Spirulina has been reported to inhibit tumorigenesis in experimental animals and to induce the regression of existing tumors.1-4 There are indications that one of the mechanisms involved in this protective effect is the stimulation of innate immune responses, in particular macrophage phagocytosis and production of chemokines and cytokines.
In vitro, addition of Spirulina to macrophages obtained from the lung of cats significantly raised the percentage of phagocytic macrophages without affecting the number of particles that each of these macrophages engulfed.5 Similarly, in chicks that had received Spirulina as part of their diet (10-10,000 ppm) for 3 or 7 weeks, the proportion of phagocytic macrophages was significantly increased compared to unsupplemented controls.6 The number of ingested particles per phagocytic macrophage was not significantly affected. In another study, however, dietary supplementation of chicks with 0.5%, 1.0%, or 2.0% Spirulina for 14, 35, or 42 days significantly enhanced not only the percentage of macrophages involved in phagocytosis but also the number of phagocytosed particles per macrophage.7 These macrophages also exhibited significantly greater lipopolysaccharide (LPS)-induced production of nitrite than those obtained from unsupplemented animals.7 Spontaneous nitrite production (in the absence of LPS or other stimuli) was also markedly higher in macrophages from many of the Spirulina-treated groups compared to controls, although the difference did not always reach statistical significance. Nitrite measures the synthesis of nitric oxide. This molecule, along with other reactive nitrogen and oxygen species, plays an important role in the killing of the pathogens that have been engulfed through phagocytosis.
The results of another study suggest that dietary S. platensis also significantly increases the phagocytic activity of peritoneal macrophages in mice.8 Note that Spirulina constituted 10% or 20% of the diet in this experiment, and no adjustments were made for the high content of protein and other essential nutrients in Spirulina. Food intake and body weight gain were almost identical in the control group and the two groups who received different dietary levels of this alga. However, many of the individual nutrients of Spirulina are known to enhance immune functions. Therefore, it cannot be ruled out that the overall improvement in the nutrient composition of the diet rather than specific immunomodulatory substances in Spirulina were responsible for the observed effects, which included enhanced antibody production by B cells and increased proliferation of T cells (discussed below).
In contrast to whole Spirulina, perfusion of mouse liver with C-phycocyanin was associated with a concentration-dependent decrease in phagocytosis by Kupffer cells, the resident macrophages in the liver.9 There has also been a report that phy-cocyanin inhibited the respiratory burst associated with neutrophil phagocytosis.10 It is not clear, however, whether phycocyanin suppressed phagocytosis or rather neutralized the resulting reactive oxygen species through its ability to scavenge free radicals, which was demonstrated in this and other studies.10,11 In addition, it has been reported that phycocyanin induced a murine macrophage cell line to undergo apoptosis.12
Macrophages are the main source of several cytokines that promote the inflammatory response and, therefore, are collectively referred to as proinflammatory cytokines. Two of the main representatives of this group are tumor necrosis factor (TNF)-a and interleukin (IL)-1. In vitro, a hot water extract of Spirulina, containing 36% protein (compared to 60% in whole algae) and 10% polysaccharides, enhanced the IL-1 activity in mouse peritoneal macrophages.8 When human peripheral blood mono-nuclear cells, that is, immune cells obtained from the circulation, were stimulated with the soluble fraction of a Spirulina dietary supplement, IL-1^ production was increased twofold compared to incubation in medium alone.13 The combination of the mitogen PHA with Spirulina resulted in even greater stimulation of IL-1^ synthesis.
Injections of a mixture of Spirulina and Dunaliella algae into hamster buccal pouches with established tumors resulted in significant tumor regression, and this was associated with a marked increase in the number of TNF-a-producing cells in the affected pouches.3 These cells were mostly macrophages and were located primarily adjacent to the regressing carcinomas.
In vitro, a high-molecular-weight polysaccharide fraction from Spirulina-induced transcription (mRNA production) of IL-1^ and TNF-a in a human monocyte cell line.14,15 It also increased mRNAlevels of several chemokines, that is, small proteins involved in attracting specific subsets of immune cells to sites of inflammation.15 However, the authors were unable to show changes in the protein concentrations of IL-1^, TNF-a and most of these chemokines with the exception of IL-8 and macrophage inflammatory protein \j3 .15
In contrast to the results obtained with Spirulina and its polysaccharides, pretreat-ment of rats with an intraperitoneal (ip) injection of phycocyanin almost completely inhibited the 82-fold increase in serum TNF-a concentrations induced by treatment with thyroid hormone, a model of oxidative stress in the liver.9 Similarly, in mice, oral administration of phycocyanin 1 h before injection of LPS, a bacterial cell wall component that is known to induce high levels of proinflammatory cytokines, dose-dependently and significantly reduced the LPS-induced increase in serum TNF-a levels, with significant inhibition occurring at doses >100 mg/kg.16 Of note, phycocyanin alone did not markedly affect serum TNF-a concentrations.
Together these results suggest that whole Spirulina enhances macrophage functions, such as phagocytosis and production of chemokines and cytokines. This effect may be attributable at least partially to the polysaccharide fraction. In contrast, the available data suggest that C-phycocyanin down-regulates these macrophage activities.
Inflammation is characterized by pain, redness, swelling, and heat. These symptoms result from the activities of cytokines and chemokines along with a variety of other vasoactive and inflammatory mediators such as histamine, prostaglandins, and leuk-otrienes, but also reactive oxygen and nitrogen species. They mainly target local blood vessels, where they enhance blood flow, induce vasodilation, and increase the permeability of vessel walls. These changes allow fluids and plasma proteins to leak into the affected tissue. Cytokines also induce the expression of molecules that make it possible for immune cells to pass between the cells lining the blood vessels and to enter the affected tissue. Together, these alterations result in the infiltration of immune cells into the site of inflammation.
Inflammatory responses have a vital role in the protection of an organism against invading pathogens. However, many of the substances released during inflammatory responses harm not only invading microbes but also surrounding host cells, and prolonged (chronic) inflammation is generally associated with major tissue damage. Therefore, it is desirable to have substances that can minimize or prevent chronic inflammatory processes as well as inappropriate inflammatory reactions, such as those resulting from allergies against innocuous substances.
Although Spirulina can stimulate the production of some of the major proinflammatory cytokines, TNF-a and IL-1, there have been several investigations of its ability to inhibit inflammatory reactions. Even more data are available on the anti-inflammatory activities of phycocyanin.
One important group of inflammatory mediators are the prostaglandins and leuk-otrienes. They are the products of enzymatic pathways involving cyclooxygenase (COX) and lipoxygenase (LOX), respectively. It is the inducible isoform COX-2, rather than the constitutively expressed COX-1 that is responsible for the production of prostaglandins during inflammatory reactions.
A high-molecular-weight polysaccharide fraction of Spirulina induced COX-2 mRNA expression in a monocyte cell line.15 In contrast, phycocyanin was shown to selectively inhibit COX-2 activity in vitro}1 and to reduce the LPS-induced production of prostaglandin E2 in a mouse macrophage cell line, without affecting LPS-induced COX-2 protein expression.12 In addition, oral administration of phycocyanin dose dependently decreased the concentrations of leukotriene B4 and prostaglandin E2 in inflamed tissue in mouse models of inflammation.18,19 Furthermore, phycocyanin administered orally 1 h before induction of inflammation inhibited several types of acute and subchronic inflammatory responses.20 Consistent with the ability of phycocyanin to inhibit prostaglandin and leukotriene synthesis, inhibition was greatest in models where inflammation is thought to be mediated predominantly by products of the COX and LOX pathways. Studies of the anti-inflammatory activities of phycocyanin and Spirulina are summarized in Table 8.1.
Inflammatory responses are accompanied by markedly increased production of reactive oxygen species. Oxidative stress, in turn, induces the transcription of numerous genes encoding proinflammatory mediators or the enzymes producing them. Both Spirulina and phycocyanin can scavenge peroxyl, hydroxyl, alkoxyl, and superoxide radicals and have been shown to act as antioxidants in vivo and to induce enzymes that participate in the defense against oxidative damage.21
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