**Morphology and flow cytometric assay of cell surface antigens on U937 and HL-60 Cells**

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

stimulated with Phyc and Cont-CM were promyelocyte-like, while all-*trans* retinoic acid (ATRA) induced cells to differentiate into granulocytes (Figure 6b).

Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 133

the other hand, Phyco-CM increased FcγR positive cells significantly, 41%, compared with Cont and Cont-CM, 24% and 20%, respectively (Figure 8b). Further, a final concentration of 0.01 mg/mL of Phyc significantly increased the population of CD11b-antigen positive cells in U937 cells compared with Cont and Cont-CM (Figure 9a). Although the fluorescence intensity of anti-CD14 antibody per cell in U937 cells stimulated with Phyc was marginally higher than that of Cont (Figure 7a), the ratio of CD14-antigen positive cells was low (Figure 8a) and morphologically comprised 3.4% of monocytes/macrophages. This suggests that phycocyanin stimulates U937 cells to some extent to differentiate or express some CD antigens such as CD11b and CD14. CD11b as well as CD66b is specific in monocytes and granulocytes. Expression of CD11b is known to be up-regulated during granulocytic and monocytic differentiation, and is used as a marker of differentiation of myelomonocytic lineage (Lubbert et al., 1991). It has been also recognized that morphological changes of differentiating U937 cells are accompanied by cellular adherence and are paralleled by an expression of the β2 integrins, CD11a, CD11c, CD18, and particularly CD11b (Hass et al., 1989). CD11b glycoprotein represents the α-subunit of a heterodimeric association with the common β-subunit CD18 in β2 integrin, an adhesion molecule. Their extracellular domains with the CD11b/CD18 (CR3/Mac-1) β2 integrin contribute to adhesion to adjacent cells, for example, the regulation of leukocyte-endothelial cell interactions (Ebnet et al., 2004). In the study using stably-transfected U937 cells with a vector containing the β2 integrin gene in antisense orientation, Otte et al. (2011) suggested that induced adherence predominantly mediated by a functional CD11b/CD18 integrin contributed to cell cycle regulation and apoptosis during monocytic maturation. Concerning apoptotic cell death, photodynamic therapy (PDT) for tumors which is based on the tumor-selective accumulation of protoporphyrin IX (PpIX), as a photosensitizer after addition of 5-aminolevulinic acid (ALA) followed by irradiation with visible light has been demonstrated by some investigators, and it was reported that ALA-based photodynamic action (PDA) induced apoptotic cell death in U937 cells through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors (Amo et al., 2009). Furthermore pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death (Kaszuba-Zwoinska et al., 2010). They reported that U937 cells exposed to a pulsating magnetic field 50Hz, 45±5 mT three times for each 3 h with 24 h intervals induced cells death in higher cell density and conversely prevented puromycin-induced cell death. We could take advantage of the halfway differentiated U937 cells induced by phycocyanin as a cell culture model of cell differentiation and apoptotic cell

death to investigate the molecular mechanism of these various tumoricidal treatments.

stimulated with Phyc, Phyco-CM and other CMs (Figure 9b).

Both Phyc and Phyco-CM significantly increased the ratio of CD11b-antigen positive cells in U937 cells, about 30%, in comparison with those of Cont and Cont-CM, 12% and 22%, respectively, while in HL-60 neither CD11b nor CD66b cells showed significant increases in ratio when stimulated with Phyco-CM (16%, 22%) or Cont-CM (10%, 18%) cells. In contrast, the ratio of CD15-antigen positive cells in the U937 cells was low regardless of stimulation (Figure 9a). CD15-antigen is generally characteristic of granulocytes and monocytes. The ratio of CD15-antigen positive cells in the HL-60 cells showed insignificant changes when

Cell morphology was measured under light microscopy. U937 cells (a) and HL-60 cells (b), 0.5 x 106 cells/mL of medium, were cultured for 3 days with RPMI-FBS (Cont.)

(1); with Phyc (2); with PMA (3); with Phyco-CM (4); with Cont-CM (5); with ATRA (6) (Ishii et al., 2009). (Original magnification x 1000)

**Figure 9.** Morphology of U937 and HL-60 cells stimulated with Phyco-CM and others.

Flow cytometric assay was carried out using a Flow Cytometer (FCM; EPICS® ALTRA™, Beckman Coulter, Inc., Fullerton, CA). The expression of cell surface antigens, CD14, CD11b, CD66b and CD15, on U937 and HL-60 cells stimulated with various CMs, as described above, were determined by direct immunofluorescence method using appropriate fluorescent labeled monoclonal antibodies. Typical patterns of FCM analysis for CD14 antigen in both U937 and HL-60 cells stimulated with Phyco-CM were shown in Figure 7.

Ratio of CD14-antigen positive cells in U937 cells stimulated with Phyco-CM, 53%, was significantly high in comparison with those of Cont-CM and Cont without stimulation, 30% and 15%, respectively, while ratio of CD14-positive cells in HL-60 cells was originally low but was significantly increased by Phyc and Phyco-CM stimulations, about 20% (Figure 8a). Ratio of FcγR positive cells in U937 cells was originally high and those of the cells stimulated with Phyco-CM and Cont-CM were almost the same as Cont without stimulation, 65%. In HL-60, on

medium, were cultured for 3 days with RPMI-FBS (Cont.)

(Original magnification x 1000)

Figure 7.

stimulated with Phyc and Cont-CM were promyelocyte-like, while all-*trans* retinoic acid

Cell morphology was measured under light microscopy. U937 cells (a) and HL-60 cells (b), 0.5 x 106 cells/mL of

Flow cytometric assay was carried out using a Flow Cytometer (FCM; EPICS® ALTRA™, Beckman Coulter, Inc., Fullerton, CA). The expression of cell surface antigens, CD14, CD11b, CD66b and CD15, on U937 and HL-60 cells stimulated with various CMs, as described above, were determined by direct immunofluorescence method using appropriate fluorescent labeled monoclonal antibodies. Typical patterns of FCM analysis for CD14 antigen in both U937 and HL-60 cells stimulated with Phyco-CM were shown in

Ratio of CD14-antigen positive cells in U937 cells stimulated with Phyco-CM, 53%, was significantly high in comparison with those of Cont-CM and Cont without stimulation, 30% and 15%, respectively, while ratio of CD14-positive cells in HL-60 cells was originally low but was significantly increased by Phyc and Phyco-CM stimulations, about 20% (Figure 8a). Ratio of FcγR positive cells in U937 cells was originally high and those of the cells stimulated with Phyco-CM and Cont-CM were almost the same as Cont without stimulation, 65%. In HL-60, on

(1); with Phyc (2); with PMA (3); with Phyco-CM (4); with Cont-CM (5); with ATRA (6) (Ishii et al., 2009).

**Figure 9.** Morphology of U937 and HL-60 cells stimulated with Phyco-CM and others.

(ATRA) induced cells to differentiate into granulocytes (Figure 6b).

the other hand, Phyco-CM increased FcγR positive cells significantly, 41%, compared with Cont and Cont-CM, 24% and 20%, respectively (Figure 8b). Further, a final concentration of 0.01 mg/mL of Phyc significantly increased the population of CD11b-antigen positive cells in U937 cells compared with Cont and Cont-CM (Figure 9a). Although the fluorescence intensity of anti-CD14 antibody per cell in U937 cells stimulated with Phyc was marginally higher than that of Cont (Figure 7a), the ratio of CD14-antigen positive cells was low (Figure 8a) and morphologically comprised 3.4% of monocytes/macrophages. This suggests that phycocyanin stimulates U937 cells to some extent to differentiate or express some CD antigens such as CD11b and CD14. CD11b as well as CD66b is specific in monocytes and granulocytes. Expression of CD11b is known to be up-regulated during granulocytic and monocytic differentiation, and is used as a marker of differentiation of myelomonocytic lineage (Lubbert et al., 1991). It has been also recognized that morphological changes of differentiating U937 cells are accompanied by cellular adherence and are paralleled by an expression of the β2 integrins, CD11a, CD11c, CD18, and particularly CD11b (Hass et al., 1989). CD11b glycoprotein represents the α-subunit of a heterodimeric association with the common β-subunit CD18 in β2 integrin, an adhesion molecule. Their extracellular domains with the CD11b/CD18 (CR3/Mac-1) β2 integrin contribute to adhesion to adjacent cells, for example, the regulation of leukocyte-endothelial cell interactions (Ebnet et al., 2004). In the study using stably-transfected U937 cells with a vector containing the β2 integrin gene in antisense orientation, Otte et al. (2011) suggested that induced adherence predominantly mediated by a functional CD11b/CD18 integrin contributed to cell cycle regulation and apoptosis during monocytic maturation. Concerning apoptotic cell death, photodynamic therapy (PDT) for tumors which is based on the tumor-selective accumulation of protoporphyrin IX (PpIX), as a photosensitizer after addition of 5-aminolevulinic acid (ALA) followed by irradiation with visible light has been demonstrated by some investigators, and it was reported that ALA-based photodynamic action (PDA) induced apoptotic cell death in U937 cells through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors (Amo et al., 2009). Furthermore pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death (Kaszuba-Zwoinska et al., 2010). They reported that U937 cells exposed to a pulsating magnetic field 50Hz, 45±5 mT three times for each 3 h with 24 h intervals induced cells death in higher cell density and conversely prevented puromycin-induced cell death. We could take advantage of the halfway differentiated U937 cells induced by phycocyanin as a cell culture model of cell differentiation and apoptotic cell death to investigate the molecular mechanism of these various tumoricidal treatments.

Both Phyc and Phyco-CM significantly increased the ratio of CD11b-antigen positive cells in U937 cells, about 30%, in comparison with those of Cont and Cont-CM, 12% and 22%, respectively, while in HL-60 neither CD11b nor CD66b cells showed significant increases in ratio when stimulated with Phyco-CM (16%, 22%) or Cont-CM (10%, 18%) cells. In contrast, the ratio of CD15-antigen positive cells in the U937 cells was low regardless of stimulation (Figure 9a). CD15-antigen is generally characteristic of granulocytes and monocytes. The ratio of CD15-antigen positive cells in the HL-60 cells showed insignificant changes when stimulated with Phyc, Phyco-CM and other CMs (Figure 9b).

Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 135

**# # #** **# # #**

\* \* \*

\* \*

**+ + +** **# # #** **# # #**

**+ +**

**Cont Phyc Cont-CM Phyco-CM PMA ATRA**

Data analysis was based on examination of 5000 cells/sample.

**# # # # # #**

Cont-CM. Each value shows mean ± SD, n=3~7 (Ishii et al., 2009)

stimulated with Phyco-CM and other stimulants.

**# # #** **# # #**

**+ + +** **# # #**

CM and other stimulants.

**positive cells (%)**

**positive cells (%)**

++; p < 0.01, +++; p < 0.001 to each Cont, \*\*; p < 0.01, \*\*\*; p < 0.001 to each Cont-CM, ###; p < 0.001 to each Cont and

**a. U937 cells b. HL-60 cells**

**a. U937 cells b. HL-60 cells**

**Figure 11.** Percentage of CD14 and FcγR positive cells in U937 and HL-60 cells stimulated with Phyco-

**CD14+ FcγR+ CD14+ FcγR+**

**Cont Phyc Cont-CM Phyco-CM PMA ATRA**

**# # #**

> **# #**

**# #**

**# #** **# # #** **# # #** **# # #**

Data analysis was based on examination of 5000 cells/sample. Each value shows mean ± SD, n= 3 (Ishii et al., 2009) **Figure 12.** Percentage of CD11b-, CD15- and CD66b-antigen positive cells in U937 and HL-60 cells

**CD11b CD15 C11b CD15 CD66b**

Differentiation of both HL-60 and U937 cells was also assessed by cytochemical analysis with specific and non-specific esterase (SE/NSE) double staining method and Nitro-blue tetrazolium (NBT) reducing activity which is characteristic of phagocytic cells (Table 2).

**Phagocytic activity and TNF-α production, cytochemical analysis** 

**+** \*

Patterns of U937 and HL-60 cells (0.5 x 106 cells/mL of medium) stimulated with Phyc (1), Cont-CM (2), Phyco-CM (3) and PMA (4) were shown in solid lines and that of Cont was shown in dotted lines (Ishii et al., 2009).

**Figure 10.** Typical patterns of CD14-positive cells in U937 and HL-60 cells.

Data analysis was based on examination of 5000 cells/sample.

134 Blood Cell – An Overview of Studies in Hematology

Patterns of U937 and HL-60 cells (0.5 x 106 cells/mL of medium) stimulated with Phyc (1), Cont-CM (2), Phyco-CM (3)

and PMA (4) were shown in solid lines and that of Cont was shown in dotted lines (Ishii et al., 2009).

**Figure 10.** Typical patterns of CD14-positive cells in U937 and HL-60 cells.

++; p < 0.01, +++; p < 0.001 to each Cont, \*\*; p < 0.01, \*\*\*; p < 0.001 to each Cont-CM, ###; p < 0.001 to each Cont and Cont-CM. Each value shows mean ± SD, n=3~7 (Ishii et al., 2009)

**Figure 11.** Percentage of CD14 and FcγR positive cells in U937 and HL-60 cells stimulated with Phyco-CM and other stimulants.

Data analysis was based on examination of 5000 cells/sample. Each value shows mean ± SD, n= 3 (Ishii et al., 2009) **Figure 12.** Percentage of CD11b-, CD15- and CD66b-antigen positive cells in U937 and HL-60 cells

**Phagocytic activity and TNF-α production, cytochemical analysis** 

stimulated with Phyco-CM and other stimulants.

Differentiation of both HL-60 and U937 cells was also assessed by cytochemical analysis with specific and non-specific esterase (SE/NSE) double staining method and Nitro-blue tetrazolium (NBT) reducing activity which is characteristic of phagocytic cells (Table 2).

Although U937 cells were originally NSE positive, the Phyco-CM stimulated cells showed equally high ratio, while most HL-60 cells were SE positive under all stimulants. Phyco-CM stimulation significantly increased the ratio of NBT-positive cells in both U937 and HL-60 cells, while that of Cont-CM was almost the same as Cont. In addition to that, phagocytic activity in U937 cells stimulated with Phyco-CM was significantly higher than that of the cells stimulated with Cont-CM. In HL-60 cells, both Phyco-CM and Cont-CM increased phagocytic activity, as compared with Cont. Increased levels of TNF-α in both U937 and HL-60cells stimulated with Phyco-CM, were relatively high in comparison with Cont-CM but not significant, and were not synergistically increased by supplementation with LPS (1000 ng/mL) in the culture. Level of TNF-α in the cells stimulated with Phyc was under the detection limit.

Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 137

monocyte/macrophage activity (Tanaka et al., 1983), and Chiao et al. (1981) reported that conditioned medium of normal human peripheral blood lymphocytes induced HL-60 cells into macrophage and monocyte-like cell lines, but most HL-60 cells stimulated with Phyco-CM in the present study were SE positive, and only about 15% of monocytic matured cells were found in the cells after Phyco-CM stimulation. It appeared that the effects of Phyco-

CD14 antigen has been reported to be a receptor for the complex of LPS and LPS-binding protein (LBP). It is known that LPS and Gram negative bacteria as triggers (Beutler, 2000; Lu et al., 2008) can cause TNF-α release in human monocytes through TLR4 (Tudhope et al., 2008). The U937 cells stimulated with Phyco-CM, that showed high ratio of CD14-positive cells, are expected to express TLR4 in addition to CD4 because expression of TLR4 needs CD14 and LBP in response to the binding of LPS with LBP. Phyco-CM induced TNF-α production in the culture supernatants of U937 and HL-60 cells. A high molecular weight polysaccharide, Immulina, from *Spirulina* was a potent activator of nuclear factor-kappaB (NF-κB) and induced both IL-1β and TNF-α mRNAs in THP-1 human monocytes (Pugh et al., 2001), and expression of TLR2 and CD14 probably contributed to the NF-κB activation and immune enhancing activity of the Immulina in mice (Balachandran et al., 2006). The levels of TNF-α, however, were not further increased with LPS stimulation (1000 ng/mL) in the U937 cells stimulated with phyco-CM. Phagocytic activity in the stimulated U937 cells was significantly higher than that of the cells stimulated with Cont-CM, and there was no stimulatory effect in the existence of LPS. Phyc alone did not induce TNF-α in U937 and HL-

**6. Age-related changes in intestine intraepitherial lymphocyte subsets** 

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

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

**and their functional preservation by** *Spirulina* **in mice** 

minimal changes in immune response (Krause et al., 1999).

CM on HL-60 cells may insufficiently induce to matured cells.

60 cells.


Phagocytic activity was determined by ingestion of opsonin-treated latex beads. NBT reducing activity was measured as percentage of the positive cells, which contained intracellular blue-black formazan deposits. Positive cells out of 200 cells were counted under light microscopy. ++; p < 0.01, +++; p < 0.001 compared to Cont, \*\*; p < 0.01, \*\*\*; p < 0.001 compared to Cont-CM, < ; under detection limit (15.6 pg/mL), ND; not detected (values are mean ± SD, n=3)

**Table 2.** Cytochemical analysis of U937 and HL-60 cells stimulated with Phyco-CM

Phyco-CM and Phyc quite significantly increased the population of CD14-antigen positive cells in HL-60 cells, although the level was lower than that in U937 cells (Figure 8). In addition to that, Phyco-CM increased also the NBT reducing activity of HL-60 cells. Tamagawa et al. (1998) reported that NBT reducing activity was used as a marker of HL-60 cell differentiation into granulocytes and monocytes. Fontana et al. (1981) suggested that HL-60 cells were able to commit themselves to the development of two different program of hematopoietic differentiation, that is, either myeloid or macrophage depending on cytokine or stimulant. In fact, both monocytic and granulocytic cells coexisted in HL-60 cells stimulated with Phyc and Phyco-CM as the result of differentiation in two types of directions. However, NSE and SE ratios in HL-60 cells did not necessarily correspond to morphological observation. Normally NSE has been thought to be specific for monocyte/macrophage activity (Tanaka et al., 1983), and Chiao et al. (1981) reported that conditioned medium of normal human peripheral blood lymphocytes induced HL-60 cells into macrophage and monocyte-like cell lines, but most HL-60 cells stimulated with Phyco-CM in the present study were SE positive, and only about 15% of monocytic matured cells were found in the cells after Phyco-CM stimulation. It appeared that the effects of Phyco-CM on HL-60 cells may insufficiently induce to matured cells.

136 Blood Cell – An Overview of Studies in Hematology

detection limit.

U937

HL-60

Although U937 cells were originally NSE positive, the Phyco-CM stimulated cells showed equally high ratio, while most HL-60 cells were SE positive under all stimulants. Phyco-CM stimulation significantly increased the ratio of NBT-positive cells in both U937 and HL-60 cells, while that of Cont-CM was almost the same as Cont. In addition to that, phagocytic activity in U937 cells stimulated with Phyco-CM was significantly higher than that of the cells stimulated with Cont-CM. In HL-60 cells, both Phyco-CM and Cont-CM increased phagocytic activity, as compared with Cont. Increased levels of TNF-α in both U937 and HL-60cells stimulated with Phyco-CM, were relatively high in comparison with Cont-CM but not significant, and were not synergistically increased by supplementation with LPS (1000 ng/mL) in the culture. Level of TNF-α in the cells stimulated with Phyc was under the

Stimulators Phagocytic activity (%) TNF-α (pg/mL) NBT reducing activity (%)

Cont 18 ± 5.2 ND 1.3 ± 0.3 Phyc 23 ± 5.1 < 15.6 5.0 ± 3.0 Cont-CM 32 ± 5.5 ++ 46 ± 31 1.3 ± 0.8 Phyco-CM 81 ± 13 +++, \*\*\* 66 ± 15 7.5 ± 4.1\*\* PMA 96 ± 4.6 +++, \*\*\* 968 ± 150 \*\*\* 5.2 ± 2.9

Cont 17 ± 6.2 ND 0.7 ± 0.3 Phyc 16 ± 3.8 < 15.6 2.0 ± 1.0 Cont-CM 35 ± 12 ++ 32 ± 29 2.1 ± 0.7 Phyco-CM 36 ± 13 ++ 71 ± 18 5.3 ± 1.9+++, \*\* PMA 84 ± 13 +++ 585 ± 39 1.7 ± 0.8 ATRA 72 ± 5.2 +++ ND 10.8 ± 3.3 +++, \*\*\* Phagocytic activity was determined by ingestion of opsonin-treated latex beads. NBT reducing activity was measured as percentage of the positive cells, which contained intracellular blue-black formazan deposits. Positive cells out of 200 cells were counted under light microscopy. ++; p < 0.01, +++; p < 0.001 compared to Cont, \*\*; p < 0.01, \*\*\*; p < 0.001 compared to Cont-CM, < ; under detection limit (15.6 pg/mL), ND; not detected (values are mean ± SD, n=3)

**Table 2.** Cytochemical analysis of U937 and HL-60 cells stimulated with Phyco-CM

Phyco-CM and Phyc quite significantly increased the population of CD14-antigen positive cells in HL-60 cells, although the level was lower than that in U937 cells (Figure 8). In addition to that, Phyco-CM increased also the NBT reducing activity of HL-60 cells. Tamagawa et al. (1998) reported that NBT reducing activity was used as a marker of HL-60 cell differentiation into granulocytes and monocytes. Fontana et al. (1981) suggested that HL-60 cells were able to commit themselves to the development of two different program of hematopoietic differentiation, that is, either myeloid or macrophage depending on cytokine or stimulant. In fact, both monocytic and granulocytic cells coexisted in HL-60 cells stimulated with Phyc and Phyco-CM as the result of differentiation in two types of directions. However, NSE and SE ratios in HL-60 cells did not necessarily correspond to morphological observation. Normally NSE has been thought to be specific for CD14 antigen has been reported to be a receptor for the complex of LPS and LPS-binding protein (LBP). It is known that LPS and Gram negative bacteria as triggers (Beutler, 2000; Lu et al., 2008) can cause TNF-α release in human monocytes through TLR4 (Tudhope et al., 2008). The U937 cells stimulated with Phyco-CM, that showed high ratio of CD14-positive cells, are expected to express TLR4 in addition to CD4 because expression of TLR4 needs CD14 and LBP in response to the binding of LPS with LBP. Phyco-CM induced TNF-α production in the culture supernatants of U937 and HL-60 cells. A high molecular weight polysaccharide, Immulina, from *Spirulina* was a potent activator of nuclear factor-kappaB (NF-κB) and induced both IL-1β and TNF-α mRNAs in THP-1 human monocytes (Pugh et al., 2001), and expression of TLR2 and CD14 probably contributed to the NF-κB activation and immune enhancing activity of the Immulina in mice (Balachandran et al., 2006). The levels of TNF-α, however, were not further increased with LPS stimulation (1000 ng/mL) in the U937 cells stimulated with phyco-CM. Phagocytic activity in the stimulated U937 cells was significantly higher than that of the cells stimulated with Cont-CM, and there was no stimulatory effect in the existence of LPS. Phyc alone did not induce TNF-α in U937 and HL-60 cells.
