**6. Age-related changes in intestine intraepitherial lymphocyte subsets and their functional preservation by** *Spirulina* **in mice**

Age-related immune dysfunction has been reviewed by many researchers (Solana et al., 2006). The complex age-related changes in the immune system, collectively termed "immunosenescence," have been demonstrated in diverse species, including humans, and have been recognized as contributing to morbidity and mortality due to greater incidence of infectious diseases, autoimmune diseases, and cancer. The concept of age-related immunosenescence is in agreement with numerous data such as the change of cytokine balances, the decrease of interleukin (IL)-2 contrary to the increase of IL-6, and nutritional imbalance or malnutrition (Miquel, 2001; De la Fuente, 2002). It was reported that antigenspecific secretory immunoglobulin A titer in the intestinal lumen declined in senescent animals (Koga et al., 2000). Some studies have also reported that reduced bioavailability of key conditionally essential nutrients might limit immune response in aging (Cunningham-Rundles, 2004) and that well-nourished elderly people appear to have less significant or minimal changes in immune response (Krause et al., 1999).

It is generally accepted that the development of age-associated alterations occurs earlier in the mucosal immune system than in the systemic immune compartment (Schmucker et al., 2003). The mucosal immune system of the intestinal epithelia contains a functionally

specialized T-cell population known as intraepithelial lymphocytes (IELs). Because of their unique location in the mucosal epithelium, IELs are recognized as a first-line mucosal barrier against infectious diseases and food-borne allergens (Hayday et al., 2001).

Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 139

CD45+TCRαβ+ cells were observed in the aged mice, whereas neither an increase nor a decrease was observed in the aged-SP group fed with SpHW—that is, the levels were similar to those in adult mice. In particular, the proportions of CD45+CD8+ cells and CD45+TCRγδ+ cells in the aged-SP group significantly increased in comparison to the aged group. CD8+ T cells expressing αβTCR (αβT cells) are engaged in antigen-specific cell cytotoxicity mediated by major histocompatibility complex (MHC) molecules, whereas T cells expressing γδTCR (γδT cells) often manifest preliminary target cell killing without MHC restriction (Cruse and Lewis, 1995). γδT cells have also been shown to be associated with regulation of the generation and differentiation of IELs (Komano et al., 1995). These results suggest that ingestion of SpHW in the aged-SP group may contribute to the functional preservation of the intestinal epithelium as a first line of mucosal barrier against

Decreased levels of RBCs, especially the level of hematocrit, Ht, in the aged group, were also restored after ingestion of SpHW in the aged-SP group. Significant decreases in WBCs in the aged-SP group, in contrast to the increase in the aged group, may be ascribed to the antiinflammatory activity of *Spirulina* (Vila et al., 2008) and/or to the restoration of immunological function by ingesting *Spirulina*. Some reports indicated that phycocyanin and the polysaccharide isolated from *Spirulina* increased bone marrow nucleated cell and erythrocyte counts in the gamma-ray irradiated mice or dog (Zhang, 1994; Zhang et al., 2001; Verma et al., 2006). Many studies have demonstrated that *Spirulina* including phycocyanin possesses antioxidant activity, as well as an anti-inflammatory activity (Romay et al., 1998; Remirez et al., 2002), which scavenges peroxyl radicals, and also acts as an inhibitor of cyclooxygenase, like nonsteroidal anti-inflammatory drugs. In addition, a downregulation of pro-inflammatory cytokines, such as TNF-α and -γ, was observed in the aged animals on the *Spirulina*-enriched diet (Vila et al., 2008). Overexpression of MHC class Irelated chain A in the intestine of experimental transgenic mice resulted in a clonal expansion of CD4+CD8αα+ IELs and attenuated acute colitis in an experimental model of inflammatory bowel disease induced by dextran sodium sulfate administration (Park et al., 2003). CD8αα+ IELs developed along an extrathymic pathway may work as antiinflammatory regulator T cells to sustain the mucosal intranet formed by intestinal epithelial cells and IELs and to diminish the expansion of enterotoxigenic *Escherichia coli* (Kim et al., 2001). Although ingesting SpHW did not significantly increase the level of CD4+CD8+ IELs in the present study, these facts, in addition to our present results, suggest that ingestion of *Spirulina* appears to be effective for protecting immune functions or improving immune systems vulnerable to age, thereby reducing the risk of infectious and autoimmune diseases.

*Spirulina* and its extracts enhanced proliferation of hematopoietic cells and colony formation of bone marrow cell, as a marker for cell differentiation activity, in *in-vitro* and *in-vivo* study using mice. Phycocyanin, a light-harvesting pigment of *Spirulina*, also induced cell differentiation of human leukemia cell lines, U937 and HL-60 cells, into monocyte/macrophage

infectious agents through retaining the numbers of certain IELs.

However, additional detailed study is needed.

**7. Conclusions**

We have reported that ingestion of phycocyanin enhanced the antigen-specific immunoglobulin A response in the intestinal mucosa of mice (Nemoto-Kawamura et al., 2004). In this section, we investigated age-related changes in intestine IEL subsets in mice by flow cytometric (FCM) analysis and their functional preservation after the animals were fed *Spirulina*.

#### **Characterization of IELs of adult and aged mice**

IELs possess phenotypic features distinct from those of lamina propria lymphocytes in intestine. Lamina propria lymphocytes consist of predominantly activated T cells and are mainly CD4+ and CD8+ single-positive T cells in proportions of about 70% and 30%, respectively. The phenotype of lamina propria lymphocytes, in general, is similar to that of the cells in the peripheral lymphoid tissues and in the circulating blood, that is, over 95% of the cells possess a surface phenotype of T-cell receptor αβ+ (TCRαβ+), whereas less than 5% possess TCRγδ+. These cells are known to be matured in the thymus (Lydyard and Grossi, 1998). IELs, on the other hand, possess TCRγδ+ in a greater percentage (30–60%) and TCRαβ+ in a percentage of 40–70%, which might be related to their state of activation (Ewijk et al., 1999). In adult mice bred in a conventional environment, about half of the IELs have a phenotype of surface CD antigen similar to that of most peripheral T lymphocytes, that is, Thy-1+, TCRαβ+, and either CD4+ or CD8+, which are made up of heterodimers of CD8α and β chains (CD8αβ+). These cells were matured in a thymus-dependent manner (Kaminogawa and Nanno, 2004). Another major IEL population possesses the surface phenotype TCRαβ<sup>+</sup> or TCRγδ+, which expresses CD8 homodimeric α chains (CD8αα+) but does not express CD4 or CD8 heterodimeric molecules (Rocha et al., 1994). These cells are known to be of extrathymic origin. Small percentages of the TCRαβ+ and TCRγδ+ but no TCR cells are CD8– CD4–. TCRαβ+ IELs co-expressing both CD4 and CD8 molecules are rare but bear high levels of TCRαβ and CD8αα (Lefrancois, 1991). Our preliminary experiment showed that the number of CD45+ (leukocyte-common antigen-positive) cells as IELs was significantly lower in aged mice than in adult mice. Either the proportion or the number of CD8+ cells in addition to CD45+ cells of aged mice was significantly lower than that of adult mice, corresponding to the previous article by Komuro et al (1990). The proportion and number of CD4+CD8+ double-positive cells in the aged mice, on the other hand, were higher than those in adult mice. It has been reported that CD4+CD8+ T cells bearing TCRαβ in the epithelium, which were derived from thymus-dependent populations, expanded with aging at a local site of the intestine under the influence of intestinal microflora, contributing to the first line of defensive barrier in the epithelium (Takimoto et al., 1992).

Overall, increased or decreased levels of these surface antigen-positive cells observed in the aged mice tended to be restored by the ingestion of SpHW for 5 weeks in the aged-SP group. In fact, significant decreases of CD45+CD8+ cells and increases of CD8–CD4– and CD45+TCRαβ+ cells were observed in the aged mice, whereas neither an increase nor a decrease was observed in the aged-SP group fed with SpHW—that is, the levels were similar to those in adult mice. In particular, the proportions of CD45+CD8+ cells and CD45+TCRγδ+ cells in the aged-SP group significantly increased in comparison to the aged group. CD8+ T cells expressing αβTCR (αβT cells) are engaged in antigen-specific cell cytotoxicity mediated by major histocompatibility complex (MHC) molecules, whereas T cells expressing γδTCR (γδT cells) often manifest preliminary target cell killing without MHC restriction (Cruse and Lewis, 1995). γδT cells have also been shown to be associated with regulation of the generation and differentiation of IELs (Komano et al., 1995). These results suggest that ingestion of SpHW in the aged-SP group may contribute to the functional preservation of the intestinal epithelium as a first line of mucosal barrier against infectious agents through retaining the numbers of certain IELs.

Decreased levels of RBCs, especially the level of hematocrit, Ht, in the aged group, were also restored after ingestion of SpHW in the aged-SP group. Significant decreases in WBCs in the aged-SP group, in contrast to the increase in the aged group, may be ascribed to the antiinflammatory activity of *Spirulina* (Vila et al., 2008) and/or to the restoration of immunological function by ingesting *Spirulina*. Some reports indicated that phycocyanin and the polysaccharide isolated from *Spirulina* increased bone marrow nucleated cell and erythrocyte counts in the gamma-ray irradiated mice or dog (Zhang, 1994; Zhang et al., 2001; Verma et al., 2006). Many studies have demonstrated that *Spirulina* including phycocyanin possesses antioxidant activity, as well as an anti-inflammatory activity (Romay et al., 1998; Remirez et al., 2002), which scavenges peroxyl radicals, and also acts as an inhibitor of cyclooxygenase, like nonsteroidal anti-inflammatory drugs. In addition, a downregulation of pro-inflammatory cytokines, such as TNF-α and -γ, was observed in the aged animals on the *Spirulina*-enriched diet (Vila et al., 2008). Overexpression of MHC class Irelated chain A in the intestine of experimental transgenic mice resulted in a clonal expansion of CD4+CD8αα+ IELs and attenuated acute colitis in an experimental model of inflammatory bowel disease induced by dextran sodium sulfate administration (Park et al., 2003). CD8αα+ IELs developed along an extrathymic pathway may work as antiinflammatory regulator T cells to sustain the mucosal intranet formed by intestinal epithelial cells and IELs and to diminish the expansion of enterotoxigenic *Escherichia coli* (Kim et al., 2001). Although ingesting SpHW did not significantly increase the level of CD4+CD8+ IELs in the present study, these facts, in addition to our present results, suggest that ingestion of *Spirulina* appears to be effective for protecting immune functions or improving immune systems vulnerable to age, thereby reducing the risk of infectious and autoimmune diseases. However, additional detailed study is needed.

#### **7. Conclusions**

138 Blood Cell – An Overview of Studies in Hematology

**Characterization of IELs of adult and aged mice** 

of defensive barrier in the epithelium (Takimoto et al., 1992).

Overall, increased or decreased levels of these surface antigen-positive cells observed in the aged mice tended to be restored by the ingestion of SpHW for 5 weeks in the aged-SP group. In fact, significant decreases of CD45+CD8+ cells and increases of CD8–CD4– and

*Spirulina*.

specialized T-cell population known as intraepithelial lymphocytes (IELs). Because of their unique location in the mucosal epithelium, IELs are recognized as a first-line mucosal

We have reported that ingestion of phycocyanin enhanced the antigen-specific immunoglobulin A response in the intestinal mucosa of mice (Nemoto-Kawamura et al., 2004). In this section, we investigated age-related changes in intestine IEL subsets in mice by flow cytometric (FCM) analysis and their functional preservation after the animals were fed

IELs possess phenotypic features distinct from those of lamina propria lymphocytes in intestine. Lamina propria lymphocytes consist of predominantly activated T cells and are mainly CD4+ and CD8+ single-positive T cells in proportions of about 70% and 30%, respectively. The phenotype of lamina propria lymphocytes, in general, is similar to that of the cells in the peripheral lymphoid tissues and in the circulating blood, that is, over 95% of the cells possess a surface phenotype of T-cell receptor αβ+ (TCRαβ+), whereas less than 5% possess TCRγδ+. These cells are known to be matured in the thymus (Lydyard and Grossi, 1998). IELs, on the other hand, possess TCRγδ+ in a greater percentage (30–60%) and TCRαβ+ in a percentage of 40–70%, which might be related to their state of activation (Ewijk et al., 1999). In adult mice bred in a conventional environment, about half of the IELs have a phenotype of surface CD antigen similar to that of most peripheral T lymphocytes, that is, Thy-1+, TCRαβ+, and either CD4+ or CD8+, which are made up of heterodimers of CD8α and β chains (CD8αβ+). These cells were matured in a thymus-dependent manner (Kaminogawa and Nanno, 2004). Another major IEL population possesses the surface phenotype TCRαβ<sup>+</sup> or TCRγδ+, which expresses CD8 homodimeric α chains (CD8αα+) but does not express CD4 or CD8 heterodimeric molecules (Rocha et al., 1994). These cells are known to be of extrathymic origin. Small percentages of the TCRαβ+ and TCRγδ+ but no TCR cells are CD8– CD4–. TCRαβ+ IELs co-expressing both CD4 and CD8 molecules are rare but bear high levels of TCRαβ and CD8αα (Lefrancois, 1991). Our preliminary experiment showed that the number of CD45+ (leukocyte-common antigen-positive) cells as IELs was significantly lower in aged mice than in adult mice. Either the proportion or the number of CD8+ cells in addition to CD45+ cells of aged mice was significantly lower than that of adult mice, corresponding to the previous article by Komuro et al (1990). The proportion and number of CD4+CD8+ double-positive cells in the aged mice, on the other hand, were higher than those in adult mice. It has been reported that CD4+CD8+ T cells bearing TCRαβ in the epithelium, which were derived from thymus-dependent populations, expanded with aging at a local site of the intestine under the influence of intestinal microflora, contributing to the first line

barrier against infectious diseases and food-borne allergens (Hayday et al., 2001).

*Spirulina* and its extracts enhanced proliferation of hematopoietic cells and colony formation of bone marrow cell, as a marker for cell differentiation activity, in *in-vitro* and *in-vivo* study using mice. Phycocyanin, a light-harvesting pigment of *Spirulina*, also induced cell differentiation of human leukemia cell lines, U937 and HL-60 cells, into monocyte/macrophage

and granulocyte, respectively, to some extent directly and indirectly through enhancing cytokine production in human peripheral blood lymphocytes stimulated with phycocyanin. These distinguished activities of *Spirulina* as well as other certain functional foods can be preferably emphasized to be used, especially for elderly people. Recent intervention study showed that 6- and 12-week supplementation of *Spirulina* increased mean hemoglobin level and indoleamine 2,3-dioxygenase activity, as a sign of immune function, in the elderly subjects, suggesting that *Spirulina* may ameliorate anemia and immunosenescence in elderly people (Selmi et al., 2011). Pentón-Rol et al. (2011) demonstrated that phycocyanin triggered preventing or downgrading experimental autoimmune encephalitis (EAE) expression in rats, and that ingestion of phycocyanin induced a regulatory T cell (Treg) response in peripheral blood mononuclear cells from the patients with multiple sclerosis (MS). The authors suggested that phycocyanin may act as a neuroprotector and thereby may restore the functional damage in neurodegenerative disorders of the central nervous system (CNS). Another animal model in rats showed that *Spirulina* promoted stem cell genesis and protected against LPS-induced declines in neural stem cell proliferation, and that cytokines did appear capable of regulating several phases of the neurogenesis process, supporting their hypothesis that a diet enriched with *Spirulina* may help protect the stem/progenitor cells from insults (Bachstetter et al., 2010). These studies including reports summarized in this chapter show that *Spirulina* is useful in providing complementary nutrients for modulating or maintaining the immune system and that is also may have potential therapeutic benefits for improvement of immune dysfunctions caused by, for example, radiation, chemotherapy using anti-cancer and anti-infectious drugs, and certain microorganisms such as human immunodeficiency virus (HIV) itself, other than ageing. Further research along these lines is needed to validate these evidences.

Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 141

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#### **Author details**

Osamu Hayashi *Kagawa Nutrition University, Japan* 

#### **8. References**


Balachandran, P, Pugh, ND, Ma, G, and Pasco, DS (2006). "Toll-like receptor 2-dependent activation of monocytes by Spirulina polysaccharide and its immune enhancing action in mice", *Int Immunopharmacol*, 6, 1808-1814.

140 Blood Cell – An Overview of Studies in Hematology

**Author details** 

*Kagawa Nutrition University, Japan* 

Osamu Hayashi

**8. References** 

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**Chapter 8** 

© 2012 Selz, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2012 Selz, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Spontaneous Alternation Behavior** 

**SAB:** Spontaneous alternation behavior (SAB) generally refers to the tendency of animals, even single-celled organisms, to alternate their non-reinforced (Dember & Richman, 1989) choices of T- or Y-maze arms on subsequent trials, following an initial trial or turn. First described over 80 years ago (Tolman, 1925), the phenomenon has been ascribed to the operation of a variety of mechanisms including Hullian reactive inhibition (Solomon, 1948), stimulus satiation (Glanzer, 1953), action decrement (Walker, 1958), curiosity (Dember and Earl, 1957), habituation to novelty (Carlton, 1969), foraging strategies (Estes and Schoeffler, 1955) and spatial working memory (Sarter, et al., 1988). Studies have suggested that the primary cue for alternation among invertebrates to be is the body turn. Vertebrates rely primarily on directional and odor cues. The fitness benefits associated with stimulus seeking and behavioral exploration, foraging, remain the most compelling explanation of why SAB is found ubiquitously and reliably (Richman, et al.1986). Although the underlying mechanism of SAB is open to study, there is general agreement that the ability to alternate

choices requires that the organism remember its previous choice (Hughes, 2004).

Because SAB implicates future behavior which is statistically dependent on prior behavior and accompanied by memorially-dependant loss of degrees of freedom, SAB has been used to suggest the presence of a functional short-term memory. A left or right turn in a T- or Ymaze is a statistical function of the presence and direction of the previous turn, when such a

While theories of memory are many and not the focus of this chapter, SAB is generally suggested to be recent memory dependent in animals complex enough to possess the structures postulated to underlie recent memory, and to be dependent on a more basic sensory/membrane/receptor depletion time-limited memorial mechanism in simple

and reproduction in any medium, provided the original work is properly cited.

**in Human Neutrophils** 

Additional information is available at the end of the chapter

Karen A. Selz

http://dx.doi.org/10.5772/47851

**1. Introduction** 

prior turn exists.
