SPIRULINA PLATENSIS INHIBITS ANAPHYLACTIC REACTION

LifeSciarw+ Vol. 61, No. 13, pp. 1237-1244,1997 Copyright01597 Elsder ScienceInc. Printedin the USA All rightsreserved oo24-320s/n $17.(KI+ .00

PII S0024-3205(97)00668-1

SPIRULIiVAPLATEAMS INHIBITSANAPHYLACTICREACTION Huh-Nam Yang, Eun-Hee Lee and Hyung-Min Kim Department of Oriental Pharmacy, College of Pharmacy Wonkwang University,Iksan, Chonbuk, 570-749, South Korea (Receivedin finalformJune 17,1997) Summary We investigated the effects of the powders of Spirulina platensis (SPP) on anaphylacticreactions. SPP inhibitedcompound 48180-inducedanaphylactic shock 100% with doses of 0.5, and 1.0 mg/g body weight @w). SPP significantly inhibited serum histamine levels induced by compound 48/80 in rats. SPP (0.5 mg/g BW) inhibited to 68.7°/0passive cutaneous anaphylaxis activated by antidinitrophenyl (DNP) IgE. SPP dose-dependently inhibited the histamine release from the rat peritoneal mast cells (RPMC) by compound 48/80. Moreover, SPP had a significant effect on anti-DNP IgE-induced histamine release or tumor necrosis factor-a production from RPMC. These results suggest that SPP may contain compounds with actions that inhibitmast cell degranulationin the rat. KeyWordr: anaphylacticreaetions,Spindinaplatemis,histamine,mastcells Spirdinaplatensis, one of the oldest algae species on earth, has a history of about 3 billion years. Various algae have been used as human food, and it is well known that some of them contain components possessing biological activities. In particular, the blue-green microalgae Spirrdina platensis has been used for regulation of the immune response, and it still occupies an important place in traditional Chinese medicine. The mast cell has long been thought to play a major role in the development of many physiological changes during anaphylactic and allergic responses (l). Among the preformed and newly synthesized inflammatorysubstances released on the degranulation of mast cells, histamine remains the best characterized and most potent vasoactive mediator implicated in the acute phase of type I allergic reactions (2). Mast cell degranulation can be elicited by a number of positively ~harged substances, collectively known as the basic secretagogues of mast cells (3). The most potent secretagogues include the synthetic compound 48/80, polymers of basic amino acids (4). The compound is a mixture of polymers synthesized by condensing N-methyl-p-methoxyphenyl ethykunine with formaldehyde (5), and its hypotensive effect was shown by Paton (6) to be the result of histamine release. Compared with the natural process, a high concentration of compound Corresponding author: H.M. Kim, Department of Oriental Pharmacy, College of Pharmacy, Wonkwang University, Iksan, Chonbu~ 570-749, South Korea, Tel: 82-653-50-6805, Fax: 82653-843-3421, E-mail: [email protected].

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48/80 induces almost a 90% release of histaminefrom mast cells. Thus, an appropriate amount of compound 48/80 has been used as a direct and convenient reagent to study the mechanism of anaphylactic reaction (7). The secreto~ response of mast cells can also be induced by aggregation of their cell surface-specificreceptors for IgE by the corresponding antigen (8-10), It has been established that the anti-IgE antibody induces passive cutaneous anaphylaxis (PCA) reactions as a typical model for the type I hypersensitivity (11). This paper deals with an evaluation of the effect of Spirulina platensis on the compound 48180-induced systemic anaphylactic shock and anti-IgE antibody-inducedcutaneous anaphylacticreaction.

Methods Materials Powders of Spirulina platensis (SPP) were purchased from Spirin Pharmaceutical Factory (Yunnan, China). Compound 48/80, anti-dinitrophenyl(DNP) I@, DNP-human serum albumin (HSA), and metrizarnidewere purchased iiom Sigma Chemical Co. (St. Louis, MO, USA). The et-minimalessential medium (et-MEM)was purchased from Flow Laboratories (Irvine, UK). Fetal calf serum (FCS) was purchased from Gibco Laboratories (Grand Island, NY, USA). Recombinant tumor necrosis factor-a (rTNF-et) (1 x 105 U/ml), and rabbit anti-murine TNF-ct antibody were purchased from Genzyme (Munchen, Germany). Phosphatase-labeled anti-rabbit IgG was purchased from Serotec (Oxford, England). The original stock of Wistar rats was purchased from The Korean Research Institute of Chemical Technology (Taejeon, Chungnarn, Korea), and the animals were maintainedin the College of Pharmacy, Wonkwang University. The animrds were housed five to ten per cage in a laminar air flow room maintained under a temperature of 22+1°C and relative humidityof 55*1O%throughout the study. Compound48/80-induced.ytemic anaphylaxis Rats were given an intraperitoneal injection of 8 ~g/g body weight (BW) of the mast cell degramdator compound 48/80. SPP was dissolved in saline and administered by intraperitoneal injection from 0,005 to 1.0 mg/g BW 1 hr before the injection of compound 48/80. Mortality was monitored for 1 hr after induction of anaphylactic shock After the mortality test, blood was obtained from each rat’sheart. PCA

An IgE-dependent cutaneous reaction was generated by sensitizing the skin with an intradermal injection of anti-DNP IgE followed 48 hr later with an injection of DNP-HSA into the rat’s tail vein. The DNP-HSA was diluted in PBS. The rats were injected intradermally with 100 ~g of anti-DNP IgE into each of 4 dorsal skin sites that had been shaved 48 hr earlier. The sites were outlined with a water-insoluble red marker. Forty-eight hours later each rat received an injection of 1 mg of DNP-HSA in PBS containing4!%.Evans blue (1:4) via the tail vein. SPP (O.1, 0.5 mg/g BW) was orally administered 1 hr before the challenge. Thirty min after the challenge, the rats were sacrificed and the dorsrd skin was removed for measurement of the pigment area. The amount of dye was then determined calorimetricallyafler extraction with 1 ml of 1.0 N KOH and 9 ml of a mixture of acetone and phosphoric acid (5:13) based on the method of Katayama et al. (12). The absorbant intensity of the extraction was measured at 620 nm in a spectrofluorometer, and the amount of dye was calculated with the Evans blue measuring-line.

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Preparation of serum and histaminedetermination The blood was centrifuged at 400 x g for 10 min. The serum was withdrawn and the histamine content was measured by the o-phthalaldehydespectroflurometric procedure of Shore etal. (13). The fluorescent intensitywas measured at 438 nm (excitation at 353 nm) in a spectrofluorometer. Preparation of rat peritoneal mast cells (RPMC) RPMC were isolated as previously described (14). In brief, rats were anesthetized by ether, and injected with 20 ml of Tyrode buffer B (NaCl, glucose, NaHC03, KCI, NaH2P04) containing 0.1% gelatin (Sigma Chemical Co,), into the peritoneal cavity, and the abdomen was gently massaged for about 90 sec. The peritoneal cavity was carefilly opened, and the fluid containing peritoneal cells was aspirated by a Pasteur pipette. Thereafter, the peritoneal cells were sedimented at 150 x g for 10 min at room temperature and resuspended in Tyrode buffer B, Mast cells were separated from the major components of rat peritoneal cells, i.e. macrophages and small lymphocytes, according to the method described by Yurt et a/. (15), In brief, peritoneal cells suspended in 1 ml Tyrode buffer B were layered on 2 ml of 22.5°/0w/v metrizamide (density, 1.120 ghnl, Sigma Chemical Co.) and centrifuged at room temperature for 15 min at 400 x g. The cells remaining at the buffer-metrizamideinterface were aspirated and discarded; the cells in the pellet were washed and resuspended in lml Tyrode buffer A containing calcium. Mast cell preparations were about 95% pure as assessed by Toluidine Blue staining. More than 97% of the cells were viable as judged by Trypan Blue uptake. Inhibition of histamine release Purified mast cells were resuspended in Tyrode buffer A containing calcium for the treatment of compound 48/80. Mast cell suspensions (1 x 106cells/ml) were preincubated at 37°C for 10 min before the addition of compound 48/80 (5 @ml). The cells were preincubated with SPP preparations, and then incubated (10 rein) with compound 48/80. Mast cells (1 x 106 cells/ml) were sensitized with 10 I.@mlanti-DNP IgE for 2 hr and preincubated with SPP at 37°C for 10 min prior to the challenge with DNP-HSA (1 ~g/ml). The reaction was stopped by cooling the tubes in ice. The cells were separated from the released histamineby centrifugation at 400 x g for 5 min at 4°C, Residual histamine in the cells was released by disrupting the cells with perchloric acid and centrifhgation at 400 x g for 5 tin at 4“C. Assay of histaminerelease The inhibitionpercentage of histaminerelease was calculated using the followingequation : 0/0 Inhibition = Histamine release without SPP- Histamine release with SPP x 100 Histamine release without SPP Assay of 7iYF-aproduction TNF-Ct secretion was measured by modification of an enzyme linked immunosorbent assay (ELISA) as described (16). The ELISA was sensitiveto TNF concentrations in the medium above 40 pg/ml. The ELISA was devised by coating 96-well plates with 6.25 ng/well of murine monoclinal antibody with specificity for murine TNT-a. Before use and between subsequent steps in the assay, the coated plates were washed twice with PBS containing 0.05°/0Tween-20

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(PBS-tween) and twice with PBS alone. All reagents used in this assay and the coated wells were incubated for 1 hr at room temperature For the standard curve, r~-a was added to serum previously determined to be negative for endogenous TNF-ct Alter exposure to the medium, the assay plates were sequentially exposed to rabbit anti-TNF-a, phosphatase-conjugated goat antirabbit IgG, and 2, 2’-azinobis, Optical density readings were made within 10 min of the addition of the substrate on a Titertek Muitiscan (F1owLaboratories) with a 405 run filter. Appropriate specificitycontrols were included. ,Vtatistical analy.fis

The results obtained were expressed as mean + S.E for the number of animals. The Student’s ttest was used to make a statistical comparison between the groups, Results with P
TABLE I Effects of SPP on Compound48/80-Induced SystemicAnaphylaxis

SPP addition (mg/g BW)

Compound 48/80 (8 ~g/g BW)

Mortality (%)

None (Saline) 0.005 0,01 0,05 0.1 0.5 1.0 1,0

+ + + + + + + —

100 100 80 60 30 0 0 0

Group of rats were intraperitoneally pretreated with 200 @ srdine or SPP was given at various doses 1 hr before (n=10/group) the compound 48/80 injection. The compound 48/80 solution was intraperitoneally given to the group of rats. Mortality (?’.) within 1 hr followingthe compound 48/80 injection is presented as the No. of dead rats x 100/total No. of experimentalrats.

The ability of SPP to influence compound 48180-induced serum histamine release was investigated. SPP was given with 1.0 mg/g BW 1 hr before (n=7/group) the compound 48/80

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injection. The serum from the rats was unitiormiygathered with a heart puncher 15 tin after the compound 48/80 injection. The inhibitionrate was significant(FwO.01)at the dose of 1.0 mg/g BW (Table II).

TABLEII Effects of SPPon Compound 48/80-Induced Serum Histamine Release

SPP addition (mg/g BW)

Compound48/80 (8 @g BW)

Inhibition(??)

None (Saline) 1.0

+ +

o 62.7*

Groups of rats were intraperitoneally pretreated with 200 I.L1 saline or SPP. The compound 48/80 solution was intraperitoneally given to the group of rats. The drug was given 1 hr before (n=7/group) the compound 48/80 injection. *P43.O1; significantlydifferent from the salinevalues. Another way to test anaphylactic reactions is to induce PCA (17). As described in the Experimental Procedures, local extravaaation is induced by a local injection of anti-DNP IgE followed by an intravenous antigenic challenge. Anti-DNP IgE was injected into the right dorsal skin sites. As a control, the left dorsal skin site of these rats was injected with saline alone. After 48 hr, all animals were injected intravenouslywith DNP-HSA injected with Evans blue dye. The cutaneous anaphylactic reaction was best visualized by the extravasation of the dye. OraJ administration of SPP (0.5 mg/g BW) showed a marked inhibitionrate (68.70/o)in PCA reactions (Table 111),

TABLE111 Effects of SPPon the 48-hr PCAin Rats

SPP addition (mg/g BW)

Amount of dye (8 ~g/g BW)

None (Saline) 0.1 0.5

14.128H.165 8.369MI.074* 4.426k0.046*

Inhibition(%)

40.8 68.7

The drug was administered orally 1 hr prior to the challenge with antigen. Each amount of dye is presented as the mean k S. E. of four independent experiments. *P
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vol. 61,No. 13,199’7

100 *

*

80-

/i!!1

------------

ft /f IJ If 11

60If 11 II (J

40. 20- ij-----” ------

0 1 ()-2

10-1

100

I 01

SPP (~g/ml) FIG. 1 Effects of SPP on compound 48/80-induced or IgE-mediated histamine release from RPMC. RPMC (1 x 106cellshnl) were preincubated with the drug at 37°C for 10 min prior to incubation with compound 48/80 (.) or prior to the challenge with DNP-HSA (0). *P
TABLE IV Effects of SPP on IgE-Mediated TNF-a. Production from RPMC

SPP addition (~timl)

TNF-a production (rig/ml)

None (Saline) None (Anti-DNP IgE plus DNP-HSA) 0,01

0.23+0.02 0.75M.05 0.53k0.06*

RPMC (2 x 106 cells/ml) were incubated for 4 hr in the absence or presence of SPP before stimulation with DNP-HSA (100 rig/ml). TNF-ct released into the medium is presented as the mean * S. E, of three independent experiments. *P< 0,01; significantlydifferentfrom the salinevalue.

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Discussion Experimental studies in animal models have demonstrated an inhibitory effect of SPP on compound 48/80-induced systemic anaphylaxis and anti-DNP I@-induced PCA reactions. SPP inhibited the serum histamine levels in rats. SPP also inhibited the compound 48/80 or anti-DNP I@-induced histamine release from RPMC. Therefore we simply speculate that these results indicate that anaphylactic degranulation of mast cells is inhibited by SPP. There is absolutely no doubt that stimulation of mast cells with compound 48/80 initiates the activation of a signaltransduction pathway which leads to histamine release. Some recent studies have shown that compound 48/80 and other polybasic compounds are able, apparently directly, to activate Gproteins (18,19). The evidence indicates that the protein is Gi-like and that the activation is inhibited by benzalkoniumchloride (20) Tasaka et al. (21) reported that compound 48/80 increased the permeability of the lipid bilayer membrane by causing a perturbation of the membrane. This result indicates that the membrane permeability increase may be an essential trigger for the release of the mediator from the mast cells. SPP might act on the lipid bilayer membrane afecting the prevention of the perturbation being induced by compound 48/80. The SPP-adrninisteredrats are protected from I@-mediated anaphylaxis. The possible mechanism of protection against anti-DNP I@, while not clear at present, may be evidenced only in particular conditions. Our data demonstrated that SPP inhibited anti-DNP I@-induced TNF-(s production from RPMC. The effect of SPP on mast cell cytokine production in vivo and the relative importance of mast cells as a source of TNF-a during inflammatory and immuneresponses are important areas for tlture studies. The amounts of SPP which have been used (in vitro or in vivo) in this study are quite large, raising the possibility that the active agent or agents in the powder represent a small component of the total mass. In addition, we did not confirmin this study whether SPP can mast cell degradation, including cytotoxic degranulation. Further studies are required to resolve these questions. In conclusion, the results obtained suggest that SPP may contain compoundswith actions that inhibit mast cell degranulation in the rat. Therefore, further work should address the possibilitythat such compounds may rdsobe active in the inhibitionof human mast cell degranulation and, therefore, in the treatment of human allergic disorders. Acknowledgements We wish to thank Kathryn Elliston, an English Instructor at Wonkwang University, for editing this manuscript. This paper was supported by Wonkwng Universityin 1997. References 1 2 3 4 5 6

S.1. WASSERMAN, and D.L. MARQUARDT, Ancphylaxis in Allergv: Principles and Practice. 3rd Ed., 1365, C. V. Mosby Co., St. Louis (1988). L.J. PETERSEN, H. MOSBECH, and P. SKOV, J. Allergy Clin. hmmmol. 97672-679 (1996). D. LAGUNOF, T.W. MARTIN, and G. READ, Annu. Rev. Pharmacol. Toxicol. 23331-351 (1983). M. ENNIS, F.L. PEARCE, and P.M. WESTON, Br. J. Phamacol. 70329-334 (1980). R. BALTZLY, J.S. BUCK, E.J. DE BEER and F.S. WEBB, J. Am. Chem. Soc. 71 13011305 (1949). W.D.M. PATON, Br. J. Phamacol. 6499-508 (1951).

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