**4. Enhancement of proliferation and differentiation of bone marrow cells stimulated with** *Spirulina* **and its extracts**

Immunomodulation properties of *Spirulina* have been widely studied in chickens, prawns and fish, other animals, and humans. Generally, *Spirulina* and its extracts, such as hot-water extracts and phycocyanin, tended to enhance immune functions including mucosal or innate immunity through macrophage and secretions of the related cytokines (Belay, 2002; Hirahashi et al., 2002; Nemoto-Kawamura et al., 2004). Mao et al. (Mao et al., 2000) demonstrated that *Spirulina* stimulated the secretion of IL-1ß and IFN-γ in human peripheral blood mononuclear cells (PBMC) examined to nearly 2.0 and 3.3 times basal levels, respectively, and suggested that *Spirulina* helped balance the production of Th1 and Th2 cytokine stimulation. Phycocyanin, a characteristic photosynthesis pigment protein and an antioxidant in *Spirulina*, has been known to promote the growth of a human myeloid cell line, RPMI 8226 (Shinohara et al., 1988). Liu et al. (2000) reported that phycocyanin inhibited growth of human leukemia K562 cells and enhanced the arrest of the cell growth at G1 phase, suggesting enhancement of differentiation of the cells.

We have reported that *Spirulina* and its extracts enhanced immune responses in mice, mainly through increased production of interleukin-1 (IL-1) in macrophages (Hayashi et al., 1994; Hayashi et al., 1998). In the mice which ingested phycocyanin for 6 weeks, a marked increase of OVA antigen-specific IgA, as well as total IgA level was observed in the Peyer's patches, mesenteric lymph nodes and intestinal mucosa, as well as in the spleen cells (Nemoto-Kawamura et al., 2004). These findings suggest that *Spirulina* or its components such as phycocyanin, affects immune functions by promoting immune competent-cell proliferation or differentiation in lymphoid organs.

Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 127

SP cs PP cs PE cs

**Figure 6.** Bone marrow-cell proliferation by *Spirulina* extracts, SpHW, Phy, and SpCW, and by culture supernatant (CS) of lymphoid organ, spleen (SP) and Peyer's patch (PP), and peritoneal-exudates (PE) cells stimulated with the *Spirulina* extracts (Hayashi et al., 2006) (values are mean ± SD, n = 6)

Medium SpHWPhyc SpCWCont. SpHWPhyc SpCWCont. SpHWPhyc SpCWCont. SpHWPhyc

A.

Colony Cluster

Cont.

0

10

20

30

Number of colonies & clusters per well

40

50

Number of colonies & clusters per well

PWM

B.

SpHW 0.5

Colony Cluster

SpHW 1.0

*Spirulina*

SpHW 2.0

SpHW 4.0

Phyc 0.5

Phyc 1.0

extracts as a stimulant (mg mL-1)

Phyc 2.0

\*

\*

SpCW 1.0

\*

\* \* \*

SpCW 2.0

SpCW 4.0

Fluorescence intesity

**Figure 7.** Bone marrow-cell colony and cluster formation in soft agar assay with the culture supernatant of the spleen cells (a) and peritoneal-exudates cells (b) stimulated with Spirulina extracts. Spleen cells were stimulated with 0.5, 1.0, 2.0 and 4.0 mg Spirulina extract/mL. Peritoneal-exudates cells were stimulated with 2.0 mg Spirulina extract /mL (Hayashi et al., 2006) (values are mean ± SD, n = 3).

Cont. LPS SpHW Phyc SpCW

PE cell culture supernatant

We first investigated the effects of *Spirulina* and its extracts on proliferation of hematopoietic cells of mice and induction of colony-forming activity.

## **Colony-formation of bone marrow cells in** *in-vitro* **study**

*Spirulina* extracts such as a hot-water extract (SpHW), phycocyanin (Phyc), and cell-wall fraction (SpCW) recovered from *Spirulina* treated with 0.1 % sodium dodecyl sulfate to remove cytoplasmic material were used in this study. Culture supernatants of spleen (SP), Peyer's patch (PP), and peritoneal-exudated (PE) cells cultured with 20 μg/mL of the *Spirulina* extracts significantly enhanced proliferation of bone marrow cells (Figure 3). Each of the *Spirulina* extracts, SpHW, Phyc, and SpCW, itself, also directly enhanced proliferation of bone marrow cells in the concentration of 100 μg/mL of culture medium. In addition to that, colony- and cluster-formations of the bone marrow cells supplemented with culture supernatants of the spleen cells stimulated with *Spirulina* extracts, 50–400 μg/mL, were measured by soft agar method. The supernatants of cells cultured with Phyc and SpCW significantly increased the colony- and cluster-formations of the bone marrow cells in comparison to that of control or of the smallest concentration of each extract (Figure 4a). Culture supernatants of PE cells, which consisted of macrophages and lymphocytes in a ratio of about 50 % each and a small ratio of mast cells and neutrophils, also enhanced colony- and cluster-formations (Figure 4b). The numbers of these colonies, however, were almost the same as that by each other culture supernatant. Furthermore, Both granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 3 (IL-3) contents in the culture supernatant or the serum as colony-forming activities were measured by commercially supplied ELISA assay kits. High amounts of GM-CSF or IL-3 were detected in the culture supernatants of the spleen and peritoneal-exudates cells stimulated with the *Spirulina* extracts, especially those with SpCW (Table 1). The amounts of IL-3 in the culture supernatants of the cells stimulated with SpHW and Phyc were relatively high, although colony formation by the supernatant was not so high. Culture supernatant of the cells stimulated with SpCW contained high amounts of GM-CSF but not of IL-3.


**Table 1.** GM-CSF and IL-3 contents in the culture supernatants (SC) of the spleen (SP)and the peritoneal-exudates (PE) cells stimulated with *Spirulina* extracts (values are mean ± SD, N = 3)

not of IL-3.

stimulated with

proliferation or differentiation in lymphoid organs.

cells of mice and induction of colony-forming activity.

**Colony-formation of bone marrow cells in** *in-vitro* **study**

Colonies/well GM-CSF

Control 0.5 ± 0.7 2.0 ± 2.3 <4 <4 47.3 ± 4.0 <3 SpHW 2.8 ± 2.6 33.0 ± 7.1 <4 7.1 76.7 ± 8.0 10.7 Phycocyanin 14.0 ± 5.9 37.3 ± 9.3 9.2 ± 0.7 4.3 94.7 ± 10.8 11.0 SpCW 28.2 ± 5.5 32.2 ±4.6 1,206 ± 333 104.7 481.7 ± 144.4 <3

**Table 1.** GM-CSF and IL-3 contents in the culture supernatants (SC) of the spleen (SP)and the peritoneal-exudates (PE) cells stimulated with *Spirulina* extracts (values are mean ± SD, N = 3)

pg/mL of CS

SP PE SP PE SP PE

IL-3 pg/mL of CS

(Nemoto-Kawamura et al., 2004). These findings suggest that *Spirulina* or its components such as phycocyanin, affects immune functions by promoting immune competent-cell

We first investigated the effects of *Spirulina* and its extracts on proliferation of hematopoietic

*Spirulina* extracts such as a hot-water extract (SpHW), phycocyanin (Phyc), and cell-wall fraction (SpCW) recovered from *Spirulina* treated with 0.1 % sodium dodecyl sulfate to remove cytoplasmic material were used in this study. Culture supernatants of spleen (SP), Peyer's patch (PP), and peritoneal-exudated (PE) cells cultured with 20 μg/mL of the *Spirulina* extracts significantly enhanced proliferation of bone marrow cells (Figure 3). Each of the *Spirulina* extracts, SpHW, Phyc, and SpCW, itself, also directly enhanced proliferation of bone marrow cells in the concentration of 100 μg/mL of culture medium. In addition to that, colony- and cluster-formations of the bone marrow cells supplemented with culture supernatants of the spleen cells stimulated with *Spirulina* extracts, 50–400 μg/mL, were measured by soft agar method. The supernatants of cells cultured with Phyc and SpCW significantly increased the colony- and cluster-formations of the bone marrow cells in comparison to that of control or of the smallest concentration of each extract (Figure 4a). Culture supernatants of PE cells, which consisted of macrophages and lymphocytes in a ratio of about 50 % each and a small ratio of mast cells and neutrophils, also enhanced colony- and cluster-formations (Figure 4b). The numbers of these colonies, however, were almost the same as that by each other culture supernatant. Furthermore, Both granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 3 (IL-3) contents in the culture supernatant or the serum as colony-forming activities were measured by commercially supplied ELISA assay kits. High amounts of GM-CSF or IL-3 were detected in the culture supernatants of the spleen and peritoneal-exudates cells stimulated with the *Spirulina* extracts, especially those with SpCW (Table 1). The amounts of IL-3 in the culture supernatants of the cells stimulated with SpHW and Phyc were relatively high, although colony formation by the supernatant was not so high. Culture supernatant of the cells stimulated with SpCW contained high amounts of GM-CSF but

**Figure 6.** Bone marrow-cell proliferation by *Spirulina* extracts, SpHW, Phy, and SpCW, and by culture supernatant (CS) of lymphoid organ, spleen (SP) and Peyer's patch (PP), and peritoneal-exudates (PE) cells stimulated with the *Spirulina* extracts (Hayashi et al., 2006) (values are mean ± SD, n = 6)

**Figure 7.** Bone marrow-cell colony and cluster formation in soft agar assay with the culture supernatant of the spleen cells (a) and peritoneal-exudates cells (b) stimulated with Spirulina extracts. Spleen cells were stimulated with 0.5, 1.0, 2.0 and 4.0 mg Spirulina extract/mL. Peritoneal-exudates cells were stimulated with 2.0 mg Spirulina extract /mL (Hayashi et al., 2006) (values are mean ± SD, n = 3).

#### **Colony-formation activity in the mice fed** *Spirulina* **in** *in-vivo* **study**

As a preliminary experiment for *in-vivo* study, we measured next colony forming activity in the mice fed with the *Spirulina* extracts, SpHW, Phyc and SpCW, for 5 consecutive days or in the mice treated with an intra-peritoneal single injection of the extracts.

Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 129

in various period of usage in their daily life, and measured correlation between the salivary IgA level and the amount of *Spirulina* ingested (Ishii et al., 1999). Total S-IgA level of the group ingesting *Spirulina* for more than one year was significantly increased (p < 0.01) in comparison to the group ingesting *Spirulina* for less than half a year, and statistically significant correlation between S-IgA levels in the saliva and total amount of *Spirulina* ingested by the subjects was observed (correlation coefficient R = 0.288, n = 72, p < 0.05).

**Figure 8.** Bone marrow-cell colony and cluster formation in soft agar assay with the serum from the mice fed with *Spirulina* extracts for 5 consecutive days (Hayashi et al., 2006) (values are mean ± SD,

Cluster \*

Colony

Normal serum OVA SpHW Phyc SpCW

serum samples

It is known that multi-potent colony-stimulating factors such as G- and GM-CSF and IL-3, which are produced by a variety of cells including monocytes and lymphocytes can support proliferation of immature hematopoietic cells (Ihle, 1992). Liu et al. (2000) reported that phycocyanin from *Spirulina platensis* inhibited growth of human leukemia K562 cells in a dose-dependent manner, arresting them at the G1 phase with increased level of c-*myc* expression, suggesting that phycocyanin may enhance differentiation of the leukemia cells. Seya et al. (Hirahashi et al., 2002; Akao et al., 2009) reported that hot-water extract of *Spirulina* when taken orally in adult human enhances NK activation through the MyD88 pathway via Toll-like receptor (TLR) 2 and TLR4 on myeloid dendritic cells. From these findings, it appeared that *Spirulina*, including its components such as phycocyanin can affect enhancing proliferation or differentiation of immune competent-cells including bone marrow cell, which may cause normally sustaining or enhancing immune functions. Colony-stimulating activity other than IL-3 or GM-CSF, for example arginase and G-CSF, in

the serum may contribute to the cell differentiation, although this is still not clear.

Zhang et al. (Zhang, 1994) found that C-phycocyanin and polysaccharide isolated from *Spirulina* increased leukocyte and bone marrow nucleated cell counts as well as colony formation of colony forming unit-granulocyte and macrophage (CFU-GM) in the gammaray irradiated mice, and also found that C-phycocyanin possessed high erythropoietin activity. Some institution facilities have reported the potential radiation protection effects of *Spirulina* against radiation-induced membrane damage and cellular dysfunction by reactive

n = 3). \*; p<0.05 compared to Normal

Number of colonies & clusters per well

The sera from the mice which ingested Phyc or SpCW (1 mg/0.2 mL) for 5 consecutive days with feeding catheter enhanced colony formation of bone marrow cells (Figure 5). The serum from Phyc-feeding group significantly increased it in comparison to controls in which normal serum was added. All of the sera obtained from the mice which were treated with intra-peritoneal single injection of the *Spirulina* extracts (10 mg/0.5 mL) also showed significantly high colony formation in comparison to control of normal serum, although levels of the activities of the sera were almost the same each other (data were not shown). Colony-stimulating factors, GM-CSF and IL-3, in the sera from the mice which were either fed or intra-peritoneally injected with the extracts, however, were under detection limit (<4 and 3 pg/mL serum, respectively). Concentration of GM-CSF in the LPS serum obtained by *i.p*. injection was 50.1 pg/mL.

For longer-period experiment in *in-vivo* study, colony- and cluster-formation in the bone marrow cells with culture supernatants of the spleen (SP), Peyer's patch (PP), and peritoneal-exudated (PE) cells from the mice, which ingested the *Spirulina* extracts, SpHW, Phyc, and SpCW, for 5 weeks were then measured to confirm the former results. Culture supernatants of each lymphoid-organ, SP, PP, and PE cells from the groups were prepared under stimulation with or without phycocyanin. Colony formation by the culture supernatant of SP cells from the mice of SpHW group, as well as by that of PE cells from Phyc or SpCW group, under stimulation with phycocyanin, was significantly higher than that by each culture supernatant of cells from control group, and thus colony-forming activity was also significantly induced in the blood, spleen, and Peyer's patch cells in the mice which ingested *Spirulina* extracts for 5 weeks although neither significant amount of GM-CSF nor IL-3 was detected in the blood (data not shown). On the other hand, ratios of neutrophils in the SpHW-ingesting group and of lymphocytes in the SpCW-ingesting group were significantly higher than in controls, while ratios of lymphocytes, neutrophils, and monocytes in the peripheral blood of control group were in the normal range. A significant increase in ratio of lymphocytes was also observed in bone marrow cells in Phyc-ingesting group, although the number of cells was small. In addition, increased ratio of reticulocytes was observed in the bone marrow of the mice fed with SpHW.

In the mice ingested 0.05% phycocyanin solution for 6 weeks, a marked increase in the antigen-specific IgA antibody level as well as the total IgA antibody level was observed in the intestinal mucosa, the Peyer's patches and mesenteric lymph nodes, which comprise a major part of the gut-associated lymphoid tissues (GALT), whereas neither IgG1 nor IgE was affected in the spleen cells (Nemoto-Kawamura et al., 2004). Phycocyanin ingestion for 8 weeks, on the other hand, suppressed the production of antigen-specific IgG1 and IgE antibody in the serum. Further, we investigated the effect of *Spirulina* on salivary IgA antibody level of the subjects who customarily ingested the *Spirulina* tablets as health food in various period of usage in their daily life, and measured correlation between the salivary IgA level and the amount of *Spirulina* ingested (Ishii et al., 1999). Total S-IgA level of the group ingesting *Spirulina* for more than one year was significantly increased (p < 0.01) in comparison to the group ingesting *Spirulina* for less than half a year, and statistically significant correlation between S-IgA levels in the saliva and total amount of *Spirulina* ingested by the subjects was observed (correlation coefficient R = 0.288, n = 72, p < 0.05).

128 Blood Cell – An Overview of Studies in Hematology

*i.p*. injection was 50.1 pg/mL.

**Colony-formation activity in the mice fed** *Spirulina* **in** *in-vivo* **study** 

the mice treated with an intra-peritoneal single injection of the extracts.

was observed in the bone marrow of the mice fed with SpHW.

As a preliminary experiment for *in-vivo* study, we measured next colony forming activity in the mice fed with the *Spirulina* extracts, SpHW, Phyc and SpCW, for 5 consecutive days or in

The sera from the mice which ingested Phyc or SpCW (1 mg/0.2 mL) for 5 consecutive days with feeding catheter enhanced colony formation of bone marrow cells (Figure 5). The serum from Phyc-feeding group significantly increased it in comparison to controls in which normal serum was added. All of the sera obtained from the mice which were treated with intra-peritoneal single injection of the *Spirulina* extracts (10 mg/0.5 mL) also showed significantly high colony formation in comparison to control of normal serum, although levels of the activities of the sera were almost the same each other (data were not shown). Colony-stimulating factors, GM-CSF and IL-3, in the sera from the mice which were either fed or intra-peritoneally injected with the extracts, however, were under detection limit (<4 and 3 pg/mL serum, respectively). Concentration of GM-CSF in the LPS serum obtained by

For longer-period experiment in *in-vivo* study, colony- and cluster-formation in the bone marrow cells with culture supernatants of the spleen (SP), Peyer's patch (PP), and peritoneal-exudated (PE) cells from the mice, which ingested the *Spirulina* extracts, SpHW, Phyc, and SpCW, for 5 weeks were then measured to confirm the former results. Culture supernatants of each lymphoid-organ, SP, PP, and PE cells from the groups were prepared under stimulation with or without phycocyanin. Colony formation by the culture supernatant of SP cells from the mice of SpHW group, as well as by that of PE cells from Phyc or SpCW group, under stimulation with phycocyanin, was significantly higher than that by each culture supernatant of cells from control group, and thus colony-forming activity was also significantly induced in the blood, spleen, and Peyer's patch cells in the mice which ingested *Spirulina* extracts for 5 weeks although neither significant amount of GM-CSF nor IL-3 was detected in the blood (data not shown). On the other hand, ratios of neutrophils in the SpHW-ingesting group and of lymphocytes in the SpCW-ingesting group were significantly higher than in controls, while ratios of lymphocytes, neutrophils, and monocytes in the peripheral blood of control group were in the normal range. A significant increase in ratio of lymphocytes was also observed in bone marrow cells in Phyc-ingesting group, although the number of cells was small. In addition, increased ratio of reticulocytes

In the mice ingested 0.05% phycocyanin solution for 6 weeks, a marked increase in the antigen-specific IgA antibody level as well as the total IgA antibody level was observed in the intestinal mucosa, the Peyer's patches and mesenteric lymph nodes, which comprise a major part of the gut-associated lymphoid tissues (GALT), whereas neither IgG1 nor IgE was affected in the spleen cells (Nemoto-Kawamura et al., 2004). Phycocyanin ingestion for 8 weeks, on the other hand, suppressed the production of antigen-specific IgG1 and IgE antibody in the serum. Further, we investigated the effect of *Spirulina* on salivary IgA antibody level of the subjects who customarily ingested the *Spirulina* tablets as health food

**Figure 8.** Bone marrow-cell colony and cluster formation in soft agar assay with the serum from the mice fed with *Spirulina* extracts for 5 consecutive days (Hayashi et al., 2006) (values are mean ± SD, n = 3). \*; p<0.05 compared to Normal

It is known that multi-potent colony-stimulating factors such as G- and GM-CSF and IL-3, which are produced by a variety of cells including monocytes and lymphocytes can support proliferation of immature hematopoietic cells (Ihle, 1992). Liu et al. (2000) reported that phycocyanin from *Spirulina platensis* inhibited growth of human leukemia K562 cells in a dose-dependent manner, arresting them at the G1 phase with increased level of c-*myc* expression, suggesting that phycocyanin may enhance differentiation of the leukemia cells. Seya et al. (Hirahashi et al., 2002; Akao et al., 2009) reported that hot-water extract of *Spirulina* when taken orally in adult human enhances NK activation through the MyD88 pathway via Toll-like receptor (TLR) 2 and TLR4 on myeloid dendritic cells. From these findings, it appeared that *Spirulina*, including its components such as phycocyanin can affect enhancing proliferation or differentiation of immune competent-cells including bone marrow cell, which may cause normally sustaining or enhancing immune functions. Colony-stimulating activity other than IL-3 or GM-CSF, for example arginase and G-CSF, in the serum may contribute to the cell differentiation, although this is still not clear.

Zhang et al. (Zhang, 1994) found that C-phycocyanin and polysaccharide isolated from *Spirulina* increased leukocyte and bone marrow nucleated cell counts as well as colony formation of colony forming unit-granulocyte and macrophage (CFU-GM) in the gammaray irradiated mice, and also found that C-phycocyanin possessed high erythropoietin activity. Some institution facilities have reported the potential radiation protection effects of *Spirulina* against radiation-induced membrane damage and cellular dysfunction by reactive

oxygen species in mice and against reduced levels of the leukocytes in the blood and nucleated cells in the bone marrow in dog (Zhang et al., 2001; Verma et al., 2006). Doctors in Belarus reported that ingestion of 5 g of *Spirulina* a day resulted in the reduction of Cesium-137 in urine by 50%, in children subjected to low level of radiation over a long period of time (Loseva and Dardynskaya, 1993). Rahadiya and Patel in India (Rabadiya and Patel, 2010) also reported radiation-effect-reducing activity of *Spirulina* in their review. Anti-oxidant and anti-inflammatory effects as well as proliferation and differentiation activity of *Spirulina* possibly contribute to the radiation protection effects.

Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 131

**Preparation of Conditioned Medium (CM) of peripheral blood mononuclear** 

Human peripheral blood mononuclear cells (PBMCs) from 7 healthy volunteers were separated by density centrifugation (800 g, 30 mins) using a Lymphoprep™ (Density 1.077 g/mL, NYCOMED) under approving by the Institutional Review Board of our University. Cells from each subject were suspended in RPMI-FBS medium and adjusted to 1 x 106 cells/mL and were cultured with and without 2 mg/mL of phycocyanin (Phyc) using 24-well cultured plates. The conditioned mediums (CMs), both with and without Phyc (Phyco-CM and Cont-CM, respectively), were harvested on the 7th day and filtered through an 0.45

Cell growth of U937 as well as HL-60 cells supplemented with Phyco-CM resulted in significant inhibition during the 7-day culture in comparison with those of control without supplementation, while supplementation with 2 mg/mL Phyc itself had no effect on growth in these cells. Both U937 and HL-60 cells stimulated with Phyc and Phyco-CM were more than 80% viable, as were those cells stimulated with phorbol-12-myristate-13-acetate (PMA)

**Morphology and flow cytometric assay of cell surface antigens on U937 and HL-**

U937 cells cultured in the medium supplemented with Phyco-CM morphologically changed into the cells with large cytoplasm with vacuoles, non-condensed nuclear chromatin, and a persistence of nucleolus that resembled to those stimulated with PMA as a positive control (Figure 6a) while control U937 cells, without stimulation, were promonocyte-like with variable nuclear shapes and regular indentations and comprised moderate cytoplasm containing numerous small eosinophilic granules and a few vacuoles. Cont-CM or conditioned medium of lymphocytes cultured without phycocyanin, only partially changed U937 cells into monocytic cells comprising moderate cytoplasm with large indented nucleus (Figure 6a). U937 cells stimulated with Phyco-CM and Cont-CM consisted of monocytes/macrophages in the ratio of 57% and 21%, respectively, and each ratio was significantly higher than that of control (1.4%) without stimulation. Stimulation by Phyc changed U937 cells partially to promonocytes

with indents on the nuclei. The ratio of monocytes/macrophages was only 3.4 %.

Control HL-60 cells, without stimulation, was predominantly promyelocytes with azurophilic granules, large round nuclei, and prominent nucleoli. Morphological classification of the cells, especially those stimulated with Phyco-CM, was relatively difficult because various features of promyelocytes coexisted. The Phyco-CM-stimulated HL-60 cells showed a morphologically matured monocytic cell lineage (about 15.4%), that is, with decreased nuclear/cytoplasmic ratio and a paler cytoplasm with vacuoles (Figure 6b). The cells (about 80%) other than monocytic cells consisted of granulocytes, including promyelocytes and myelocytes, with large nuclei, less prominent cytoplasmic granules and a marked decrease or complete disappearance of nucleoli. Almost of all HL-60 cells

**cells cultured with phycocyanin** 

μmφ filter to remove cell debris.

**60 Cells** 

0.02 μg/mL as a positive control of differentiation.
