In vitro modulation of inflammatory cytokine and IgG levels by extracts of Perna canaliculus

Background Inflammation is a predominant characteristic of autoimmune diseases which is characterized by the increased expression of pro-inflammatory cytokines. Soon to be published work from our laboratory has shown that ingestion of Perna canaliculus prevents the development of autoimmune diseases such as Systemic Lupus Erythematosus and rheumatoid arthritis in laboratory animals. The current paper attempts to illustrate how Perna can alleviate inflammation by modulating inflammatory cytokines, cyclooxygenase enzymes and Immunoglobulin-G (IgG) levels. Methods In the present study, hydrochloric acid [HCl] and Tween-20 were used to develop extracts of Perna. These extracts were assayed for protein content. Increasing concentrations of these extracts were then tested in cell culture for modulation of inflammatory cytokine, cyclooxygenase enzymes and IgG levels. Parallel tests were run using an available glycogen extract of Perna as a comparison to our in-house laboratory preparations. Results Tween-20 Perna extracts were found to be more stable and less toxic in cell culture than HCl digest of Perna. They also assayed higher in protein content that HCl extracts. Although both extracts inhibited IgG production in V2E9 hybridomas, Tween-20 extracts were more consistent in IgG suppression than HCl extracts. Overall Tween-20 extracts effectively decreased levels of TNF-α, IL-1, IL-2 and IL-6 as observed using cytokine bioassays. Twenty micrograms of Tween-20 Perna extracts induced such significant decreases in inflammatory cytokine production that when tested on sensitive cell lines, they very nearly abolished the decrease in viability induced by these cytokines. Tween-20 extracts effectively inhibited both COX-1 and COX-2 cyclooxygenase activity. As a comparison, the glycogen extract also demonstrated a similar though weaker effect on COX-1 and COX-2 enzymes. The active components of both extracts (Tween-20 and glycogen) were observed to possess molecular weights above 100 kDa. Although the anti-cytokine activity of the Tween-20 extract was destroyed by Proteinase-K treatment, the anti-COX-1 and anti-COX-2 activity of both the extracts were not sensitive to protease treatment. Conclusion We have successfully demonstrated modulation in the levels of inflammatory cytokines, cyclooxygenase enzymes and immunoglobulins by our in-house laboratory preparations of Perna canaliculus, whereby suggesting an immunomodulatory role of Perna canaliculus in regulating inflammation.


Background
Autoimmune diseases include a wide spectrum of systemic diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [1,2]. The important characteristic of all autoimmune syndromes is inflammation and the damage inflicted upon self-tissues mediated by host cellular and/or humoral responses. Autoantibodies are fundamental to all autoimmune diseases and are products of auto-reactive B cells and plasma cells, which in turn, are driven by a T helper subset 1 (Th1) immune response [3]. T lymphocytes play a role in the disease process by the stimulation of other immune cells via T helper subset 2 (Th2) and/or direct cell-cell interaction [4][5][6]. Cytokines also play a role in autoimmunity by stimulating or regulating B and T cells, macrophages, and/or dendritic cellular responses to antigenic stimulation [7]. It is well established that inflammatory cytokines play important roles in the development and pathogenesis of autoimmune diseases such as RA and SLE [8][9][10][11]. These factors overcharge or overwork the immune networks and must be regulated by modulation or suppression. The New Zealand green-lipped mussel (Perna canaliculus) contains components that can act as natural immunomodulators. Laboratory investigations of the freeze-dried extract of Perna canaliculus have demonstrated that Perna extracts decreased experimentally induced inflammation in carra-geenan-induced edema of the rat hind footpad [12]. In our laboratory, Perna has been shown to suppress the development of collagen-induced arthritis (CIA) in the rat, and has even reversed inflammation associated with CIA in mice. Perna has been employed as a therapeutic agent in arthritis with some degree of success [13][14][15]. However, evidence from other clinical trials utilizing Perna products for rheumatoid arthritis (RA) and osteoarthritis (OA) have been conflicting with respect to the therapeutic potential of Perna [14,16].
The exact mechanism of Perna's anti-inflammatory action is unknown. There is some evidence that certain components in Perna may inhibit prostaglandin synthesis via production of prostaglandin synthetase inhibitors [17][18][19]. Perna also contain the histamine blocker lysolecithin that may contribute to its anti-inflammatory activity [20]. Omega 3 fatty acids in Perna canaliculus have been shown to decrease inflammation [21,22]. Although many authors have demonstrated various anti-inflammatory mediators in Perna canaliculus, inconsistencies in therapeutic activity levels persist. These inconsistencies may be due to the lack of standardized extraction / fractionation protocols and the possible presence of many anti-inflammatory mediators in Perna canaliculus. Different Protocols for preparing extracts of Perna powder, a freeze dried preparation of Perna canaliculus using either concentrated HCl or 0.1% Tween-20   [20][21][22][23][24].
Our paper discusses two extraction techniques for obtaining Perna extracts and explores the effects of these extracts on inflammatory cytokine production, cyclooxygenase activity and IgG modulation in both lymphocytic cell lines and B cell hybridomas.

Preparation of Perna canaliculus (Perna) extracts
We used two protocols to create extracts of the lyophilized Perna powder (FoodScience Corp., Essex Jct., VT), the commercially available freeze-dried powder of the New Zealand green lipped mussel, Perna canaliculus. The first method used concentrated hydrochloric acid (1 M HCl) while the second employed Tween-20 ( Figure 1). Acid extraction proceeded by placing 1 g samples of freezedried Perna powder in 250 ml beakers along with 5 ml of concentrated HCl solution (pH 1.0) in a 37°C water bath for 5 hours with constant agitation. The extracts were neutralized with either 0.1 N or 1.0 N sodium hydroxide (NaOH) and filtered through a series of filters leading to 0.22µ. The filtrate was then analyzed and adjusted for protein content by the Bradford assay prior to use in cell culture. Tween-20 extraction of Perna was performed by treating 1 g samples of freeze-dried Perna with 5 ml of 0.1% Tween-20 solution for 24 hours at 4°C with constant agitation. The samples were then filtered and assayed for protein content as above before use in cell culture. Both HCl and Tween-20 extracts were adjusted to yield a range of concentrations for use in cell culture assays based on their protein content.

Cell Lines and Cell Culture
The cell lines used in this study were V2E9, THP-1, L-929, U-937, A375.S2, Jurkat E6-1, EL-4, CTLL-2, LS174T, and 7TD1. Characteristics of each cell line including cytokine production and/or sensitivity are presented in Table 1. All the cell lines, with the exception of V2E9, were obtained from the American Type Culture Collection (ATCC). V2E9 cell line, a B cell hybridoma constitutively secreting mouse IgG against the β1 subunit of chicken integrin, was obtained courtesy of Ellis Kline, Clemson University.
V2E9 cells grown in Ex-Cell 610 medium (JRH Biosciences) supplemented in initial studies with 10% fetal bovine serum (FBS, Gibco BRL) and 1% Antibiotic-Antimycotic (Gibco BRL) and later adapted to grow in serum-free media. All the other cell lines were cultured as per ATCC recommendations with regard to media and supplements. CTLL-2 and 7TD1 are cytokine-dependent cell lines requiring the addition of Interleukin-2 (IL-2) and IL-6 respectively. All cell lines were grown at 37°C in a humidified 5% CO 2 incubator, except for EL-4 mouse thymoma cells which required 10% CO 2 .

Modulation of IgG production in V2E9 hybridoma cells (ELISA)
V2E9 cells were harvested, enumerated, and 1 ml of cells in Ex-cell 610 media at a concentration of 5e 6 cells/ml were transferred into 24 well plates. One hundred µl of varying dosages (containing 0 µg, 5 µg, 10 µg, 15 µg, 20 µg, and 25 µg protein) of Perna extract were added. An additional 900 µl of Ex-cell media were added to bring the total volume to 2 ml. After incubation at 37°C for 48 hours, supernatants of Perna-treated and untreated control cells were drawn off and assayed for IgG levels using ELISA.
Goat affinity purified antibody to mouse IgG (Cappel, Organon Teknika Corp.) was diluted in phosphate buffered saline [PBS] (pH 7.4) to a concentration of 0.1 µg/µl. Ninety-six well, high-affinity binding ELISA plates (Corning, NY) were coated with 100 µl of this buffered suspension for an hour at 37°C. The plates were placed for an additional hour at room temperature (RT). The plates were washed twice with PBS, blocked with freshly prepared Blotto (Skim milk in PBS) for 60 minutes at 37°C, rinsed twice with PBS, and blotted. One hundred µl of the supernatants from the treatment and control wells were added to the plates and allowed to incubate for an hour at 37°C. Following incubation, plates were rinsed twice with Blotto and PBS. One hundred µl of peroxidase conjugate goat affinity-purified antibody to mouse IgG (Cappel, Organon Teknika Corp.) diluted 1:100 in PBS were added to each well. Plates were incubated for 30 minutes at 37°C and rinsed four times with Blotto and twice with PBS. One hundred micro liters of fresh color developer [24.3 ml of

Modulation of cytokine production in cell lines (Cytokine Bioassays)
Modulation of inflammatory cytokine production by Perna was determined by cytokine bioassays [25][26][27][28]. hours at 37°C in a humidified 5% CO 2 incubator. Supernatants from these cells were then added to the indicator cell line L929 that is sensitive to TNF-α and incubated for 24 hours at 37°C in a humidified 5% CO 2 incubator [29][30][31]. After incubation, MTS-PMS solution, (Cell Titer 96 Aqueous Kit, Promega, Madison, WI) was added to each well as recommended by the manufacturer [32]. Detection of cell proliferation or inhibition is based on a colorimetric assay system utilizing the tetrazolium reagent, Dose response effects of Perna (HCl extract) on IgG secretion in V2E9 hybridoma cells The experiments were carried out in triplicate and repeated three to six times for each cytokine. Similarly, U-937 cells which secrete Interleukin-1 (IL-1) constitutively were treated with Perna and the supernatants added to the sensitive cell line A375.S2 [33,34]. Likewise, Jurkat E6-1 and EL-4 mouse thymoma cells which when stimulated with Ionomycin and phorbol myristate acetate (PMA) respectively secrete IL-2, were treated with Perna and the super-natants were added to the responder cell line CTLL-2 [35][36][37][38]. Finally, LS174T colon adenocarcinoma cells that constitutively secrete IL-6 were treated with the Perna extracts and the supernatants added to the responder 7TD1 cell line [39][40][41]. further to derive the appropriate concentrations for testing. The assay was then performed as described in the assay kit booklet.

Statistical analysis
The results were expressed as the means ± SEM of the number of measurements (independent experiments) shown. Statistical comparisons between dosage groups were done using the paired t-test and a one-way ANOVA, with P < 0.01 indicating significance.

Perna canaliculus (Perna) extracts
Extracts of Perna was prepared using two procedures, one, which uses concentrated HCl and the other a cationic detergent, Tween-20 ( Figure 1). Hydrochloric acid was chosen initially for preparing an extract of Perna to simulate the acidic gastric conditions of Perna when it is taken orally. Tween-20 which draws out membrane-bound proteins was used on the premise that the active component in Perna might be a protein moiety [22]. It was found that Tween-20 extract yielded higher protein recovery levels than HCl extract. Protein estimation was used as a quantitative measure of extract recovery at the end of HCl digestion and Tween-20 treatment and also as a standard for measuring activity. The low amount of protein in HCl extracts could be based on either the dilution effect of neutralizing HCl with NaOH or the inability of HCl to extract active proteins efficiently. Since protein content was used as the standard to measure activity, HCl extracts had to be concentrated prior to use in cell culture assays. Extracts in the range of 0 µg, 5 µg, 10 µg, 15 µg, 20 µg, and 25 µg protein content were used in assays to determine activity. The neutralized HCl extracts were toxic to the cells at concentrations approaching 20 µg (~70-80% viability), whereas Tween-20 extracts did not affect viability of cells (>90% viability) even at concentrations greater than 25 µg.
As a comparison to our in-house laboratory prepared extracts, a commercially available extract of Perna canaliculus was obtained from Aroma New Zealand Limited, Christchurch, New Zealand. This extract is rich in glycogen and obtained by enzymatic treatment and extraction with aqueous ethanol. Insoluble components were separated by centrifugation and the extract isolated from the supernatant by lyophilization [42].

Modulation of IgG production in V2E9 hybridoma cells
Both HCl and Tween-20 extracts were compared for the ability to modulate IgG antibody production in V2E9 hybridoma. Both extracts significantly decreased antibody expression in the hybridoma (Figure 2, Figure 3). Maximum decreases of antibody levels (21%) were observed for HCl extract containing 20 µg of protein, whereas Tween-20 extract decreased antibody levels by 26% at similar protein concentrations. To determine whether the active component of Perna was associated with a protein moiety, protease digestion with Proteinase-K of Tween-20 extracts from Perna was carried out over different time periods. On addition of these digests to V2E9 cells, no decrease in antibody production was observed (Figure 4). Furthermore the decrease in inhibitory activity of Proteinase-K treated Perna extract was time dependent. IgG secretion levels increased in parallel with the length of protease pretreatment. This correlated with an increase in IgG lev-els over baseline levels from -18.5% to -1.6%, over a 48hour protease treatment period. The proteolytic enzyme apparently eliminated the component in the Perna extract, which inhibited antibody production.

Modulation of inflammatory cytokine expression
Tween-20 extract of Perna was tested for its ability to modulate cytokine expression in cell lines. Cytokine bioassay results ( Figure 5, Figure

TNF-α α α α Secretion Levels in Perna (Tween-20 Extract) Treated THP-1 Cells
5µ µ µ µg 10µ µ µ µg 15µ µ µ µg 20µ µ µ µg Control Perna-treated Jurkat E6-1 and EL-4 cells, which secrete IL-2. Supernatants from these cell lines, when added to the responder cell line CTLL-2, triggered a decline in proliferation of CTLL-2 due to decreased levels of IL-2. In Jurkat E6-1, secretion levels of IL-2 as evidenced by the proliferation of CTLL-2 cells dropped from a high of 23.56% to a low of 2.98% (Figure 7). This translates to a drop of 21% with 20µg of Perna extract. In EL-4 mouse thymoma cells, IL-2 secretion levels as measured by the growth rate of CTLL-2 cells reduced from 15.38% to 0.89% with the highest growth rate drop of 14% observed with 20 µg Perna (Figure 8). Likewise IL-6 production levels in LS174T were also depressed in the presence of Perna extract, leading to a drop in proliferation of 13% in 7TD1 cells (Figure 9).

Cyclooxygenase-1 (COX-1) and Cyclooxygenase-2 (COX-2) Assay
We examined the effects of the Tween-20 extract of Perna on the activity of both COX-1 and COX-2 enzymes.
Included in parallel studies was the glycogen rich commercial extract derived from Aroma New Zealand. As observed in figure 10, COX-1 activity was inhibited by over 50%, when Perna extracts were tested. The magnitude of COX-1 inhibition was higher in dilutions from Tween-20 extracts (1:200 and 1:400 dilutions) compared to the Glycogen extracts (1:10 and 1:100 dilutions). Inhibition of both the COX-1 and COX-2 enzyme activity was consistently higher in all Tween-20 preparations ( Figure  10).

Discussion
Inflammation is a prominent feature of autoimmunity leading to tissue injury. Cytokines play an important role in inflammatory autoimmune diseases such as RA and SLE [43]. Elevated local and systemic levels of inflammatory cytokines are often associated with depressed levels of anti-inflammatory cytokines, which suppress inflammatory pathways suggesting an aberrant Th1/Th2 regulation. Current research on immunomodulation focuses on the

IL-1 Secretion Levels in Perna (Tween-20 Extract) Treated U-937 Cells
ability of agents to regulate or modify the immune network via regulation of Th1/Th2 immune responses. Natural substances such as hormones, vitamins, and amino acids have been used to achieve this modulation [44,45].
Perna canaliculus has been a popular nutritional supplement for treating arthritis. Oral consumption of Perna stems from the observation that native New Zealanders (Maoris) who lived near the sea had a much lower incidence of arthritis than their relatives who lived inland [46]. Both the diets were similar with the exception that the Maoris on the coast consumed large amounts of greenlipped mussel. Various researchers have experimented with a range of methodologies for generating Perna canaliculus extracts and have demonstrated the presence of various anti-inflammatory mediators [21,22,47,48]. Inconsistencies in the therapeutic availability of the active components in these preparations still persist and may be due to the presence of many anti-inflammatory mediators in Perna canaliculus. Different approaches to extract and/or fractionate Perna have been employed albeit with mild success [20][21][22][23][24]. Hence, we developed simple extraction protocols using HCl and Tween-20 for generating soluble extracts of the commercially available freeze-dried powder of Perna canaliculus as a means to isolate, test and identify some of the active components. Our rationale behind using Hydrochloric acid (HCl) as an extraction tool stems from our understanding of the primary route of Perna intake which is ingestion. This is based primarily on our laboratory studies related to collagen induced arthritis (CIA) experiments involving rats and mice, where we observed a delay in the onset of arthritis in animals that were given Perna orally (manuscript in preparation). Similar observations have been made by other investigators

IL-2 Secretion Levels in Perna (Tween-20 Extract) Treated Jurkat E6-1 Cells
5µ µ µ µg 10µ µ µ µg 15µ µ µ µg 20µ µ µ µg Control with the oral administration of either Perna canaliculus or Perna extracts in alleviating symptoms of arthritis and other autoimmune diseases [47][48][49][50][51] These observations led to the assumption that the active principle may be facilitated by the digestive process. Hence we simulated the acidic environment of the stomach by using hydrochloric acid (HCl) to extract Perna. Tween-20 is a cationic detergent and was used based on the assumption and prior evidence from Miller et al., that the anti-inflammatory principle may be associated with a protein moiety [22]. We found that Tween-20 extraction of Perna was more stable and less toxic in cell culture than HCl digest of Perna. The total protein content of HCl extract was lower and more variable than Tween-20 extract due to the elaborate nature of the HCl extraction process. Neutralization with NaOH after HCl treatment dilutes this extract, requiring removal of water by lyophilization to achieve parity in protein content prior to use in cellular assays. This concentration process led to an increase in osmolarity and contributed towards decreased cellular viability. Tween-20 being a cationic detergent and when used at a concentration of 0.1% did not affect the viability of cells (>90% viability) even at standard protein concentrations as high as 25 µg. Thus, Tween-20 extracts was the preferred method of extraction in all cytokine bioassays. Although the extract derived from HCl digestion of Perna was not as active as that obtained from similar treatment with Tween-20, our results demonstrate that the anti-inflammatory properties were conserved in both extraction processes.
Perna extracts inhibited IgG production in the V2E9 hybridoma cell line. Tween-20 extract was more consistent in IgG suppression than the HCl fraction. In RA and SLE, the pathological antibody associated with disease manifestations is of the IgG2a isotype which is produced Dose response effects of Perna (Tween-20 extract) on IL-2 secretion levels in EL-4 mouse thymoma cells Figure 8 Dose response effects of Perna (Tween-20 extract) on IL-2 secretion levels in EL-4 mouse thymoma cells. Each bar represents the mean ± SEM of data points from three independent experiments with triplicate wells. Columns representing the different concentrations are statistically different from the stimuated but untreated baseline control (no Perna). P ≤ 0.01.  This observed decrease in IL-1 expression although small as measured by cytokine bioassays was repeatable in at least four independent experiments. Minor cytokine changes tend to become significant when complex networked systems in living organisms are involved. Similar changes in cytokine profiles that are enough to alter the therapeutic outcome have been observed in mouse models of rheumatoid arthritis and Systemic lupus erythematosus [52]. This decrease in inflammatory cytokines may help to explain the delayed onset of arthritis and the reversal of arthritic inflammation observed in the collagen induced arthritis (CIA) experiments involving rats and mice in our laboratory (manuscript in preparation). We used a commercially available extract of Perna canaliculus obtained from Aroma New Zealand Limited, Christchurch, New Zealand as a comparison to our inhouse laboratory preparations. This extract is reported to be rich in glycogen and has been obtained by enzymatic treatment followed by extraction with aqueous ethanol and is now called the Glycogen extract [42]. This glycogen extract was found to decrease foot pad edema (inflammatory edema) which had been induced by carrageen in rats [22]. In preliminary studies we found that the active component of both the Tween-20 and glycogen extracts had molecular weights above 100 kDa. Anti-inflammatory cytokine activity was destroyed on treatment of the Tween-20 extract with Proteinase-K. A similar digestion eliminated the anti-inflammatory activity of the glycogen extract in the Carrageen induced edema model in rats [22].

Inhibition of Ovine Cox-2 by Tween-20 and Glycogen Extracts
In evaluating the anti-inflammatory potential of our Perna extracts, we have performed the colorimetric Ovine Cyclooxygenase (COX) assay. We have successfully demonstrated that Tween-20 extracts of Perna canaliculus was effective in inhibiting both COX-1 and COX-2 cyclooxygenase activity. As a comparison, the glycogen extract also demonstrated a similar though weaker effect on COX-1 and COX-2 enzymes. COX enzymes are essential in the inflammatory process and control the downstream regulation of immune cell activation and inflammatory cytokine induction [53]. Currently a great deal of attention is being paid to understanding the multifaceted nature of the cyclooxygenase and the lipooxygenase pathways in inflammation and cytokine induction. Our results demonstrating the inhibition of Ovine COX-1 and COX-2 have supported the role played by Perna extracts in modulating inflammation and inflammatory cytokine response.

Conclusion
In summary, we have prepared two distinct extracts of the freeze dried Perna powder. The Tween-20 extract was determined to be more potent and less toxic than HCl extract in decreasing IgG expression in hybridoma cell culture. Consequently we used the Tween-20 extracts in all subsequent experiments involving modulations of inflammatory cytokine expression and cyclooxygenase activity. Tween-20 Perna extracts were shown to suppress pro-inflammatory cytokines expression, inhibit COX-1 and COX-2 activity and decrease IgG levels. In parallel studies using glycogen extracts, we observed similar decline in COX-1 and COX-2 activity. Although, both the extracts (glycogen and Tween-20) possessed active components that appeared to be larger than 100 kDa, the component effective against inflammatory cytokines was sensitive to proteolysis using Proteinase-K and Pronase, whereas the component effective against COX enzymes was resistant to proteolysis. Based on all the above observations, we suggest a possible immunomodulatory role for Perna canaliculus in mediating Th1/Th2 regulation as it relates to inflammation.
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