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Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand and * Fonterra Marketing and Innovation Centre, Palmerston North, New Zealand
2To whom correspondence should be addressed. E-mail: glen.buchan{at}stonebow.otago.ac.nz.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and interleukin-10) associated with APC activation, we identified a number of compounds that are immunoactive. The mouse macrophage cell line MH-S offered a simple and robust target for identification of immunoactives. The assay was shown to be adaptable for measuring immunoenhancing or immunosuppressive substances. Large-scale screening of milk extracts using this bioassay has the potential to identify substances that could be developed into nutraceuticals or pharmaceutical-grade immunotherapeutics.
KEY WORDS: • antigen-presenting cell • milk • immunoactive • cytokines • CD markers
To achieve maximum reproductive success, mammals make a large physiological investment in their offspring. Colostrum and milk are complete nutritional substances that provide biologically active factors that enhance the survival of infants (1). These are particularly important for the poorly prepared immune system of the neonate (2) and help the naïve immune system while it develops in the environment into which the infant was born (3). Bovine colostrum is ideally suited for supporting the neonatal immune system of calves. Not only does it have high nutritional value, but it contains immunoglobulins (4) and other immune-enhancing factors (3,5). Many as yet undiscovered immune-regulating factors may be present in bovine colostrum and milk.
New immunomodulatory roles for milk proteins are steadily being discovered. For example, a bovine milk–derived protein with growth factor–like activity and homology to the cytokine transforming growth factor ß (TGFß)3 was reported; this protein can suppress human lymphocyte proliferation (6). In mice, TGFß was studied in animals genetically deficient for this growth factor (7); neonatal mice developed chronic inflammation of the lower intestine, although this remained suppressed for longer in mice that received maternal milk containing immunosuppressive TGFß analogs.
In addition to whole bioactive proteins and lipids, milk and colostrum also contain peptides, which are activated only upon digestion and cleavage from the parent molecule. These are known as "encrypted peptides." Many bioactive peptides with immunomodulatory effects are encrypted in bovine milk proteins (8).
The discovery of new, biologically active products in milk or colostrum has become a major area of research in the dairy industry. Platform technologies have been developed by many companies to produce libraries of potentially biologically active compounds. To identify the novel, commercially viable compounds within the large number of compounds produced in a library, robust screening procedures are required. Simple bioassays can be developed to screen the library for promising products, which can then be subjected to more specific, labor-intensive functional assays, in vivo studies, and clinical trials.
The present study aimed to develop such a bioassay, with the ability to detect immunomodulatory activity of protein extracts from bovine milk. The desired target compounds were those with the potential to have an effect on multifunctional cells of the immune system, and consequently, the immune response as a whole. To study whether a compound is a modulator of the immune system, the ideal place to start is with antigen-presenting cells (APCs). The 2 primary APCs of the immune system are dendritic cells (DC) and macrophages. APCs are the multifunctional directors of the immune response. They are highly responsive to their environment and relay messages instructing other arms of the immune system e.g., T cells and B cells, to respond in an appropriate way. These messages can be membrane-anchored cell surface molecules that facilitate direct interactions between APCs and T cells (costimulatory molecules), or soluble proteins (cytokines) that act like hormones and can signal systemically. Costimulatory molecules such as cluster designation (CD)40, CD80, and CD86 are produced by the APC to aid their interaction with the T cell upon antigen presentation (9). Low levels of these molecules may be present on the APC before antigen presentation. After antigen processing, gene expression is upregulated and the number of molecules present on the cell surface increases markedly. In addition to upregulating the expression of cell surface molecules, APCs can secrete soluble protein cytokines. Interleukin (IL)-10 and tumor necrosis factor- (TNF) are examples of anti-inflammatory and proinflammatory cytokines, respectively, expressed by APCs after activation. The cell surface markers and cytokines act together to carefully control all parts of the immune system as well as the type of response generated.
IL-10 production by APCs inhibits the generation of cell-mediated immune responses and is considered anti-inflammatory. It is associated with humoral or Type 2 immune responses. TNF is a strong inducer of inflammatory responses and is associated with cell-mediated immunity (CMI, or Type 1) responses. Immunopathology can arise when the balance between Type1 and Type 2 responses is not tightly controlled, for example, in chronic inflammation (10).
Several immortalized macrophage cell lines are available that proliferate rapidly and remain stable over many passages. These cells are relatively easy to use and could potentially provide an experimentally accessible "read out" via the cell surface markers and cytokine secretion. Here we describe the use of a macrophage cell line (MH-S), in addition to murine bone marrow-derived DCs (BMDCs), to screen compounds isolated from bovine milk/colostrum for immune-regulatory activity.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Culture of the macrophage cell line MH-S. The cell line MH-S (ATCC number CRL-2019) was obtained from ATCC. A total of 5 x 106 MH-S cells were seeded in a 75-cm2 falcon flask (BD Falcon) with 20 mL warm DMEM + 5% fetal calf serum (FCS) + L-glutamine (200 mmol/L) (all from Life Technologies/Gibco) and incubated at 37°C with 10% CO2. Cells were routinely maintained at <80% confluence. To harvest cells for use in bioassays supernatants were removed from the adherent cells before the addition of 2 mL PBS + 2 mmol/L EDTA, and the culture was left at 37°C for 10 min. Vigorous pipetting helped dislodge cells, which were then washed in fresh serum-containing medium before counting.
Preparation of murine BMDCs.
BALB/c mice (6–8 wk old) were obtained from the University of Otago Department of Animal Laboratory Sciences. These mice were used to produce a source of bone marrow cells, under approval from the University of Otago Animal Ethics Committee. Mice were killed humanely and bone marrow was flushed from dissected femurs and tibias with 5% FCS in PBS. The RBCs were lysed with ammonium chloride and the remaining cells washed 3 times in PBS. Cells were plated at 2 x 106 cells/mL in 6-well plates and cultured in DMEM containing 20 µg/L of granulocyte macrophage colony-stimulating factor (GM-CSF; derived from an Escherichia coli expression system). The cultures were fed on d 4 and 6 by replacing 
75% of the medium. On d 6–7, nonadherent cells were harvested as BMDCs.
Culture of APCs with bovine milk extracts.
Harvested cells were counted and resuspended in culture media at 9 x 105 cells/mL. Milk protein extracts in powder form were diluted to a concentration of 2 g/L in appropriate cell culture media immediately before use. Serial dilutions were made to give appropriate concentrations. Cell suspensions were added 1:1 to media containing diluted test extract or control, and cultured in a 12-well tissue culture plate (Nunc). Control treatments included cells in medium only (negative) and a positive control of interferon 
(IFN-) at a concentration of 5 µg/L plus LPS from E. coli 026:B6 (Sigma) at a concentration of 1 mg/L. Cells were incubated at 37°C with 10% CO2 for 24 h before being harvested for cell surface marker staining.
Detection of cell surface markers by fluorescence activated cell scanning (FACS). Cell suspensions were transferred to tubes for FACS analysis. Cell suspensions were pelleted and resuspended in 1 mL ice-cold FACS Buffer (Dulbecco’s PBS containing 5% FCS and 0.01% sodium azide). Aliquots (100 µL) of diluted fluorescence-labeled anti-murine antibodies [anti-CD40, anti-CD86, anti-major histocompatibility complex (MHC) II] or appropriate isotype controls (cat #553930 and 553989) were added to separate tubes (all antibodies PharMingen). Cells were incubated on ice for 30 min. Cells were washed in FACS buffer and then resuspended in 200–400 µL FACS Buffer. The presence of labeled cell surface markers was detected using a FACScalibur flow cytometer (Becton Dickinson). Results were analyzed using CellQuest or CellQuestPro software (Becton Dickinson).
Preparation of supernatants from pulsed cells.
Cells were suspended in media at 9 x 105 cells/mL. Media containing milk extracts (2 g/L) or control treatments were then added to the cells in a 1:1 ratio in a 12-well plate (Nunc). Control treatments included cells in medium only (negative) and a positive control of IFN- (5 µg/L) plus LPS (1 mg/L). Cells were incubated at 37°C with 10% CO2 for 6 h after which media was removed from the wells, leaving the adherent cell layer behind. Wells were then rinsed with medium, and 2 mL of fresh medium was replaced into each well. Samples were then incubated for a further 24 h at 37°C with 10% CO2 after which the supernatants were harvested and prepared for cytokine analysis using an ELISA. Cytokines assayed for were IL-10 and TNF.
Cytokine detection by ELISA.
Rat anti-mouse cytokine capture antibody pairs (rat anti-mouse IL-10 #551215, rat anti-mouse TNF #551225; PharMingen) were diluted to 1–4 mg/L (1/5000) in coating buffer. ELISA plates (BD Falcon) were coated with 50 µL/well of diluted antibody and incubated overnight at 4°C. Plates were then washed 4 times in wash buffer to remove unbound antibody. Nonspecific protein binding was prevented by adding 200 µL blocking buffer (PBS containing 10% FCS) per well and incubating for a further 2 h at room temperature. Blocking buffer was removed by washing 4 times in wash buffer. Standard cytokines [IL-10 and TNF (PharMingen)] and samples were diluted in blocking buffer and 100 µL added per well before plates were covered and incubated overnight at 4°C. Plates were then washed 4 times in wash buffer before the addition of 100 µL/well of biotinylated detection antibodies (rat anti-mouse IL-10 #554423, rat anti-mouse TNF cat #554415; PharMingen), which were diluted 1:500 in block buffer. The plates were incubated at room temperature for 45 min, then washed 4 times in wash buffer. Streptavidin horseradish peroxidase (Zymax GradeTM, ZYMED Labs) was diluted 1:3000 in blocking buffer and 100 µL was added per well. After 30 min of incubation at room temperature, plates were washed 4 times in wash buffer. Trimethylbenzidine substrate (TMB kit, ZYMED) was added at 100 µL/well and left for color to develop; the reaction was stopped by the addition of 100 µL of 1 mol/L H2SO4 to each well, and the optical densities were read at 540 nm using a Microplate Reader (Model 550, BioRad).
Assays for suppressive activity of milk compounds on stimulated APCs.
To determine whether any of the milk extracts could have a suppressive effect on stimulated APCs, 9 x 105 cells/mL were first stimulated by incubation in media containing IFN- (5 µg/L) at 37°C with 10% CO2 for 6 h. Following this, medium only or media containing milk extracts (2 g/L) were added 1:1 per well and incubated at 37°C with 10% CO2 for 24 h. Cells were harvested and prepared for FACS analysis as described above.
Data interpretation and statistical analysis. For the interpretation of qualitative data (such as cell staining after surface fluorescence labeling), a sample was considered positive if the expression of molecules induced by a treatment was at least 2 SD above the standard negative control (skim milk). For the statistical evaluation of numerical data, such as the quantitative effect of different treatments on cell responses, one-way ANOVA was used (SPSS). Dunnett’s post hoc test was employed to identify significant differences between controls (no treatment) and the various treatments. In all cases, P < 0.05 was taken to identify significant differences.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and milk compounds.
Changes in expression of the APC costimulatory molecule CD40 were determined in MH-S cells in the absence (Fig. 1A) and presence (Fig. 1B) of stimulation with IFN, and compared against the effects of a milk compound previously found to have immunostimulatory activity (Fig. 1C). The milk compound activated APCs, causing upregulation of CD40 expression on cells in a manner similar to that of immune stimulant IFN-, as shown by a shift to the right in the fluorescence intensity histogram.
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or LPS) or to 2 milk extracts (1 with immunomodulatory activity [Product 2] and 1 without [Product 1]) indicated that these response patterns were broadly similar in MH-S cells (Fig. 2, upper panel) and mouse BMDCs (Fig. 2, lower panel). Results are representative of all positive samples tested; no sample was found to give positive results with one cell type and not the other. Although MH-S cells showed negligible expression of activation markers in response to the negative control, they strongly upregulated expression of CD40 (P < 0.001), CD86 (P < 0.001), and MHC II (P < 0.001) on the cell surface when treated with milk Product 2 or the positive control (IFN-) (Fig. 2, upper panel). There was no significant upregulation of expression of any marker after treatment with Product 1. Similarly, BMDCs were hyporesponsive to Product 1 and the negative control (Fig. 2, lower panel), with a positive staining frequency of < 5% of total cells for CD40. As with the MH-S cells, the known immunomodulatory milk extract (Product 2) caused significant increases in CD40, CD86 and MHC II expression on BMDCs (P < 0.001) (Fig. 2, lower panel).
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0.001). No upregulation of CD40 or CD86 occurred after treatment with any of the other milk extracts. Extracts E and I also induced the upregulation of MHC II (P < 0.05). MHC II was not upregulated after treatment with any of the other milk extracts.
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was detected in supernatants of cells treated with any of the milk extracts. The MH-S cells treated with IFN + LPS did, however, produce a large amount of TNF (5.4 ± 0.4 µg/L), indicating that these cells were capable of producing this cytokine.
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for 6 h before coculture for a further period of 24 h with milk extracts, demonstrated a degree of regulation of cell surface molecule expression (Fig. 6). After stimulation with IFN-, 6 of the milk extracts (A, D, E, G, I, and K) induced some degree of synergistic upregulation of CD40 (Fig. 6, upper panel). CD86 expression on preactivated cells was also strongly induced by extracts A, E, I, and K. None of the extracts upregulated MHC II above the level induced by pretreatment with IFN. Only 1 compound (C) showed significant suppressive activity of MHC II expression on preactivated APCs (Fig. 6, lower panel).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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APCs are ideal cells to use as biosensors because they respond to their surroundings in an easily measured manner. APCs recognize and take up antigens from their environment, and present these to T cells to direct the appropriate immune response. To achieve successful T-cell activation, the APC is required to deliver 2 signals to the T cell. The first results from engagement of the T-cell receptor by antigen fragments presented on molecules of the MHC II. To facilitate this process, activated APCs greatly increase the number of MHC II molecules on their surface in response to certain environmental signals. The second signal is delivered by a range of costimulatory molecules. These can be either surface bound as with CD40 and CD86 or secreted, as is the case with cytokines such as TNF, IFN-, and IL-10. By monitoring the expression of these "activation markers" by APCs, we were able to identify a number of milk extracts with the ability to activate these cells.
A macrophage-derived murine cell line, MH-S, was compared with primary DCs, cultured from mouse bone marrow, to determine whether they responded in a similar manner. MH-S cells are a self-renewing, immortalized cell line that is simple to grow in culture. DCs are considered to be highly sensitive, professional antigen-presenting cells (15), but they are difficult to isolate and require careful handling to prevent them becoming activated before treatment with bioactives. Thus, they can produce variable results, making them less appropriate for a large-scale screening process in a biotechnology setting than a cell line such as MH-S. Our data suggest that, under the conditions tested for in these experiments, the MH-S cell line can be substituted for BMDCs without loss of assay sensitivity.
The response of APCs to particular milk extracts might give an indication of the type of condition/disease that may be treatable with particular extracts. For example, extracts with immunosuppressive activity could potentially be developed into a nutraceutical or pharmaceutical to aid in suppression of inflammatory autoimmune diseases such as rheumatoid arthritis or asthma. Extracts that stimulate CMI could be developed as treatments for infections, some types of cancer, and conditions in which people suffer from immunosuppression.
Four extracts in this study had immunoactivity. Although cytokines such as IFN- are active in nanogram to picogram amounts, milk extracts are required in the microgram to milligram range. This may be due to the activity residing in a minor subfraction of these complex extracts; on the other hand, the immunoactive molecules could be relatively nonspecific or target low-affinity receptors. Further subfractionation of positive compounds to obtain pure fractions will elucidate which option is correct. The relative expression of surface activation markers differed between samples, with CD40 appearing to give the clearest and most consistent differences between foreground and background.
Although none of the extracts induced TNF production by MH-S cells, 2 induced IL-10, which downregulates activation markers on APCs (16). It is interesting to note that sample F induced IL-10 production in the MH-S cells but little upregulation of CD40, CD86, or MHC II, whereas sample A induced IL-10 production but also strongly upregulated other activation markers. Sample C had a potent suppressive activity on MHC II expression but appeared to induce little IL-10 expression. This suggests that these complex compounds contain numerous and possibly antagonistic activities that will become obvious only with further purification (3).
We also adapted the assay to use activated APCs as the starting point and then looked for substances that would suppress or further enhance (act synergistically on) the expression of activation markers. Interferon- is an inducer of APC activation markers; thus, it was used to preactivate target cells. One milk-based product (C) had strong suppressive activity on IFN- preactivated cells, whereas extracts A, E, I, and K appeared to act synergistically with IFN- to upregulate the expression of activation markers. It is not surprising that these same 4 extracts were identified as being active on unstimulated APCs as well. Two samples (D and G) were able to upregulate CD40 but not CD86. The importance of differential expression of these markers will require further investigation.
Although there are many other activation markers that could be used to measure APC activation, this would invariably increase the complexity of the assay without providing a corresponding increase in information. For example, CD80 expression tended to mimic CD86 and CD40 expression in this study (data not shown). Because time and expense can be saved by minimizing the number of markers measured, assaying for 1 or 2 representative markers is likely to provide sufficient information to identify most compounds with immunoactivity on APCs.
Although food-grade cow’s milk is pasteurized before the separation of protein extracts, the possibility of bacterial products such as LPS being present in the samples must be considered. LPS is an endotoxin and is a potent activator of APCs. Indeed, a recent study indicated that bacterial LPS contamination can be a major contributor to the perceived immunoactivity of bovine milk proteins (17). However, several lines of evidence suggest that microbial products such as LPS were not an important factor in our experiments. First, despite a common separation technique only a few (4 of 12) of the products showed any ability to activate target APCs. If microbial contamination was a major issue, we would have expected the majority of samples to have been positive in our assay system. LPS is a potent inducer of TNF production by APCs. In our experiments, no TNF production by MH-S cells was detected in response to coculture with milk proteins alone, whereas the LPS positive control induced large amounts. We found that the induction of cell surface, costimulatory molecules, in the MH-S cell line, is insensitive to LPS unless IFN- is also present. This argues against LPS being responsible for the activation of MHC II, CD40, or CD86. Finally, samples were tested independently at ESR (the National Testing Laboratory in New Zealand) for endotoxin contamination, and the concentration of endotoxin was <0.15 ng/g. When we tested these concentrations of LPS experimentally, as well as at 10-fold higher concentrations, on both MH-S and bone marrow dendritic cells, no stimulation of costimulatory molecules occurred.
The results of this study suggest that the expression of activation markers on APCs can be used reliably for testing milk extracts for activity, which could potentially have downstream effects on the immune response by virtue of the multifunctional action of APCs in vivo. Expression of these markers can be used to identify milk extracts with stimulatory or suppressive activities. Application of this process will facilitate the discovery of novel molecules in milk or colostrum before in vivo studies in animals and humans. Large-scale screening of milk extracts using the bioassay developed in this study has the potential to identify substances that could be developed into nutraceuticals or pharmaceutical-grade immunotherapeutics.
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FOOTNOTES |
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3 Abbreviations used: APC, antigen-presenting cell; BMDC, bone marrow–derived dendritic cells; CD, cluster designation; CMI, cell-mediated immunity; DC, dendritic cell; EU, endotoxin unit; FACS, fluorescence-activated cell scanning; FCS, fetal calf serum; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin; IFN-, interferon-; LPS, lipopolysaccharide; MHC, major histocompatibility complex; TGFß, transforming growth factor ß; TNF, tumor necrosis factor-.
Manuscript received 8 June 2005. Initial review completed 5 July 2005. Revision accepted 11 August 2005.
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