Flavanols and procyanidins isolated from cocoa have been reported to possess multiple activities potentially relevant to oxidant defenses, vascular function, and immune function. In a combination of in vivo and in vitro studies, we and others have observed that cocoa can be an anti-inflammatory modulator and that compounds in cocoa are capable of modulating eicosanoid production, platelet aggregation, and the pool size of nitric oxide. The present study extends these findings by examining the in vitro effects of cocoa procyanidins on polymorphonuclear cells (PMNs). PMNs, part of the innate arm of the immune system, represent 50-60% of the total peripheral white blood cells and are the first cells to be recruited to the sites of inflammation or injury secondary to bacterial infections. Herein, we demonstrate that certain flavanols and procyanidins isolated from cocoa can moderate a subset of signaling pathways derived from lipopolysaccharide (LPS) stimulation of PMNs, mainly, PMN oxidative bursts and activation markers, and they can influence select apoptosis mechanisms. We hypothesize that flavanols and procyanidins can decrease the impact of LPS on the N-formyl-Met-Leu-Phe-primed PMN ability to generate reactive oxygen species by partially interfering in activation of the mitogen-activated protein kinase pathway.
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There has been considerable work on the relationships between nutrition and the immune response, particularly on studies that have focused on adaptive responses. There is increasing recognition of the importance of innate immunity in host protection and initiation of cytokine networks. In this study, we examined the effect of select cocoa flavanols and procyanidins on innate responses in vitro. Peripheral blood mono-nuclear cells (PBMCs), as well as purified monocytes and CD4 and CD8 T cells, were isolated from healthy volunteers and cultured in the presence of cocoa flavanol fractions that differ from another by the degree of flavanol polymerization: short-chain flavanol fraction (SCFF), monomers to pentamers; and long-chain flavanol fraction (LCFF), hexamers to decamers. Parallel investigations were also done with highly purified flavanol monomers and procyanidin dimers. The isolated cells were then challenged with lipopolysaccharide (LPS) with quantitation of activation using CD69 and CD83 expression and analysis of secreted tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-10, and granulocyte macrophage colony-stimulating factor (GM-CSF). The chain length of flavanol fractions had a significant effect on cytokine release from both unstimulated and LPS-stimulated PBMCs. For example, there was a striking increase of LPS-induced synthesis of IL-1beta, IL-6, IL-10, and TNF-alpha in the presence of LCFF. LCFF and SCFF, in the absence of LPS, stimulated the production of GM-CSF. In addition, LCFF and SCFF increased expression of the B cell markers CD69 and CD83. There were also unique differential responses in the mononuclear cell populations studied. We conclude that the oligomers are potent stimulators of both the innate immune system and early events in adaptive immunity.
Epidemiological reports have suggested that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavanols and their related oligomers, the procyanidins CFP, isolated from cocoa can modulate the production and level of several signaling molecules associated with immune function and inflammation in vitro, including several cytokines and eicosanoids. To further elucidate the potential immuno-modulatory functions of flavanol-rich cocoa, the present investigation examined whether isolated CFP fractions (monomers through decamers) influence the secretion of tumor necrosis factor-alpha (TNF-alpha) from resting and phytohemagluttinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). We used an in vitro culture system where PBMC from 14 healthy subjects were introduced to individual CFP fractions for 72 h prior to measuring the levels of TNF-alpha released. The intermediate-sized CFP fractions (tetramers through octamers) were the most active on resting cells, causing a 3-4 fold increase in TNF-alpha relative to media baseline. The monomers and dimers were the least stimulatory of the fractions tested, displaying a 42 and 31% increase, respectively, over media control, whereas the trimers, nonamers and decamers showed an intermediate stimulation of this cytokine. In the presence of PHA, the intermediate-sized CFP fractions again were the most active, enhancing TNF-alpha secretion in the range of 48-128% relative to the PHA control. The monomers and dimers were slightly inhibitory (-1.5 and -15%, respectively), while trimers, nonamers and decamers stimulated moderate increases in TNF-alpha levels (13, 19 and 15%, respectively). The above results lend support to the concept that CFP can be immunomodulatory. The stimulation of TNF-alpha secretion may contribute to the putative beneficial effects of dietary flavanoids against microbial infection and tumorigenesis.
We examined whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate cytokine production at the levels of transcription and protein secretion in both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). In resting cells, interleukin (IL)-1beta and IL-4 gene expression from cocoa-treated cells varied markedly among the subjects tested. However, at the protein level, the larger fractions (pentamer through decamer) stimulated a dramatic increase in IL-1beta concentration (up to ninefold) with increasing degree of polymerization. Similarly, these larger fractions augmented IL-4 concentration by as much as 2 pg/ml, whereas the control displayed levels nearly undetectable. In the presence of PHA, gene expression also seemed to be most affected by the larger procyanidin fractions. The pentameric through decameric fractions increased IL-1 beta expression by 7-19% compared with PHA control, whereas the hexameric through decameric fractions significantly inhibited PHA-induced IL-4 transcription in the range of 71-86%. This observation at the transcription level for IL-1 beta was reflected at the protein level in PHA-stimulated PBMC. Significant reductions in mitogen-induced IL-4 production were also seen at the protein level with the hexamer, heptamer and octamer. Individual oligomeric cocoa fractions were unstimulatory for IL-2 in resting PBMC. However, when induced with PHA, the pentamer, hexamer and heptamer fractions caused a 61-73% inhibition in IL-2 gene expression. This study offers additional data for the consideration of the health benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.
Chocolate and cocoa are extensively used in many cultures. Although their composition has been studied, the functional significance of the components has not been as well defined. There are indications that cocoa constituents exert beneficial effects on human health, and therefore cocoa and chocolate may be considered functional foods. The use of functional foods to modulate human health has gained greater significance in recent years, and chocolate is widely consumed throughout society. We performed an extensive review of literature in both animal and human systems with respect to composition, bioavailability, comparative analysis with other food products and, especially, implications for cardiovascular disease and the human immune system. Although chocolate contains a high amount of saturated fats, the two major fatty acids are palmitic and stearic acid, which appear to have fewer implications for progression of coronary artery disease than other saturated fatty acids. In addition, the implications of flavonoids and other polyphenols in chocolate as antioxidants are significant, and their ability to modulate the immune system may also be applicable to infection and neoplasia. In this review, we attempt to place these issues in perspective and to provide the reader with an extensive summary of the literature on chocolate and cocoa and their potential mechanisms of action with respect to human health.
Given the widespread ingetion of cocoa in many cultures, we investigated whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate synthesis of the antiinflammatory cytokine, interleukin (IL-4). Both resting and phytohemagluttinin (PHA)- stimulated peripheral mononuclear blood cells (PMBC) were investigated at the protein secretion level. The smaller-sized cocoa fractions (tetramer or less) were unable to induce an IL-4 response (i.e. values fell below the detection limit of 0.25 pg/ml). The larger oligomeric procyanidines (penatmer or greater) stimulated secretion of IL-4 in resting PBMC by as much as 1.42 pg/ml, as shown by the nonamer. However only the hexameric, heptameric and decameric fractions proved to be statistically significant. Cells co-incubated with PBA showed an immense increase in IL-4 (21.1 ± 1.1 pg/ml). Only the monomeric fraction was able to induce the PHA-induced secretion of 48%. The other procyanidin oligomers supressed IL-4 production; in particular the hexameric, heptameric and octameric fractions significantly inhibited mitogen-stimulated secretion of IL-4 by 55%, 61% and 71%, respectively. This study offers additional data for the consideration of the health benefits of polyphenols from a wide variety of foods, including those benefits associated specifically with cooca and chocolate consumption.
