of inflammatory cytokines and chemokines21,31,32. In this study, we used specific inhibitors for JNK, p38 MAPK and Raf/MEK/ERK members of the MAPK kinase family to identify those that may play a role in SC stimulation of leukocytes. Both JNK and p38 MAPK signaling appear to be important signaling molecules in TNF-a production in SCstimulated leucocytes, whereas the inhibition of the Raf/ MEK/ERK pathway did not affect cytokine production in responding cells. Results show that there are several, likely interrelated, signaling pathways activated in response to SC and that at least two are members of the MAPK kinase family. Determining the clinical significance of in vitro cytokine and chemokine induction by SC prompts, the question of whether the dietary intake of SC can induce similar immunomodulating effects in vivo at localized or systemic sites. One relevant point to consider is the extent to which ingestion of cartilage in carnivorous animals is capable of stimulating cells lining the gut. This would depend on the nature of cartilage fragments (i.e. large/small, glycosylated/ unglycosylated, proteins/peptides) released by digestion and to what extent they are absorbed to induce a systemic effect, through circulation33–35. Recently collagen type II, alpha 1 peptide has been identified as the TNF-a-inducing component of commercial SC8. Collagen type II, alpha 1 protein, purified from fetal bovine articular cartilage, has been experimentally used to induce arthritis in a mouse RA model36. Since the cytokine-inducing property of commercial SC is retained in acid extracts prepared with either weak (0.05 M acetic acid) or strong (1.0 N hydrochloric acid) acids indicates that the bioactivity will not be affected by the low pH of the stomach. Furthermore, 20% of the activity of acid extracts was retained following treatment with digestive enzymes, chymotrypsin and trypsin (2 mg of each enzyme added directly to 10 ml SC extract and incubated for 3 h; digestion was stopped by the addition of 4 mg of trypsin–chymotrypsin inhibitor directly to the mixture)22. Given that the diverse cellular milieu of the gut mucosa provides a rich environment of reactive immune cells one can propose a model whereby sufficient bioactive proteins/peptides released from SC during the digestive
Pro-inflammatory properties of SC supplement
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process are sufficiently bioactive to stimulate immune cells lining the gut to induce similar responses with localized inflammatory cytokine production in the gut and exacerbation of inflammatory gastrointestinal disease35,37. Furthermore, SC-derived peptides small enough to be absorbed through the mucosa could, upon access to the circulation, stimulate circulating leucocytes and induce responses similar to those demonstrated in vitro. Thus, if through intestinal absorption the active component(s) of SC 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 would affect the health of individuals suffering from conditions where the underlying pathology involves inflammation and the up regulation of Th1 cytokines is undesirable. However, the property of SC to up-regulate a Th1-type response can be used to develop preparations for topical application where an enhanced cellular immune response is advantageous. Alternatively, in the absence of Th2 stimulation (involving enhanced antibody production) by SC in which IL-4 and IL10 production is restricted, the down regulation of the IgE antibody response could be a benefit to the hypersensitive individuals whose allergic state is often associated with production of IgE.
Conclusion Extracts of dietary SC contain bioactive component(s), which stimulate immunoreactive cells to produce inflammatory cytokines and chemokines and activate specific signaling pathways. It is reasonable to assume that the bioactive molecules released from the digestion of dietary cartilage, when absorbed, can potentially modulate innate immune function of leukocytes in vivo, thereby causing at either a localized or systemic level, exacerbation of conditions where inflammation is the underlying pathology.
Acknowledgements The authors thank volunteers who donated blood and the Comparative Immunology core laboratory for use of equipment. The protocol for the collection and isolation of leucocytes from peripheral blood of healthy human donors was approved by FIU’s Institutional Review Board (IRB).
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. L. M. nor S. L. S. are associated with the manufacturers of commercial shark cartilage. This study was supported, in part, by a summer research award to L. M. (NIH/NIGMS R25 GM061347) and a Faculty Research Enhancement Award to S. L. S. funded by the NICHD/EARDA program (G11HD038341).
Author contributions L. M. and S. L. S. contributed equally in different ways to the design, development and support of this study, the analysis and interpretation of data and the preparation of this manuscript.
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References 1. Conti F, Ceccarelli F, Massaro L, et al. Biological therpies in rheumatic diseases. La Clinica Terapeutica 2013;164:e413–e428. 2. Demoruelle MK, Deane KD, Holers VM. When and where does inflammation begin in rheumatoid arthritis? Curr Opin Rheumatol 2014;26:64–71. 3. Taylor KR, Trowbridge JM, Rudisill JA, et al. Hyaluronan fragments stimulate endothelial recognition of injury through TLR4. J Biol Chem 2004;279:17079–17084. 4. Taylor KR, Yamasaki K, Radek KA, et al. Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2. J Biol Chem 2007;282:18265–18275. 5. Matzinger P. An innate sense of danger. Sem Immunol 1998;10: 399–415. 6. Korb-Pap A, Stratis A, Muhlenberg K, et al. Early structural changes in cartilage and bone are required for the attachment and invasion of inflamed synovial tissue during destructive inflammatory arthritis. Ann Rheum Dis 2012;71:1004–1011. 7. Noble PW, Liang J, Jiang D. Hyaluronan as an immune regulator in human diseases. Physiol Rev 2011;91:221–264. 8. Merly L, Smith, SL. Collagen type II alpha 1 protein: a bioactive component of shark cartilage. Int Immunopharmacol 2013;15: 309–315. 9. Backlund J, Treschow A, Bockermann R, et al. Glycosylation of type II collagen is of major importance for T cell tolerance and pathology in collagen-induced arthritis. Eur J Immunol 2002;32: 3776–3784. 10. Gahche J, Bailey R, Burt V, et al. Dietary supplement use among U.S. adults has increased since NHANES III (1988–1994). NCHS data brief, no 61. Hyattsville, MD: National Center for Health Statistics; 2011. 11. Suarez-Almazor M, Kendall CJ, Dorgan M. Surfing the Net – Information on the World Wide Web for persons with arthritis: patient empowerment or patient deceit? J Rheumatol 2001;28: 185–191. 12. Brem H, Folkman J. Inhibition of tumor angiogenesis mediated by cartilage. J Exp Med 1975;141:427–439. 13. Lee A, Langer R. Shark cartilage contains inhibitors of tumor angiogenesis. Science 1983;221:1185–1187. 14. Gonzalez RP, Leyva A, Moraes MO. Shark cartilage as source of antiangiogenic compounds: from basic to clinical research. Biolog Pharmaceut Bull 2001;24:1097–1101. 15. Ostrander GK, Cheng KC, Wolf JC, Wolfe MJ. Shark cartilage, cancer and the growing threat of pseudoscience. Cancer Res 2004; 64:8485–8491. 16. Patra D, Sandell LJ. Antiangiogenic and anticancer molecules in cartilage. Expert Rev Molecul Med 2012;14:1–27. 17. Finkelstein JB. Sharks do get cancer: few surprises in cartilage research. J Natl Cancer Inst 2005;97:1562–1563.
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18. Habermann TM, Thompson CA, LaPlant BR, et al. Complementary and alternative medicine use among long-term lymphoma survivors: a pilot study. Am J Hematol 2009;84:795–798. 19. Spelman K, Burns JJ, Nichols D, et al. Modulation of cytokine expression by traditional medicines: a review of herbal immunomodulators. Altern Med Rev 2006;11:128–150. 20. Romagnani S. Regulation of the T cell response. Clin Exp Allergy 2006;36:1357–1366. 21. Seger R, Krebs EG. The MAPK signaling cascade. FASEB J 1995; 9:726–735. 22. Merly L, Simjee S, Smith SL. Induction of inflammatory cytokines by cartilage extracts. Int Immunopharmacol 2007;7: 383–391. 23. Shahrara S, Proudfoot AEI, Park CC, et al. Inhibition of monocyte chemoattractant protein-1 ameriolates rat adjuvant-induced arthritis. J Immunol 2008;180:3447–3456. 24. Appay V, Rowland-Jone SL. RANTES: a versatile and controversial chemokine. Trends Immunol 2001;22:83–87. 25. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunolog Meth 1983;65:55–63. 26. Strober W. Trypan blue exclusion test of cell viability. Curr Protocols Immunol 2001;Appendix 3B. 27. Melgarejo E, Medina MA, Sanchez-Jimenez F, Urdialis JL. Monocyte chemoattractant protein-1: a key mediator in inflammatory processes. Int J Biochem Cell Biol 2009;41:998–1001. 28. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 1995;9:484–496. 29. Newton AC. Protein kinase C: poised to signal. Am J Physiol Endocrinol Metab 2010;298:E395–E402. 30. Corvera S. Phosphatidylinositol 3-kinase and the control of endosome dynamics: new players defined by structural motifs. Traffic 2001;2:859–866. 31. Aderem A, Ulevitc RJ. Toll-like receptors in the induction of the innate immune response. Nature 2000;406:782–787. 32. Yang SH, Sharrocks AD, Whitmarsh AJ. MAP kinase signaling cascades and transcriptional regulation. Gene 2013;513:1–13. 33. Heinegard D. Proteoglycans and more – from molecules to biology. Int J Exp Pathol 2009;90:575–586. 34. Stern R, Asari AA, Sugahara KN. Hyaluronan fragments: an information-rich system. Eur J Cell Biol 2006;85:699–715. 35. Sanos SL, Diefenbach A. Innate lymphoid cells: from boarder protection to the initiation of inflammatory diseases. Immunol Cell Biol 2013;91:215–224. 36. Hegen M, Keith JC, Collins M, Nickerson-Nutter CL. Utility of animal models for identification of potential therapeutics for rheumatoid arthritis. Ann Rheum Dis 2008;67:1505–1515. 37. Schenk M, Mueller C. The mucosal immune system at the gastrointestinal barrier. Best Pract Res Clin Gastroenterol 2008; 22:391–409.
RESEARCH ARTICLE
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Pro-inflammatory properties of shark cartilage supplement Liza Merly* and Sylvia L. Smith Department of Biological Sciences, Florida International University, Miami, FL, USA
Abstract
Keywords
The erosion and breakdown of cartilage is generally recognized to be an integral manifestation of arthritic disease, which is often accompanied by the development and progression of inflammation associated with it. Commercial shark cartilage (SC) is a popular dietary supplement taken for the prevention and/or control of chronic disease, including arthritis. The efficacy of SC in maintaining joint health remains questionable; there is a lack of sufficient reliable information on its effect on immunocompetent cells, and the potential health risks involved have not been adequately assessed. Our earlier in vitro studies showed that SC extracts induce a Th1-type inflammatory cytokine response in human leucocytes, and collagen type II alpha 1 protein was shown to be an active cytokine-inducing component in SC. In this study, we further define the cellular response to SC stimulation by classifying leucocytes into primary and secondary responders employing enriched leucocyte subpopulations. Inhibitors of specific signaling pathways were used to verify the functional effect of SC on specific pathway(s) utilized. Results indicate the monocyte/macrophage as the initially responding cell, followed by lymphocytes and the production of interferon-g. Chemokines, MCP-1 and RANTES, were produced at significant levels in stimulated leucocyte cultures. Initial cellular activation is likely followed by activation of Jun Kinase and p38 mitogen-activated protein kinase signal transduction pathways. This study presents evidence of significant immunological reactivity of components of commercial SC supplement, which could pose a potential health risk for consumers, particularly those with underlying inflammatory disease such as irritable bowel syndrome and arthritis.
Immunomodulation, innate immunity, leucocyte response, pro-inflammatory cytokines, shark cartilage
Introduction Cytokines are involved in the development and progression of inflammation associated with chronic arthritic disease1. The cause of the initial lesion and factors involved in the initiation of an inflammatory response remains a matter of discussion2. However, the erosion and breakdown of cartilage is generally recognized to be an integral manifestation. Cartilage is mostly composed of a single cell type, the chondrocyte, and an extracellular matrix of collagen, proteoglycans and water. Fragments of glucosaminoglycans of proteoglycans when released from the ECM following injury to bones and joints can act as innate immune ‘‘danger’’ signals indicative of tissue injury/trauma/distress and induce inflammatory responses3–5. Thus, it appears that components of cartilage ECM play a role in maintaining the integrity of healthy tissue through surveillance by initiating innate immune responses
*Present address: Department of Marine Biology and Ecology, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149, USA Address for correspondence: Sylvia L. Smith, PhD, Department of Biological Sciences, OE 243, Florida International University, 11200 SW 8th St, Miami, FL 33199, USA. Tel: 305-348-3183 or 305-858-5485. Fax: 305-858-2151. E-mail: [email protected]
History Received 28 August 2014 Revised 1 December 2014 Accepted 12 December 2014 Published online 20 January 2015
following tissue stress. Consequently, it is reasonable to consider that cartilage-derived factors might represent a group of molecules that are able to activate antigen-presenting cells, such as tissue macrophages, leading to the modulation of overall cellular activity in such microenvironments resulting in disruption of normal physiology at localized and/or systemic sites6,7. Recently, an immune-active component was identified in a commercial preparation of shark cartilage (SC) as collagen type II, alpha 1 protein, which was shown to stimulate human leukocytes to produce significant levels of tumor necrosis factor (TNF)-a in culture8. Collagen type II, alpha 1 protein, purified from fetal bovine articular cartilage, has been experimentally used to induce arthritis in a mouse rheumatoid arthritis (RA) model referred to as collageninduced arthritis9. Commercial, over-the-counter products, such as chondroitin sulfate, glucosamine and cartilage, from a variety of sources, are taken by members of the public as therapeutic and/or preventative measures, and their use in the practice of complementary alternative medicine has increased significantly over the past decade10,11. SC, a dietary supplement, is one such product. To date, in vivo and in vitro studies on the biological and pharmacological properties of SC mostly focus on its antiangiogenic properties and effectiveness as an anticancer drug12–18. Most studies investigating its role as a
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modulator of immune function use laboratory-purified SC, which does not represent the potential bioactivity of commercial SC, which is the focus of this study. Immunomodulators often act through the induction of specific patterns of growth factors, cytokines and chemokines which contribute significantly to regulating and controlling immune responses19,20. Cellular responses include a cascade of intracellular signaling events, often involving members of the kinase family of proteins that lead to the production of immune mediators21. Merly et al.22 showed that SC extracts induce, preferentially, a Th1-type inflammatory cytokine response in human leukocytes and suggested that the cytokine pattern was the cumulative effect of several successive cellular responses of different leucocyte populations involving inter-cell communication through cytokines and chemokines. The question remained as to the role played by different leucocyte subpopulations in the Th-1 cytokine response and which signal transduction networks might be involved. The aim in this study was to test the hypothesis that the Th-1 cytokine response, consisting of an upregulation of TNF-a, interleukin (IL)-1b, IL-6 and IL-8, was the functional effect of distinct leucocyte types responding in a differential manner to SC stimulation. The objective was to stimulate leucocyte subpopulations and, using the production of TNF-a as an indicator of cytokine induction, to determine which cell type(s) was initially responding to SC and which leucocyte subpopulation was a secondary responder that produced cytokine in response to mediators released by primary responders and not the result of direct stimulation by immunomodulating factor(s) in SC extract. A simple fourstep cell separation protocol was employed to obtain highly enriched leucocyte subpopulations from peripheral blood leukocytes. SC-stimulated leukocytes were also examined for production of MCP-1 and RANTES, which have been shown to play a role in the development of arthritic lesions23,24. To further our understanding of the signaling networks that are most likely activated following SC stimulation, specific inhibitors of signaling pathways were used to determine the effect of inhibition on subsequent cytokine response.
Methods Cartilage extracts An acid extract of shark cartilage (SCE; 0.575 mg protein/ml) was prepared from commercial SC capsules (Solgar Vitamin and Herb, Leonia, NJ) of a single lot (Lot #47580)22. SCE was further subjected to solid-phase extraction (SPE)8 on a prepacked SPE C18 column (Waters, Sep-Pak 6 g 1 cc, Miliford, MA). Bound material was eluted with 50% acetonitrile with 0.1% TFA, and acetonitrile was removed by vacufugation. The remaining solid extract (SC-SPE) was re-suspended in culture medium (CM) consisting of RPMI-1640 supplemented with 0.3 mg/ml glutamine and 25 mM HEPES, 100 mg/ml of streptomycin and 100 U/ml of penicillin. All extracts were assayed for endotoxin using the E-Toxate assay (Limulus polyphemus amoebocyte lysate assay, Sigma catalog #210-500, St. Louis, MO), and only endotoxin-free extracts were used to stimulate cells. The detection limit of the endotoxin assay was 0.015 EU/ml.
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Leucocyte subpopulations Employing a combination of dextran sedimentation, Ficoll Paque separation and adherence to plastic highly enriched subpopulations of leucocytes were isolated from peripheral blood. Peripheral blood leucocytes (PBL) were separated from heparinized blood from healthy donors by dextran sedimentation22. An aliquot of the same blood sample used to isolate PBL was used to prepare mononuclear cell-enriched leucocyte populations (peripheral blood mononuclear cell; PBMC) by density-gradient centrifugation. Blood was diluted 1:1 with sterile, physiological saline and layered onto MonoPoly Ficoll-Paque Resolving medium (Sigma, Cat#171440-02). The mixture was centrifuged immediately at 250 g for 30 min and the buffy layer containing the mononuclear cell-enriched fraction was aspirated, washed several times in phosphate-buffered saline and suspended in serum-free CM. Further separation of mononuclear leucocytes into sub-populations was achieved by adding 1 ml of PBMC suspension (1 106cells/ml) per well of a 24-well culture plate and incubated in complete culture medium (CCM) consisting of RPMI-1640 medium supplemented with 10% fetal bovine serum and incubated for 2 h at 37 C in 5% CO2 with high humidity. The monocyte/macrophage-enriched (MME) subpopulation (approximately 5 105 cells) adhered to the bottom of wells, whereas non-adherent lymphocyteenriched (LE) cell population was harvested and standardized to 2.5 105cells/ml. CCM was added to the remaining adherent monocyte/macrophage (MME) sub-population and cultures stimulated as described below. The LE were re-plated on 24-well plates by adding 200 ml of the standardized suspension to each well and stimulated. For each leucocyte population, cytospin monolayers were examined to confirm the homogeneity of leucocyte type(s) in each subpopulation. Final cell suspensions were prepared in serum-free culture medium and standardized to desired concentrations. Cell viability was maintained at 495% and was required for subsequent cell culture (Mosmann, 1983; Strober, 2001)25,26. A differential count was performed on PBL to determine the proportion of leucocyte types present. Induction of cytokine and chemokine response in leucocytes Concurrent leucocyte cultures of PBL, PBMC, MME and LE cells, similarly treated, were set up in triplicate in 24-well flat-bottom tissue culture plates. Each culture contained 50 ml of cartilage extract or control stimulant, 100 ml of CM and 200 ml of a standardized cell suspension (2.0–2.5 105 cells/ ml) except for MME cultures, which contained approximately 3–5 105 adherent cells per well. PBMC, MME and LE cultures were stimulated with SC-SPE (1.5 mg protein/ml), lipopolysaccharide (LPS; 5 mg/ml) or CM for 24 h. Previous studies had shown that collagen type II, alpha 1 protein is the component in SC-SPE that induces TNF-a in stimulated leucocytes (Merly and Smith, 2013). LPS (Escherichia coli, Difco Laboratories) served as the positive-control stimulant. Unstimulated cultures contained CM in lieu of stimulant. Cultures were incubated in a humid chamber at 37 C in 5% CO2. Culture supernatants were harvested at 24 h from the four sets of leucocyte subpopulations and assayed for TNF-a.
DOI: 10.3109/08923973.2014.999160
Table 1. Protein kinase inhibitors of specific signaling pathways. Inhibitory molecule
Signaling pathway
BI 78D3 Wortmannin Rottlerin SB202190
C-Jun N-terminal kinase (JNK) Phosphatidylinositol 3-kinase (PI-3) Protein kinase C (PKC) p38 Mitogen-activated protein kinase (p38 MAPK) Raf/MEK/ERK2 MAPK kinase
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GW 5074
To determine production of interferon (IFN)-g, LE cultures were stimulated with SC-SPE, LPS, PHA or medium and supernatants collected at 72 h and assayed for IFN-g. Release of RANTES and MCP-1 by PBMC was measured following SCE stimulation by assaying PBMC culture supernatants, collected at 4–24 h intervals for 96 h, for chemokines. Since it was possible that a component of cartilage other than collagen type II, alpha 1 protein (the cytokine-inducing component of SC-SPE) could induce leucocytes to produce RANTES and MCP-1, PBMC were stimulated with SCE in lieu of SC-SPE. Cultures stimulated with LPS or CM served as controls. Harvested supernatants representing 0 h were estimated to be samples collected within 5 min immediately following culture set-up. Analysis of intracellular signaling A series of inhibitors of specific signaling pathways were used to verify the functional effect of SC and to validate the utilization of a specific pathway (Table 1). For each pathway studied, two concentrations of each inhibitor were used in the pre-treatment of leucocytes (PBL) prior to stimulation. Triplicate cultures were established in a 24-well tissue culture plate by adding 200 ml of cell suspension (2.5 105 cells/ml) to each test well. An additional 100 ml of CM was added to each well. Cell cultures were pre-incubated with 50 ml of either a specific inhibitor or a DMSO/PBS vehicle control for 60 m. The two negative control cultures consisted of: preincubation with CM alone and pre-incubation with DMSO/ PBS vehicle with no inhibitor. Following pre-treatment, leucocytes were stimulated with 50 ml of SC-SPE, LPS (5 mg/ml) or medium alone and incubated for 24 h. All cultures were incubated in a humid chamber at 37 C in 5% CO2. Culture supernatants were harvested at 24 h and assayed for TNF-a to determine the effect of kinase inhibition on TNF-a production. The concentrations of each test kinase inhibitor used for specific signaling pathways are listed in Table 1. Statistical analysis Leucocyte cultures were set up in duplicate and cytokine/ chemokine assays performed in triplicate. Experiments for each treatment were repeated at least twice. The student t test and ANOVA was used to identify significant differences between untreated and treated cell cultures. A p value of less than 0.05 was considered statistically significant.
Results PBLs represent a heterogeneous population of cells that play a significant role in orchestrating and coordinating host
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immune response (Figure 1a). Using a combination of separation methods, highly enriched leucocyte sub-populations were isolated from PBLs: the mononuclear cell-enriched fraction (PBMC) composed of monocytes and lymphocytes (Figure 1b), which upon further fractionation yielded the MME subpopulation (Figure 1c) and the LE subpopulation (Figure 1d). Cytospin monolayers confirmed the relative homogeneity of leucocytes in each subpopulation where other leukocyte types were either absent or accounted for less than 3% of preparations (triplicate counts of 200 cells/cytospin performed). Cytokine and chemokine response Cultures of PBL, PBMC, MME and LE were concurrently stimulated with SCE or a solid-phase extract of SCE (SCSPE), LPS (positive control) or CM (unstimulated control). Supernatants were assayed for TNF-a, MCP-1 or RANTES. PBL and MME cultures produced significant levels of TNF-a in response to both LPS and SC-SPE stimulation (Figure 2a and b). The level of TNF-a was considerably higher in MME cultures than that in PBL cultures, which was not unexpected since the relative numbers of monocytes/macrophages contained in PBL cultures was proportionately less. The TNF-a response of LE cultures to SC-SPE was insignificant, whereas the response to LPS stimulation, although low, was significant (Figure 2c). Supernatants from SC-SPE-stimulated MME and LE cultures were also assayed for IFN-g. At 24 and 72 h, no significant level of IFN-g was detected in MME culture supernatants (results not shown). Furthermore, SC-SPEstimulated LE cultures did not produce significant levels of IFN-g when compared with the IFN-g response of control cultures stimulated with LPS or CM (Figure 3). However, the IFN-g response of LE cultures to PHA stimulation (positive control) was significant indicating that the IFN-g response is absent when lymphocytes are stimulated in the absence of monocytes and granulocytes, further suggesting that the release of IFN-g by PBLs is a response to a mediator produced by a primary responding cell, probably, the monocyte/macrophage, rather than a direct effect of SCE stimulation on LE subpopulation. Supernatants from SCE-stimulated PBMC cultures were assayed for MCP-1 and RANTES: MCP-1 was produced through 96 h, at levels significantly higher than that produced in response to LPS or by unstimulated cells (Figure 4a). The level of MCP-1 was significant as early as 8 h following stimulation and continued to rise through 96 h, unlike the response to LPS, which remained relatively constant between 24 and 96 h (p ¼ 0.0004). Monocyte activation is likely maintained in an autocrine fashion by MCP-1 following initial SC stimulation. In contrast, a threshold level of RANTES was present in PBMC cultures for the first 12 h in both unstimulated and stimulated cultures, which was not significantly different (Figure 4b). However, after 24 h, RANTES levels in unstimulated cultures decreased steadily through 96 h, while the level in stimulated cultures increased and remained elevated (p ¼ 0.003) albeit at significantly different levels. This could be a reflection of changes in lymphocyte function as a consequence of the culture environment, although cell viability remained relatively
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Figure 1. Cytospin monolayers of leucocyte subpopulations. (a) Heterogeneous mixture of several types of leucocytes (PBL), (b) peripheral blood mononuclear cells (PBMC), (c) monocyte/macrophage-enriched leucocyte sub-population (MME) and (d) lymphocyte-enriched leukocyte (LE) subpopulation. The relative homogeneity of leucocytes in each subpopulation was confirmed by either the absence of other leucocyte types or accounted for less than 3% of preparations (triplicate counts of 200 cells/cytospin performed). (M, monocyte/macrophage; L, lymphocyte; and N, neutrophil).
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stable through 96 h (495%). Alternatively, there may be protection of RANTES expression patterns in leucocytes cultured in a stimulated environment that is lacking in leucocytes cultured in medium alone.
Signaling pathways Leucocyte cultures were pretreated with either low or high concentrations of a specific inhibitor (Table 1) for Jun Kinase
Pro-inflammatory properties of SC supplement
DOI: 10.3109/08923973.2014.999160
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Figure 3. Interferon-g produced by LE subpopulation. IFN-g production by LE cells was measured following stimulation by SC-SPE, LPS, PHA and medium alone. IFN-g was not detected in supernatants of cultures stimulated with SC-SPE at 72 h. Significant levels of IFN-g were produced by lymphocytes stimulated by PHA (positive control). **Indicates statistical significance.
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Figure 5. TNF-a production following inhibition of the PI-3 kinase and PKC pathway. PBLs were pre-treated with either Wortmannin or Rottlerin, CM or DMSO/PBS control for 1 h prior to stimulation with either SC-SPE, LPS or medium alone for 24 h. (a) At both high and low PI-3 kinase inhibitor concentration (2 mM and 0.2 mM, respectively), TNF-a production was not blocked and an increased cytokine response seen in SC-stimulated cultures (p50.002). (b) There was no significant inhibition of TNF-a production in response to SC-SPE by blocking PKC. TNF-a response was significantly inhibited following LPS stimulation (p50.02). **Indicates statistical significance.
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Figure 4. MCP-1 and RANTES response of PBMC to SCE stimulation. Supernatants of PBMC cultures stimulated with SCE, LPS or CM for 96 h were harvested at intervals and assayed for (a) MCP-1 or (b) RANTES. MCP-1 was induced in response to SCE unlike cultures stimulated with CM or LPS (p50.005). There was no significant difference in the level of RANTES through 12 h; after 12 h, it decreased in CM-stimulated cultures and increased in response to LPS. RANTES response to SCE was constant over 96 h and did not decrease over duration of the experiment (p50.004). **Indicates statistical significance.
(JNK), p38 mitogen-activated protein kinase (MAPK) and Raf/MEK/ERK members of the MAPK kinase family prior to SC or LPS (control) stimulation. Control cultures pre-treated with DMSO served as vehicle control. When phosphoinostidol-3 (PI-3) kinase was blocked, TNF-a production was not inhibited in SC-SPE-stimulated cultures. Interestingly, inhibition of the PI-3 kinase significantly up-regulated the cytokine
response to SC, while the LPS response remained unchanged (p50.002; Figure 5a). When the protein kinase C (PKC) signaling pathway was inhibited, the production of TNF-a in response to LPS was significantly reduced compared with pre-treated leucocyte controls (p ¼ 0.019), while its production in response to SC was not affected. Results indicate that while PKC is a likely mediator of the response to LPS, it is not essential for the cytokine response to SC-SPE (Figure 5b). When the JNK pathway was blocked, cytokine production was completely inhibited in SC-SPE-stimulated leucocytes, suggesting that JNK is an important intracellular signal transduction mediator for TNF-a induction by SC-SPE (p50.02; Figure 6a). In addition, the TNF-a response to both LPS and SC-SPE stimulation was reduced (p50.05) when the p38 MAPK kinase pathway was blocked suggesting that p38 MAPK is a critical mediator in TNF-a response to cartilage (Figure 6b). However, when another member of the MAPK family of kinases, the Raf/MEK/ERK2 pathway, was
6
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Figure 6. TNF-a production following inhibition of the MAPK pathways. PBLs were pre-treated with either BI 78D3, SB202190, GW 5074, CM or DMSO/PBS control for 1 h prior to stimulation with either SC-SPE, LPS or CM for 24 h. (a) When JNK was blocked, there was complete inhibition of TNF-a production (p50.02). (b) When p38 MAPK was inhibited at both high and low concentrations, TNF-a response to both LPS and SC-SPE was significantly reduced (p50.05). (c) When the Raf/MEK/ERK2 MAPK kinase pathway was blocked, TNF-a response to neither LPS nor SC-SPE was inhibited. **Indicates statistical significance.
blocked, no significant difference in the TNF-a-inducing activity of either LPS or SC-SPE was noted indicating that inhibiting the Raf/MEK/ERK2 pathway does not affect the leukocyte TNF-a response to cartilage stimulation (Figure 6c). Both JNK and p38 MAPK signaling appear to be important signaling molecules in TNF-a production in SC-stimulated leucocytes, whereas the inhibition of the Raf/ MEK/ERK pathway does not affect cytokine production in responding cells. Results show that there are several, likely interrelated, signaling pathways activated in response to SC and that at least two are members of the MAPK kinase family.
Discussion This study demonstrates that the cytokine response pattern expressed in cartilage-stimulated leucocytes is the cumulative result of successive leukocyte responses involving the monocyte/macrophages initially responding to SC with the release of TNF-a, followed by lymphocytes as secondary responders producing IFN-g. Although the contribution of neutrophils in the initial response cannot be ruled out, a significant amount of TNF-a was produced by monocytes alone (in MME cultures) when compared to TNF-a produced by heterogeneous hPBL cultures. The difference can be explained by the relative number and proportion of MM responding in hPBL
cultures. Lymphocytes do not appear to contribute to this initial response. They appear to be activated later, producing significant levels of IFN-g after 72 h. The lymphocyte IFN-g response is absent in the absence of other cell types (such as in LE cultures). This suggests that lymphocytes do not respond directly to SC stimulation, but instead produce cytokine in response to early mediators like TNF-a released by primary responding cells present in PBMC cultures. MCP-1 is a potent recruiter of monocytes and macrophages in inflamed tissues27. It is produced by chondrocytes in articular joints in response to factors present in cartilage and thought to play a critical role in the underlying pathology of arthritis by initiating monocyte infiltration. RANTES is a potent pro-inflammatory chemokine for monocytes and T lymphocytes24. The MCP-1 and RANTES response of leucocytes to SC or LPS stimulation is distinctly different; suggesting that stimulation by these two substances is via distinct cell receptors and/or employs different activation/ signaling pathways. This is particularly evident with the MCP-1 response where SC induces at least a 10-fold higher level of cytokine production when compared to LPS. Differences in activation pathways for SC and LPS are further borne out by results obtained using kinase inhibitors. When the PKC signaling pathway was inhibited, the production of TNF-a in response to LPS was significantly reduced,
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DOI: 10.3109/08923973.2014.999160
whereas its production in response to SC was not affected. This may be due at least, in part, to lipid signaling within the cell membrane associated with LPS stimulation that is absent when leucocytes are stimulated with SC28,29. The results indicate that PKC is not essential for SC-stimulated cytokine production. A slightly different phenomenon was observed for PI-3 kinase where inhibition significantly up-regulated the cytokine response to SC, while the LPS response remained relatively unchanged. PI-3 kinase is involved in endosomal trafficking, particularly with regards to the recycling of receptors to the cell surface30. It may be that blocking the PI-3 kinase pathway increases signaling in response to SC because the receptor remains on the cell surface, thereby increasing its initial activation. Alternatively, it might be that there is another regulatory protein that normally attenuates the cellular response to SC that is governed by PI-3 kinase. When PI-3 kinase is inhibited, this regulatory protein is absent, and the response to SC is up-regulated. MAPK signaling is involved in almost every aspect of innate immune responses and is responsible for signaling events that lead to cytokine gene expression, especially for the production of inflammatory cytokines and chemokines21,31,32. In this study, we used specific inhibitors for JNK, p38 MAPK and Raf/MEK/ERK members of the MAPK kinase family to identify those that may play a role in SC stimulation of leukocytes. Both JNK and p38 MAPK signaling appear to be important signaling molecules in TNF-a production in SCstimulated leucocytes, whereas the inhibition of the Raf/ MEK/ERK pathway did not affect cytokine production in responding cells. Results show that there are several, likely interrelated, signaling pathways activated in response to SC and that at least two are members of the MAPK kinase family. Determining the clinical significance of in vitro cytokine and chemokine induction by SC prompts, the question of whether the dietary intake of SC can induce similar immunomodulating effects in vivo at localized or systemic sites. One relevant point to consider is the extent to which ingestion of cartilage in carnivorous animals is capable of stimulating cells lining the gut. This would depend on the nature of cartilage fragments (i.e. large/small, glycosylated/ unglycosylated, proteins/peptides) released by digestion and to what extent they are absorbed to induce a systemic effect, through circulation33–35. Recently collagen type II, alpha 1 peptide has been identified as the TNF-a-inducing component of commercial SC8. Collagen type II, alpha 1 protein, purified from fetal bovine articular cartilage, has been experimentally used to induce arthritis in a mouse RA model36. Since the cytokine-inducing property of commercial SC is retained in acid extracts prepared with either weak (0.05 M acetic acid) or strong (1.0 N hydrochloric acid) acids indicates that the bioactivity will not be affected by the low pH of the stomach. Furthermore, 20% of the activity of acid extracts was retained following treatment with digestive enzymes, chymotrypsin and trypsin (2 mg of each enzyme added directly to 10 ml SC extract and incubated for 3 h; digestion was stopped by the addition of 4 mg of trypsin–chymotrypsin inhibitor directly to the mixture)22. Given that the diverse cellular milieu of the gut mucosa provides a rich environment of reactive immune cells one can propose a model whereby sufficient bioactive proteins/peptides released from SC during the digestive
Pro-inflammatory properties of SC supplement
7
process are sufficiently bioactive to stimulate immune cells lining the gut to induce similar responses with localized inflammatory cytokine production in the gut and exacerbation of inflammatory gastrointestinal disease35,37. Furthermore, SC-derived peptides small enough to be absorbed through the mucosa could, upon access to the circulation, stimulate circulating leucocytes and induce responses similar to those demonstrated in vitro. Thus, if through intestinal absorption the active component(s) of SC 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 would affect the health of individuals suffering from conditions where the underlying pathology involves inflammation and the up regulation of Th1 cytokines is undesirable. However, the property of SC to up-regulate a Th1-type response can be used to develop preparations for topical application where an enhanced cellular immune response is advantageous. Alternatively, in the absence of Th2 stimulation (involving enhanced antibody production) by SC in which IL-4 and IL10 production is restricted, the down regulation of the IgE antibody response could be a benefit to the hypersensitive individuals whose allergic state is often associated with production of IgE.
Conclusion Extracts of dietary SC contain bioactive component(s), which stimulate immunoreactive cells to produce inflammatory cytokines and chemokines and activate specific signaling pathways. It is reasonable to assume that the bioactive molecules released from the digestion of dietary cartilage, when absorbed, can potentially modulate innate immune function of leukocytes in vivo, thereby causing at either a localized or systemic level, exacerbation of conditions where inflammation is the underlying pathology.
Acknowledgements The authors thank volunteers who donated blood and the Comparative Immunology core laboratory for use of equipment. The protocol for the collection and isolation of leucocytes from peripheral blood of healthy human donors was approved by FIU’s Institutional Review Board (IRB).
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. L. M. nor S. L. S. are associated with the manufacturers of commercial shark cartilage. This study was supported, in part, by a summer research award to L. M. (NIH/NIGMS R25 GM061347) and a Faculty Research Enhancement Award to S. L. S. funded by the NICHD/EARDA program (G11HD038341).
Author contributions L. M. and S. L. S. contributed equally in different ways to the design, development and support of this study, the analysis and interpretation of data and the preparation of this manuscript.
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L. Merly & S. L. Smith
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