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Phycocyanin Enhances Secretary IgA Antibody Response and Suppresses Allergic IgE Antibody Response in Mice Immunized with Antigen-Entrapped Biodegradable Microparticles

- Jan 25, 2018 -


Chinami NEMOTO-KAWAMURA1, Tomohiro HIRAHASHI2, Takayuki NAGAI3, Haruki YAMADA3, 

Toshimitsu KATOH2, Osamu HAYASHI1


1Department of Health and Nutrition, Kagawa Nutrition University

2Biochemical Division, Dainippon Ink & Chemicals Inc.

3Kitasato Institute for Life Sciences, Kitasato University




In the present study, we have investigated the effects of phycocyanin, a biliprotein of Spirulina platensis, on mucosal and systemic immune responses and allergic inflammation in C3H/HeN and BALB/cA mice. To induce the antigen-specific antibodies in the peripheral lymphoid tissues such as Peyer's patches and mesenteric lymph nodes, biodegradable ovalbumin-entrapped poly (DL-lactide-co-glycolide) particles were used as an antigen. Two weeks after the onset of phycocyanin ingestion, mice were immunized with an aqueous ovalbumin (OVA) solution. Starting at one week after the primary immunization, the mice were subjected to oral immunization with the biodegradable OVA microparticles twice a week. IgA, IgE and IgG 1 antibodies were determined by ELISA. The OVA icroparticles of 4-μm diameter successfully induced antigen-specific antibodies. In the mice that received phycocyanin treatment for 6 wk, a marked increase in the antigen-specific, as well as the total, IgA antibody level was observed in the Peyer's patches, mesenteric lymph nodes and intestinal mucosa as well as in the spleen cells. Both antigen-specific IgG 1 and IgE antibody levels in the serum were suppressed by ingestion of phycocyanin for 8 wk. However, inflammation of the small intestine, monitored as vascular permeability by the Evans blueleaking method was reduced by phycocyanin at 6 wk, which preceded the suppression of antigen-specific IgG 1 and IgE antibody production by 2 wk. These results suggest that phycocyanin enhances biological defense activity against infectious diseases through sustaining functions of the mucosal immune system and reduces allergic inflammation by the suppression of antigen-specific IgE antibody.

Key Words:phycocyanin, biodegradable microparticles, mucosal immune system, secretory IgA antibody, IgE antibody


The mucosal surface is constantly exposed to food, microorganisms and other foreign objects that function as extrinsic antigens. The mucosal immune system serves as the frontline of defense, and the secretory IgA plays an important role in preventing allergies and infectious diseases (1).

Spirulina platensis, which belongs to the class Cyano phyceae or Cyanobacteria, is used as a health food be cause it is rich in high-quality proteins, vitamins and minerals (2, 3). Oral intake of Spirulina controls blood sugar levels and improves hyperlipidemia and anemia (2, 4), Ingestion of either intact or a hot water extract of Spirulina promotes phagocytic capacity of macro phages, production of interleukin-1, and both cell growth and antibody production in the spleen of mice (5, 6). Moreover, calcium spirulan, one of the compo nents of the hot water extract of Spirulina, can inhibit virus entry (7) and metastasis of melanoma cells to the lung (8).

Spirulina contains phycocyanin, a blue, 270-kDa photosynthetic pigment protein, which accounts for approximately 15% of the dry weight of Spirulina (3). We have previously investigated the effect of phycocya nin ingestion on the immune response of the intestinal mucosa, which plays an important role as one of the body's defense mechanisms against food allergies and infectious diseases. Ingestion of phycocyanin promoted production of total IgA antibody in the intestinal mucosa of the mice immunized with soluble ovalbumin (OVA) as an antigen (9). In Peyer's patches that com prise the peripheral lymphoid tissues, the production of total IgA antibody was also promoted by phycocyanin. However, the antigen-specific IgA antibodies in both Peyer's patches and mesenteric lymph nodes were undetected, probably because an aqueous solution of OVA was used as an antigen. The aqueous OVA antigen could be too easily degraded in the digestive tract to retain functional antigenicity. An antigen that has been entrapped in biodegradable microparticles may circumvent the problem of antigen degradation (10-12).

In the present study, we focused on the study of immune responses in the intestinal mucosa in mice after ingestion of phycocyanin. OVA entrapped in biode gradable microparticles made of poly (DL-lactide-co-gly colide) was used as an antigen, which successfully induced antigen-specific IgA antibodies in the periph eral lymphoid tissues and enabled us to examine the effects of phycocyanin on the mucosal immune re sponse. IgA antibody responses were monitored in the intestinal mucosa, mesenteric lymph nodes, Peyer's patches and spleen cells. The effects of phycocyanin on type I allergieees were studied by measuring serum IgE antibody levels. In addition, the relationship of phyco cyanin ingestion to inflammation as a factor in intesti nal vascular permeability was also investigated.



Preparation of phycocyanin solution. Phycocyanin was extracted from spray-dried Spirulina platensis with 50mm sodium-phosphate buffer (pH 6.0). The crude extract was partially purified by DE-52 ion-exchange chromatography (13). The eluate was dialyzed against distilled water (DW) and lyophilized. Phycocyanin con tents of the resultant powder were over 80%, and the recovery from the crude extract was approximately 6%. The phycocyanin powder was dissolved in DW to a con centration of 0.05%. The solution was then centrifuged, and the supernatant was sterilized by filtration through a 0.20-μm-pore filter.

Preparation of OVA-entrapped microparticles. The OVA - entrapped biodegradable microparticles (OVA micropar ticles) were prepared using the water-in-oil-in-water emulsion solvent evaporation technique according to the method of Jeffery et al. (14). OVA (Albumin, chicken egg, 5×crystalline; Calbiochem, San Diego, CA, USA) was dissolved in DW to a concentration of either 7.5% or 15%, and filter-steril ized. Poly (DL-lactide-co-glycolide, 50:50) (PLG; Sigma Aldrich, St. Louis, MO, USA) was dissolved in dichlo romethane (DCM) to a concentration of 6%. Polyvinyl alcohol (PVA; Sigma-Aldrich) was dissolved in DW to a concentration of either 5% or 10% and filtered. Two milliliters of aqueous OVA solution and 5mL of PLG solution were homogenized in a microhomogenizer (Ultra-Turrax Tl8; IKA, Nara, Japan) at 4,000 to 10,000rpm for 5 to 25s, followed by an additional 50mL of aqueous PVA solution and a second homoge nization at the same rotation speed and duration as the first homogenization. After stirring for 15h to evapo rate DCM, the homogenate was centrifuged at 170×g for 10min. The sediment was rinsed three times with sterile DW by centrifugation and lyophilized. To observe the shape of microparticles and obtain the average value of their diameter, more than 400 microparticles in each batch were observed by scanning electron microscopy. Protein contents of OVA microparticles were deter mined using the method of Jeffery et al. (14). Lyo philized OVA microparticles (5mg) were dissolved in 0.1M NaOH containing 5% sodium dodecyl sulfate. The solution was centrifuged, and the amount of protein in the supernatant was measured using a bicinchoninic acid kit (BCA-kit; Sigma-Aldrich).

Animals, immunization grouping and immunization schedule. Four-week-old female C3H/HeN mice (CLEA Japan Inc., Tokyo, Japan) were divided into 3 groups of 10 animals each. All experiments were performed under the guidelines of the animal usage committee of Kagawa Nutrition University. OVA antigen solution of 0.5mL phosphate-buffered saline (PBS, pH 7.4) con taining 0.5mg of OVA and 1×1010 of inactivated Bor detella pertussis cells (Wako Pure Chemical Industries, Ltd., Osaka, Japan) as an adjuvant was administered to each animal through the intraperitoneal route for the primary immunization. For oral administration, the OVA microparticles were suspended in 0.5mL of PBS so that each animal was given 1mg of OVA antigen in each administration. All animals were fed standard lab oratory chow (Oriental Yeast Co., Tokyo, Japan) ad libi tum and housed at a temperature of 25±1℃, 40-50% relative humidity, and a 12h-light period from 8:00 to 20:00.

The schedules for phycocyanin feeding and OVA-anti gen administration were as follows. OVA-phycocyanin groups The animals were given sterile 0.05% phycocyanin solution ad libitum for 2wk prior to the primary immunization, and then a suspen sion of OVA microparticles was administered orally twice a week for 3wk starting at one week after the pri mary immunization. The period of the OVA-microparti cle administration was determined by monitoring OVA -specific IgA antibody levels in the feces, which began to increase at 2wk after the OVA-microparticle adminis tration. The animals were also given phycocyanin solu tion continuously instead of drinking DW during the entire 6wk experimental period.

OVA-H2O group: The animals were given sterile DW ad libitum, but without phycocyanin. The primary and oral immunizations were the same as for the OVA-phy cocyanin group (OVA-Phyc group).

PBS-H2O group (control group).The animals were given sterile DW without phycocyanin ad libitum but no other experimental treatment. PBS was used instead of OVA antigen solution or OVA-microparticle suspen sion.

The three groups of mice consumed almost the same volume of drinking water, that is, 2.3mL per mouse per day on average, which is equivalent to approximately 57.5mg/kg body weight of the ingested phycocyanin. During the preliminary phycocyanin ingestion period, neither diarrhea nor soft stool was observed and the weight gains of mice were identical in all 3 groups. BALB/cA mice (4-wk-old female; CLEA Japan Inc.), which have been often used for the study of allergies such as food allergies and bronchial asthma, were also used to confirm the results on OVA-specific IgGl and IgE in the serum of C3H/HeN mice, Phycocyanin was given for 6 or 8wk to the mice. The other experimental conditions were the same as those described for the treatments of C3H/HeN mice.

Preparation of serum and small intestine mucous. 

Serum: After the final week of phycocyanin ingestion, blood samples were collected from the artery in the left inguinal region of the ether-anesthetized mice and the serum was separated by centrifugation.

Small intestine mucous: After collecting the blood, the entire small intestine from the duodenum to the ileum was excised and severed, on ice, in the longitudi nal direction. After rinsing with ice-chilled PBS to re move the intestinal contents, the mucous was gently scraped off from the inner surface of the severed intes tine. The isolated mucouououous (0.5g of wet weight) was homogenized in 2mL of cold PBS, and the homogenate was centrifuged at 40,000×g for 20min to obtain the supernatant.

Culture supernatants of spleen, mesenteric lymph node and Peyer's patch cells. After collecting the blood, the spleen, mesenteric lymph nodes and Peyer's patches were excised under an aseptic condition. Each specimen was aseptically suspended in Hank's balanced salt solu tion (HBSS; Gibco Co., Grand Island, NY, USA), and the cells were gently dissociated using a homogenizes. The cell suspensions were centrifuged with refrigeration for 5min at 1,300×g. Spleen cell preparation was treated with 0.16M NH4Cl in 0.17M Tris-HCI buffer (pH 7.6) to remove contaminating erythrocytes. After rinsing the cells 3 times with HBSS by centrifugation, each speci men was adjusted to 2×106cells/mL by dilution with RPMI 1640 (Nikken Biomedical Laboratory, Kyoto, Japan) containing 10% FBS (Gibco). Ten microliters of 10% FBS/RPMI 1640 containing 5μg OVA was added to 1mL of each of the cell suspensions, which were then cultured for 4d in a CO2 incubator. Following the incu bation period, the culture supernatants were collected by filtration (0.20μm).

Determination of antibody levels by ELISA. IgA and IgGl antibodies were measured using the method of Takahashi et al. (15). Wells of microtiter plates were coated either with rabbit anti-mouse IgA or IgG 1 (Zymed Lab. Inc., South San Francisco, CA, USA) for total antibody assay, or with OVA solution for assay of antigen-specific antibody. Horseradish peroxidase-con jugated goat anti-mouse IgA or IgGl (Zymed) was used as the secondary antibody. Mouse myeloma IgA or IgGl (Zymed) was used as a standard for the total antibody. Absorbance at 492nm was measured with a micro plate reader, Assays of each sample were performed in duplicate. 

Antigen-specific IgE antibody. Measurement of IgE antibody was performed using the method described previously by Nagai et al. (16). Briefly, wells of micro titer plates were coated with a rat anti-mouse IgE (Pharmingen Inc., Omaha, NE, USA), followed by incu bation for 3h at 37℃. Blocking was performed for 1h at 37℃ with 1% skim milk. The diluted serum sample was added, followed by an overnight incubation at room temperature. The resulting mixture was then incubated with biotin-labeled OVA for 1h at room tem perature. Streptavidin-conjugated β-galactosidase (Calbiochem) was added, followed by incubation for 1h at room temperature. The enzyme substrate, 4-methylum belliferylβ-D-galactopyranoside (Sigma-Aldrich) was added, followed by incubation for 3h at 37℃. Fluores cence (Ex. 355nm, Em. 460nm) was measured with a spectrofluorometer. Assays of each sample were per formed in duplicate. 

Intestinal vascular permeability test using pigment-leak age method. Intestinal vascular permeability was examined according to the method of Kataoka et al. (17) with minor modifications. A solution of 0.5% Evans blue (wako Pure Chemical Industries) in saline was filtered through a 0.2-μm filter for sterilization. After injecting 0.2L of the Evans blue solution into the caudal vein of each C3H/HeN mouse (5 mice per group) in the absence of anesthesia at the completion of the OVA-antigen administration schedule, 0.5mL of 0.1% OVA aqueous solution was immediately adminis tered orally. Thirty minutes later, the animals were sac rificed by cervical dislocation, and the entire small intestine was excised. The isolated intestine was trans ferred to a tube containing 4mL of 100% formaide (wako Pure Chemical Industries) and incubated for 3d at 37℃. The supernatant was collected by centrifuga tion, and the absorbance at 637nm was measured with a colorimeter. 

Statistical analysis. StatView J-5.0 (SAS Institute Inc., Cary, NC, USA) software was used for statistical analysis. Analysis of variance and multiple comparison tests were used for comparisons among the 3 groups, and Fisher's PLSD was used for the multiple comparison test.



OVA microparticles 

The antibody response after administration of OVA - entrapped microparticles of 1-2μm in diameter was not different from that of animals immunized with aqueous solutions of OVA. Therefore, conditions were explored for obtaining slightly larger microparticles. The diameter of the microparticles tended to depend on both the rotating speed of homogenization and the con centration of PVA composing the outermost layer of microparticles, when the concentration of PLG was kept at 6% (14). Four examples of the conditions examined are shown in Table 1. The homogenization at 6,000 rpm increased the particle diameter approximately by 1μm as compared with that at 8,000rpm (columns A and B). Homogenization speeds lower than 6,000rpm tended to increase pitted or collapsed particles and induce sheet-like structures, and a speed higher than 8,000rpm decreased the rate of particle production, in addition to the decrease in particle size. When PVA con centration was increased, the resulting particles became larger (columns A and C). OVA concentrations did not affect the particle size (columns C and D). 

When examined, the effects of the microparticle size on the mucosal responses using microparticles pre pared by the conditions A-D (Table 1), microparticles with 4-μm diameter were most effective in inducing IgA production in the intestinal mucosa and the culture supernatant of Peyer's patches of C3H/HeN mice treated with OVA-microparticles for 7wk (Fig. 1). The OVA-specific IgA antibody level in both tissues was sig nificantly higher in the mice immunized with the OVA microparticles than those immunized with soluble OVA. Especially, a remarkable increase in the OVA-specific IgA antibody level was observed in the culture superna tant of Peyer's patch cells (Fig. 1B). In both the intesti nal mucosa and the culture supernatant of Peyer's patch cells, total IgA antibody level induced by the OVA microparticles was also higher than that by the soluble OVA (Fig. 1A and B). Because the microparticles with 4μm diameter seemed suitable for studying mucosal immune response, they were used in the subsequent experiments. In the batches prepared by condition A shown in Table 1, microparticles were spherical, and their surface was smooth (Fig. 2). There was no evi dence of pitted or collapsed microparticles. More than 60% of the total number of microparticles had a diame ter of 3.5-4.4μm, and 10mg of microparticles con tained approximately 1.5mg of OVA.


Table 1. Conditions for preparation of microparticles of varying diameter.jpg


Fig. 1. Total and OVA-specific IgA antibody levels in the intestinal mucosa.jpg 


Fig. 1. Total and OVA-specific IgA antibody levels in the intestinal mucosa (A) and Peyer's patch (B) of C3H/HeN mice after oral immunization with either an aqueous OVA solution (soluble OVA) or OVA microparticles (particle OVA). In this exper iment, either soluble OVA or particle OVA was administered to the C3H/HeN mice for 7wk for the oral immunization start ing at one week after the primary immunization. The other experimental conditions were the same as in phycocyanin ingestion experiments. PBS was used instead of OVA for controls. Values of each antibody level are expressed as mean±SD (n=6). **p<0.01 compared with the control and #p<0.05, ##p<0.01 compared to the soluble OVA.


Fig. 2. Scanning electron micrograph of OVA micro particles.jpg 


Effects of phycocyanin on tissue antibody levels

IgA antibody levels in the culture supernatants of lymphoid organs and the intestinal mucosa. In the culture supernatants of the Peyer's patch and mesenteric lymph node cells which were isolated from the C3H/HeN mice treated with phycocyanin for 6 wk, high levels of OVA specific IgA antibody were observed (Fig. 3). The anti body level was nearly 8 times higher than that of OVA H2O group in the Peyer's patches. In the mesenteric lymph nodes, in particular, the antibody was detected only in the OVA-Phyc group. In the intestinal mucosa, both total and OVA-specific IgA antibody levels of the OVA-Phyc group, which was treated with phycocyanin for 6wk, were highest among the three groups, and were significantly higher than that of the OVA-H2O group (Fig. 4). In the culture supernatant of the spleen, the OVA-Phyc group demonstrated the highest levels of antigen-specific and total IgA antibodies (Fig. 5A).

Fig. 3. OVA-specific IgA antibody levels in the cell-cul ture supernatants.jpg 

Fig. 4. Total and OVA-specific IgA antibody levels in the intestinal mucosa of the C3H HeN mice treated with phycocyanin for 6wk.jpg 

Fig. 5. Total and OVA-specific IgA and IgGl antibody levels in the cell-culture supernatants of spleens from the C3H HeN mice treated with phycocyanin for 6wk.jpg 

Fig. 5. Total and OVA-specific IgA and IgGl antibody levels in the cell-culture supernatants of spleens from the C3H/HeN mice treated with phycocyanin for 6wk. Values of each antibody level are expressed as mean±SD (n=5). **p<0.01 compared to PBS-H2O and ##p<0.01 compared to OVA-H2O.



Compared with those 3wk treatments with the OVA microparticles alone, 7wk treatment with the OVA microparticles produced much higher levels of OVA-specific IgA antibody in both Peyer's patches and intestinal mucosa, and total IgA antibody in the mucosa (Figs. 1, 3, and 4). IgGl and IgE antibodies in the spleen and the serum. Both OVA-specific IgGl (Fig. 6A) and IgE antibody (Fig. 6B) levels in the serum of the OVA-Phyc group in C3H/ HeN mice, which was treated with phycocyanin for 6 wk, were almost the same as those of the OVA-H2O group. Total IgGl antibody levels in the culture super natants of the spleen and in the serum were not affected by phycocyanin (Fig, 5B and 6A). In the culture super natant of the spleen cells, however, the OVA-specific IgGl antibody level of the OVA-Phyc group was significantly higher than that of the OVA-H2O  group (Fig, 5B). The levels of both total and OVA-specific IgGl anti bodies in the culture supernatants of mesenteric lymph nodes and Peyer's patch cells were below the detection limit (data not shown).

Fig. 6, Total and OVA-specific IgGl and IgE antibody levels in the serum from the C3H HeN mice treated with phyco cyanin for 6wk.jpg 

Fig. 6. Total and OVA-specific IgGl and IgE antibody levels in the serum from the C3H/HeN mice treated with phyco cyanin for 6wk. Values of each antibody level are expressed as mean±SD (n=5), *p<0,05 compared to PBS-H2O.



Effects of phycocyanin on both IgGl and IgE antibody levels in the serum were also examined using BALB/cA mice. In the mice that were immunized with OVA microparticles and treated with phycocyanin for 6wk (Fig, 7A), both OVA-specific IgGl and IgE levels in the serum were not different form those in the OVA-H2O group, as observed in the C3H/HeN mice (Fig, 6). When phycocyanin treatment was extended to 8wk, however, both serum OVA-specific IgGl and IgE antibody levels were significantly decreased, as compared with the OVA-H2O group (Fig, 7B). In the BALB/cA mice, both total and antigen-specific IgA antibody levels in the mucosa were higher in the OVA-Phyc group than those in the OVA-H2O group in both 6 and 8wk treatments with phycocyanin (data not shown). Intestinal vascular permeability Inflammation in the small intestine of C3H/HeN mice was examined using vascular permeability to Evans blue. As shown in Fig. 8, phycocyanin ingestion for 6wk (OVA-Phyc group) lowered the leakage of the dye from the intestine to the same level as that of the PBS H2O control group.

  Fig. 7. OVA-specific IgGl and IgE antibody levels in the serum of the BALB cA mice treated with phycocyanin for 6 or 8wk. (A) 6wk and (B) 8wk..jpg

Fig. 7. OVA-specific IgGl and IgE antibody levels in the serum of the BALB/cA mice treated with phycocyanin for 6 or 8wk. (A) 6wk and (B) 8wk. Values of each antibody level are expressed as mean±SD (n=6). ** p<0,01 compared to PBS-H2O and +p<0.05,++ p<0.01 compared to OVA-H2O.


Fig. 8. Intestinal vascular permeability in the C3H HeN mice treated with phycocyanin for 6wk..jpg 



In the previous study using an aqueous solution of OVA as an antigen, we failed to induce an antigen-spe cific IgA antibody in Peyer's patches and mesenteric lymph nodes that comprise a major part of the mucosal immune system (9). In the present study, as a result of using antigen-entrapped microparticles, antigen-spe cific IgA antibody was successfully induced in those tis sues. Antigen-entrapped microparticles may be a useful tool to study the mucosal immune responses. Challa combe et al. (12) showed that microparticles of 3μm diameter activate both mucosal and systemic immunity in mice. Uchida et al. (18) reported that among several antigen-entrapped microparticles with diameters rang ing from 1.3 to 14.0μm, microparticles with a 4.0-μm diameter result in the greatest increase in serum anti gen-specific IgGl antibody levels in mice. We reported in the previous paper that microparticles having a diameter of approximately 4μm exhibit strong adhe sion to Peyer's patches (19). Microparticles with this size, in the present study, induced a remarkable increase in OVA-specific IgA antibody in the mucosal system. Therefore our data are consistent with the idea that there is an appropriate particle size that renders the microparticles effective. In addition to the enhance ment of the mucosal antibody response, the OVA micro particles increased in both total IgA and IgGl antibody levels in the spleen and the serum (Figs. 5,6A, and 7A), as well as the OVA-specific antibody. This suggests that the enhancement of the systemic immune response as well as the local immune response in mucous is caused by the particle antigen.

A marked increase in OVA-specific IgA antibody induced by phycocyanin was observed in the intestinal mucosa, and in the Peyer's patches and mesenteric lymph nodes, which comprise a major part of gut-asso ciated lymphoid tissues (GALT), suggesting that phyco cyanin stimulates the inductive sites of the GALT to induce antigen-specific IgA antibody. Contrary to OVA specific IgA antibody, OVA-specific IgE and IgGl anti bodies were decreased by the ingestion of phycocyanin. Such an antagonistic relationship between both anti gen-specific IgG and IgE antibodies to an antigen-spe cific IgA antibody is consistent with a report by Tokuyama et al. (20), in which they reported that mice treated simultaneously with retinoic acid and interleu kin-5 (IL-5) enhance IgA antibody production as a result of enhancing the class switch of B cells to IgA antibody-producing precursor cells, while IgGl anti body is strongly inhibited. The antagonistic antibody behavior produced by phycocyanin suggests that phycocyanin exerts its inhibitory effects against allergy via at least two ways: amplification of IgA production in the mucosal immunity to defend against the invasion of allergens, and suppression of IgE and IgGl production in the systemic immunity to minimize excessive responses to allergens. IL-6 and IL-10 are also known to be involved in the class switching to IgA-antibody-pro ducing precursor cells (21). The isotype class switching to IgA antibody is mediated by TGF-(3, while switching to IgGl and IgE antibodies is induced by IL-4 (21). These cytokines may also be involved in the promotion of IgA antibody production and/or the inhibition of IgGl and IgE antibody production by phycocyanin ingestion. Further experimentation is necessary to ver ify this concept. In the serum and the cultured spleen cells responsible for the systemic immune system, total IgGl-antibody response in both tissues was not affected by phycocyanin. Antigen-specific IgGl level, however, did not behave parallel in these tissues, where the anti body level in the serum was not increased, but produc tion of the antibody was enhanced by phycocyanin in the spleen cells (Figs. 5B and 6A). In the spleen cells, phycocyanin may enhance their antibody response via production of IL-4 and/or increase in the population of IL-4-forming cells since IL-4 production is stimulated in the cultured spleen cells by antigen treatment (22), but the amplification of antibody response by phycocyanin in the spleen cells appears to be not so large as to alter the antigen-specific antibody level in the serum.

In the previous papers, we reported that neither Spirulina-extract nor phycocyanin ingestion for 5wk affected IgE antibody response in C3H/HeJ mice, while IgA response in the mucosa was enhanced significantly (5,9). In the present study for both C3H/HeN and BALB/cA mice, serum OVA-specific IgE, as well as OVA specific IgGl antibody response to phycocyanin was not affected by a 6wk treatment with phycocyanin. How ever, further prolongation of phycocyanin treatment up to 8wk produced significant suppression of OVA-spe cific IgE levels in BALB/cA mice, as compared with OVA-H2O group (Fig. 7B). This suggests that the prolon gation of phycocyanin treatment contributes to the sup pression of OVA-specific IgE antibody response, because the behavior of other antibodies was similar among the strains. Another possibility that must be considered is the difference of the susceptibility to phycocyanin among the strains. That is, BALB/cA mice may be more susceptible to phycocyanin, resulting in the enhance ment of suppressor T cells or Thl functions and/or sup pression of the Th2 function. To this day, however, there has been no supportive evidence to back this claim.

Remirez et al. (23) reported that in rats phycocyanin prevents allergic dermatitis by inhibiting the release of histamine caused by compound 48/80, a histamine releaser. Romay et al. (24) also reported that phycocya nin administration before application of arachidonic acid prevents the inflammatory edema in the ears of mice, reduces the production of prostaglandin E2 and leukotriene B4, and also inhibits the activity of cycloox ygenase, an enzyme that synthesizes prostaglandins from arachidonic acid in the mast cells. In the present study, intestinal vascular permeability in C3H/HeN mice was decreased significantly by a 6-week treatment with phycocyanin, and the vascular permeability decrease proceeded by 2wk the suppression of antigen specific IgE antibody that was observed at 8wk of phy cocyanin ingestion. This suggests that phycocyanin may alleviate the inflammation independent of an IgE antibody. Specifically, phycocyanin may suppress the inflammation through a process that occurs prior to the activation of the system that results in suppression of the antigen-specific antibody production. 

The results of the present study have revealed that phycocyanin amplifies the mucosal immune response, particularly the mucosal IgA antibody response, inhib its the production of antigen-specific IgE antibodies, and reduces allergic inflammation. Spirulina products con taining phycocyanin are not only useful dietary supple ments, but also strengthen the defense mechanisms against infectious diseases, food allergies and other inflammatory diseases.



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