Inflammatory and Immunopharmacological Activities of Meta-periodate Oxidized Zymosan

Zent.bl. Baherio!. 289, 63-77 (1999) © Urban & Fischer Verlag

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Inflammatory and Immunopharmacological Activities of Meta-periodate Oxidized Zymosan Naohito Ohao l , Toshihide Miura" 2, Noriko N. Miura', Norihisa Chiba', Michiharu Uchiyama I, Yoshiyuki Adachi', and Toshiro Yadomae' J 2

School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan Department of Pharmacy, Kitasaro University Hospital, 1 15-1 Kitasato, Sagamihara , Kanagawa 229, Japan

Received May 18, 1998 . Revision received August 27, 1998· Accepted September 8, 1998

Summary Zymosan (ZYM), a strong complement-activating yeast cell preparation composed mainly of mannan and ~-glucan moieties, is a potent inflammatory substance with immunopharmacological activity. We previously analyzed the metabolism of ZYM in mice and found that it was deposited in liver and spleen for at least several months and then gradually oxidatively degraded. In this paper, we prepared oxidized ZYM by sodium metapcriodate oxidation (NaI0 4 ) and borohydride reduction (UB-ZYM) and/or limited hydrolysis of oxidized moieties (l/BIH-ZYM ). Activities of the resulting products wcre assessed by (i) vascular permeability in mice, (ii) H 20 Z synthesis by macrophages, (iii) TNF-a synthesis by macrophages, and (iv) reactivity to anti-ZYM sera. As a general trend, NaI0 4 , oxidation products exhibitcd reduced, but still significant, activity. Interestingly, the HzOz production induced by I1BIH -ZYM was significantly reduced after extensive sonication. Antagonist(s) for H 20 2 synthesis were concomitantly solubilized by sonication of UBIH-ZYM. On the countrary, TNF-u production induced by UBIH-ZYM was comparable with that of ZYM. These facts strongly suggest that highly branched 1,3 -~- and 1,6-~-glucosidic linkages resistant to NaI0 4 oxidation are important for biological activity of ZYM. Further, the minimal structure in ZYM necessary for biological activity may depend on the activity tested.

0934-8840/99/28911-063 $12.00/0

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N. Ohno et al.

Introduction Beta 1,3-glucans are widely distributed in nature. The ~-glucan preparations, lentinan and sonifilan, have been applied clinically for cancer immunotherapy (11 ). Curdlan has been used as a food additive. ~-Glucans from fungal cell walls are a candidate for the causative agent for some allergic pneumonia, such as maple bark disease, farmer's lung, and sick building syndrome (3, 12). However, only limited information about the minimum structure of ~-glucans needed for biological activity is available. It has also been reported that the biological activities of soluble ~-glucans (i. e. sonifilan [SPG; from Schizophyffum commune, - 33 % branched] grifolan [GRN; from Grifola frondosa, - 33 % branched], and SSG [from Sclerotinia sclerotiorum, - 50 % branched]) and particulate ~-glucans (i. e. Zymosan [ZYM] and Zymocel) are significantly different, even though they all have beneficial immunopharmacological activities, such as antitumor activity (4). The particles induce the production of various inflammatory mediators from phagocytes in vivo and in vitro. Our research is focused on clarifying the essential structure necessary for the biological acticity of ~-glucans as well as isolation of specific ~-glucan receptors. Previously, our studies have shown that systemically administered soluble forms of ~-glucans, such as SSG, SPG and GRN, gradually disappeared from the blood stream and are deposited in the body, especially in the liver and spleen, for more than a couple of months without significant structural changes (5, 13). An increase in the glucan contents in the liver and spleen caused by multiple dosing would be due to the deposit of these glucans because of the absence of ~-glucan hydrolase in animals. Analyses of ~-glucans deposited in ICR mice revealed that the molecular weight of ~-glucans is gradually reduced and acidic groups appear in the molecules. This suggests that ~-glucans are mainly degraded by oxidative degradation mediated by the products of phagocytes, such as O 2-, H 20 2, and HOC!. Blocking the functions of phagocytes with gadolinium chloride reduced the degradation rate (14). In addition, we have demonstrated that the side chain moiety of the ~-glucan molecule degrades faster than the main chain moieties in the body. Hypochlorous acid (HOC!) is a strong oxidant derived from H 2 0 2 , with the aid of leukocytederived peroxidases, such as myeloperoxidase (MPO). Macrophages may take advantage of the MPO released from other cells at inflammatory sites to covert H 20 2 to HOCI (15). In a previous paper, a particulate ~-glucan, Zymosan (Zymosan A from Sigma, ZYM) wasoxidized by sodium hypochlorite (NaCIO ), and the oxidized products were analyzed by gel filtration and ion-exchange chromatographies and by Limulus G-test to estimate the metabolism of ~-glucans in vivo (6). ZYM is a particle suspension prepared from Saccharomyces cerevisiae and is composed mainly of ~-glucan and some mannan (12). ZYM was partially solubilized and converted into a high molecular weight polysaccharide by NaCIO treatment. The remaining particulate component of oxidized ZYM was mainly composed of 1,3 - ~- and 1,6-~-glucans with a relatively low ratio

Activity of Oxidized Zymosan

65

of branching point (manuscript in preparation). In preliminary experiments, NaClO-oxidized ZYM lost almost all of its inflammatory and/or immunopharmacological activities. Thus, milder and specific oxidation, such as periodate oxidation, was required to analyze the essential structural components of ZYM necessary for biological activity. In this paper, we present additional evidence for the essential structure of ZYM for immunopharma-/immunotoxicological activities using NaIO, oxidation.

Materials and Methods Oxidative degradation of ZYM by NaT0 4 ZYM (500 mg), suspended in 50mM sodium acetate buffer pH 5.0 (40 ml), was mixed with sodium metaperiodate to a final concentration of 20mM and incubated at 4 tiC in the dark for 1 day. After incubation, excess periodare was removed by adding ethy-

leneglycol (2 ml ). Oxidized ZYM was collected by centrifugation (2500 rpm, 15 min) and washed several times with distilled water. The resulting oxidized ZYM was sus-

pended in distilled water (40 ml) and reduced with sodium borohydride (final concen-

tration of 1 mM ) for 2 h at room temperature. Particles were collected by centrifuga-

tion and washed with 0.5 M acetic acid and distilled water, and then lyophilized (IIBZYM, yield 270 mg). The supernatant (sup-l ) was dialyzed and then lyophilized. Half of the IIB-ZYM (135 mg) was suspended in 0.1 M sulfuric acid and partially hydrolyzed at 20 °C for 15 h. The resulring particles were collected by centrifugation, washed

with 0.5 M sodium bicarbonate solution and distilled water, and then lyophilized (UB!

H-ZYM, yield 100 mg). The supernatant (sup-2) was dialyzed and then lyophilized. ZymoJyase digestion of oxidized Zymosan preparations

Aliquots of 100 mg of ZYM, IIB-ZYM, or IIB/H-ZYM were suspended in 10 ml of 50 mM acetate buffer, pH 6.0, and digested with 1 mg of zymolyase 100 T (Seikagaku Co., Tokyo, Japan ) at 37 °C for 4 h. After centrifugation , the solubilized fraction was dialyzed against distilled water using a visking tube (MW cut, 3500, SpectraJPor. Mem-

brane MWCO: 3,500, SPECTRUM, Housten, TX) overnight, and the sediment was washed with distilled water and then lyophilized (yield, ZYM; 1S mg, I/B-ZYM; 18.5 mg, I1B/H-ZYM; 22 mg). The non-dialysable fraction was lyophilized (yield, ZYM; 24.5 mg, IIB-ZYM; 20 mg, IIB/H-ZYM; 10.3 mg).

ELISA for cytokine determination TNF-a was detected using an indirect enzyme-linked immunosorbent assay (ELISA ).

Aliquots of 50 ~I (5 flg/ml each ) of anti-mouse TNF-a mAb (Pharmingen, San Diego, CA, USA ) were applied to the surface of a 96-well flat-bottomed plate (Sumitomo Bakelite Co.) by incubation at 4'C overnight in 0.1 M bicarbonate buffer (pH 9.6). The plate was washed with PBS containing 0.05 % Tween 20 (PBST) and blocked with 200 ~l of 0.5 % bovine serum albumin in PBST at 37 ' C for 40 min. After washing with PBST, the plate was incubated with rMu TNF-a (R & D Systems, Minneapolis, MN, USA) or cell culture supernatants at 37"C for 40 min. The plate was washed and .\0 ~I aliquors of a 1 :2000 dilution of anti-TNF-a rabbit polyelonal antibody (Genzyme, Boston , MA, USA) were added. After 1 h incubation, the plate was washed with the 5

Zcm.bL &ktt:riol. 28911

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N. Ohno et al.

blocking buffer and treated 1 h with 50 I each of I: 10000 diluted peroxidase-labeled anti-rabbit IgG (Organon Teknika Co.). After final washing, peroxidase-conjugated antibody was detected by addition of 50 1'1 of TMB (3,3',5,5'-tetra methylbenzidine) substrate system solution (KPL Inc., Caithersburg, MA, USA). Color development was stopped after about 10 min with 501'1 of 1 N phosphoric acid, and the absorbance at 450 om was measured with a microplate reader (MTP32, Corona Electric Co., Ltd., Tokyo, japan). Measurement of vascular permeability (9)

Each ZYM preparation (100 I'g per mouse: 500 I'glml in saline) was i. p. injected into ICR mice (provided from japan SLC, 6-8 weeks old, malc ). Thirty minutes after the injection, an aqueous solution of Evans blue (Sigma E-2 129; 10 mg/m l in saline ) was i. v. injected (0.2 m!). Thirty min. later, mice were sacrificed and the peritoneal exudate fluids were collected by washing with 4 ml of PBS. After centrifugation to remove any debris, concentration of Evans blue in the peritoneal exudate fluids was measured at 630 nm.

Preparation of macrophages Mice were injected intraperitoncally with 2 ml of proteose peptone medium (Difco Co., Detroit USA). Three days later, proteose peptone-induced peritoneal exude cells (PECs) were collected from the peritoneal cavity of mice by was hing twice wirh 5 ml of Hanks' balanced salt solution (HBSS; Nissui Seiya ku Co., Tokyo) containing hep· arin (5 V/ml). The cells were collected by centrifugation at 1200 rpm. The pellet was washed twice with HBSS and resuspended in icc-cold RPMI1640 medium (Nissui ) supplemented with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (HEI'ES, 5mM), penicillin G ( 1OOUlml) and streptomycin (100 ~glml). The cells were cultured in a 96-well flar-bottomed plate (Sumitomo Bakelite Co., Ltd) at 2 x 105 per well in 200 fll of culture medium. To ensure adherence, ce lls were cultured for at least 2 h, then washed twice with warmed RPM! 1640 medium. The obtai ned PMs were cultured for 24 h in a hum idified 5% CO 2 incubator at 37°C with glucans in the presence or abse nce of IFN-y in RPM! 1640 medium containing 10 % FCS. RAW 264.7 cells, a murine macrophage-like cclliine, were cultured in RPM! 1640 medium containing 10% fetal calf serum. The cell suspension (loS X IDS cells/ml) was placed in a flat-bottomed 48-well tissue culture plate (0.5 ml/we ll ) and cultured at 37'C in a humidified 5% CO, incubator for 24h. Cells were stimulated with ZYM or oxidized ZYM (100 I'lIml) for 24 h. The supernatant of RAW 264.7 cell cultures were screened for TNF-u. Assay of phagocytosis by macrophages

The macrophages (2.5 x 104/well) were cultured on a mu lti-well Teflon coated slide (8 mm; Bokusui Brown, New York, NY) for 3 hat 37'C in a 5% CO, incubator. The slide was rinsed twice with Krebs-Ringer phosphate buffer supplemented with 5.5mM glucosc (KRPG ), and then 50 ~l of a mixture with a suspension of 200 !1g1ml ZYM and antagonists in KRPG were added. After 30 min incubation at 37°C, the monolayers were rin sed withKRPG to remove noningested ZYM particles, and then fixed and stained with DiH-Quick solution (Kokusai Shiyaku Co., Hyogo, japan). The ingestion of ZYM panicles by 200 macrophages per well was determi ned b y v is ual countings a t400x magnification with a light microscope.

Activity of Oxidized Zymosan

67

Measurement of hydrogen peroxide production by peritoneal macrophages (10) Production of hydrogen peroxide was assayed continuously at 37 C by a micro-assay using the scopolctin technique. Briefly, the cultures were washed 3 times with 50 !II of Krebs-Ringer phosphate buffer supplemented with 5.5 mM glucose (KRPG). Then, an assay mixture consisting of 30 mM scopolctin, 1 mM NaN J , 1 purpurogallin unit/ml HPO, and ZYM (50 or 100 ~glml) in KRPG was placed in each well. For the measurement of the antagonist activity of soluble glucans, soluble fractions or laminarin (100 - 250 ~glml) were added. Immediately after the addition of the assay mixture, the plate was placed in a filter fluorometer (microplate reader MTP-32, Corona Electric Co., Ltd. Tokyo) and the fluorescence was recorded at an excitation wavelength of 365 om and an emission wavelength of 450 nm. After incubation for 30, 60, 90, or 120 min. at 37°C, the fluorescence in each well was again recorded using the microplate reader. The amount of hydrogen peroxide produced by macrophages was calculated from the fluorescence reduction using hydrogen peroxide solution as the standard. Q

FA CS analysis Flow cytometric analysis was performed with a FACSCalibur™ (Becton-Dickinson, San Jose, CAl equipped with the Cell Quest™ program. Anti-mouse IgG-FlTC and anti-rabbit IgG-FlTC were purchased from Wako Pure Chemical Co. (Tokyo, Japan ). Anti-mannan typing sera, Candida Check™, was from Iarron Laboratories, Inc., Tokyo, Japan. Anri-ZYM serum was prepared from ICR mice receiving intravenous injection of ZYM (lmglmouse, 3 times every other day) 1 month previously.

NMR analysis Solubilized fractions and authentic materials were dissolved in D20 (-1 'Yo) and IH _ NMR spectrum was measured at 70°C. Bruker DPX400 instrument equipped with a software "XWIN-NMR" were used. The IH-NMR spectrum was obtained by accumulating ca. 2000 scans.

Sonication of the oxidized ZYM preparations Suspensions of oxidized ZYM preparations (2.5 mg/ml in distilled water) were repeatedly sonicated (Otake Co., Ibkyo, Japan). The resulting suspensiuns were defined as weak (50 Hz x .10 sec xl), medium (50 Hz X 30 sec X 2), and strong (50 Hz X 30sec x3). These suspensions were centrifuged to separate the sediment from any solubilized fractions.

Statistics All experiments were run in triplicate unless otherwise stated. The values are shown as mean ± SD. Statistical evaluations in all experiments were performed by Student's t-test. A value of p < 0.05 was considered to be significant.

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N. Ohno et al.

Results Properties of NaJO, oxidized ZYM

Periodate (I) is often used in carbohydrate chemistry to oxidize nonsubstituted, vicinal hydroxide bonds to produce an aldehyde. Subsequent reduction by borohydride (B) produces a corresponding alcohol. We used IIB treatment for ZYM to modify the carbohydrate moiety. Side chain-bearing IIB-ZYM and side chain-degraded IIBIH-ZYM were prepared. I/BIH-ZYM was developed by removing oxidized sugar moieties by limited acid hydrolysis. Fractions solubilized during oxidation and mild acid hydrolysis were also collected (sup-l and sup-2). Sugar analysis of components after the complete hydrolysis by 2 M trifluoroacetic acid suggested that almost all of the mannan moieties were degraded by NaIO, oxidation (Man/Glc ratio of ZYM = 1/2.7, IIB-ZYM =1/25, IIBIH-ZYM =1/82). To further characterize the degradation products, particles were digested with an endo 1,3-~-glucanase, zymolyase 100 T, and non-dialyzable fractions were collected. Yields of the non-dialyzable fractions of ZYM, IIB-ZYM, and IIBIH-ZYM were 24.5 %, 20 %, and 10 %, respectively. Figure 1 shows partial ' H-NMR spectra of the non-dialyzable fractions. Anomeric signals around 5.3-5.0 ppm, 4.9-4.7 ppm, 4.6-4.5 ppm were assignable to mannan, 1,3 - ~-glucan, and 1.6-~-glu ­ can moieties, respectively. Signals assignable to mannan almost completely disappeared after I/B-treatment. Ratios of 1,3 - ~-glucosidic and 1 , 6-~-glu­ cosidic linkages were 112.7 (ZYM), 1/2.6 (IIB-ZYM), 110.8 (IIBIH-ZYM), respectively. Surprisingly, the ratio was similar before and after the I/Btreatment and the 1,6-~-glucosidic linkage was not completely degraded even after I/BIH-treatrnent. These facts sugested the presence of highly branched linkages in the 1,6- ~-glucan moiety. In addition, a certain steric hindrance for accessibility of the water molecule, or low molecular weight chemicals, through the cell wall might exist in the 1 ,6-~-glucan moiety. To characterize immunochemically the surface structure of oxidized ZYM, the reactivity of anti-mannan typing serum as a reagent to diagnose Candidemia, and the reactivity to anti-ZYM serum was examined by FACS. Figure 2 shows the reactivity of ZYM and oxidized ZYM to anti-mannan typing serum. Conistent with the chemical analysis, reactivity to the antimannan serum was significantly reduced after oxidative treatment. Similar to anti-mannan typing serum, the reactivity of IIB-ZYM and IIBIH-ZYM to anti-ZYM serum was reduced but a significant part of the interaction still remained (mean channels: ZYM-NMS; 40, ZYM-anti-ZYM; 115, I/B-ZYMNMS; 35; IIB-ZYM-anti-ZYM 75, IIBIH-ZYM-NMS; 35, IIBIH-ZYM-antiZYM; 80). Anti-ZYM antiserum was prepared by injecting mice with ZYM. Preliminary studies suggested that the serum mainly contained specific Ig against 1,3-~-glucan and 1,6-~-glucan moieties. Partial reactivity to IIBIHZYM strongly indicative for the only partial degradation of ~-glucan moieties.

Activity of Oxidized Zymosan

69

1.6-~-g1uc.n

mannan 1.3-~-g1ucan

a

b

c

d I

5.4

I 5.2

I 5.0

I

4 .•

I

4.6

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4.4

Chemical shift (ppm)

Fig.1. IH-NMR spectra of solubilized fractions. IH-NMR spectra were measured in D 20 at 70 "C as shown in Materials and Methods, a) zyrnolyasc digest of ZYM (non-

dialysahle fraction), b) zymolyase digest of IIB-ZYM (non-dialysable fraction), c) zymolyase digest of lIB/H-ZYM (non-dialysable fraction), d) sup-3 prepared from IIB/HZYM.

Biological activities of oxidized ZYM Biological activities of IIB- and IIBIH-ZYM are shown in Fig. 3-Fig. 6. Oxidized fractions were difficult to disperse in aqueous buffer, thus, the suspensions were ultrasonically dispersed. The vascular permeability (VP) of the peritoneal cavity of mice was examined by injecting oxidized ZYM preparations (100 f!g in 0.2 ml of saline) intraperitoneally. lIB-treatment did not modifiy VP activity. VBII-I -treatment reduced VP activity only slightly, but not in a sig-

70

N. Ohno et al. 0 0

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Fig. 2. Reactivity of NaI0 4-oxidized-ZYM to a rabbit anti-mannan typing serum (factor 1) assessed by flow cytometry. ZYM and preparations thereof were incubated with anti-mannan serum. After washing, the resulting particles were stained with the sec-

ond antibody and measured by FACS. a) ZYM, b) I/B-ZYM, c) I1BIH-ZYM, thin line, without serum; thick line, anti-mannan serum. FITC-Iabeled anti-rabbit IgG was used as the second antibody.

nificant manners [released Evans blue by OD value: saline; 0.015 ± 0.016, ZYM; 0.72 ± 0.14 (p < 0.001 vs. saline), IIB-ZYM; 0.88 ± 0.27 (p < 0.001 vs. saline), IIBIH-ZYM; 0.52 ± 0.21 (p < 0.001 vs. saline)]. Figure 3 shows receptor-mediated phagocytosis of oxidized ZYM by a murine peritoneal macrophage. About 60 % of the macrophages phagocytosed ZYM and the activity was significantly reduced to about 15 % in the presence of laminaran, a representative antagonsit. Phagocytic activity of macrophages to oxidized ZYM, both I/B- and lIB/I-!-, was similar to ZYM. The inhibitory effect of laminarin on the phagocytosis of oxidized ZYM was also similar to ZYM. Figure 4 shows TNF-a synthesis of a murine macrophage-like cell line, RAW 264.7. Similar to the activities above, TNF-a production was the same with both parent or oxidized ZYM (see below in more detail). Figure 5 shows H 2 0 2 synthesis of macro phages in the presence of oxidized ZYM prepara-

Activity of Oxidized Zymosan

71

70 60

g

50

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.c

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IIB- ZYM

IIB/ H-ZYM

Fig.3. Phagocytic activity of murine peritoneal macrophages stimulated with NaI04 oxidiud ZYM. After 3 hadherence to glass covers lips, cells were incubated with 200 ~glml Zymosan in the presence or absence of laminaran (250 Ilglml ) for 30 min. After washing, fix ing and staining, cells were visually counted by light microsocpy. This figure shows the percentage of the 200 cells tested which had ingested zymosan particles . *"" *: p < 0.001.

tions. No difference was found in each case (see also below in more detail). As described in Materials and Methods, a part of the cell wall polysaccharides was solubilized during oxidation. The solubilized fractions were abbreviated as sup-l (during liB-treatment) and sup-2 (during IIBIH-treatment). Tbe effect of these polysaccharide fractions on HzO, synthesis by peritoneal macrophages was examined (Fig.6). Both sup-l and sup-2 showed inhibitory activity against ZYM mediated HzO z synthesis, in which inhibition by sup-2 was more significant. It is to be noted that H 2 0 z production of macrophages mediated by phorbol 12-myristate 13-acetate (PMA) was unchanged by the addition of these soluble fractions (data not shown ). These facts suggest that the activities of ZYM are mainly mediated by moieties resistant to I/B/Htreatment.

Effect of sonication on activity of oxidized ZYM As shown above, oxidized ZYM preparations are difficult to suspend in aqueous buffer, thus ultrasonication was used. During this treatment, we made an interesting observation.

N. Ohno et al.

72 18000

OWeak E:!!IMedium

16000

SStrong

14000 12000

E 'bi 10000

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z

c-

8000 6000 4000 2000 0 ZYM

IIB- ZYM

IIB/ H- ZYM

Fig. 4. Effect of sonication of NaI04 -oxidized ZYM on TNf-a production by murine peritoneal macrophages. Condition of sonication (weak, medium, strong) afC described in Materials and Methods. Macrophages wefe stimulated in vitro with ZYM

or oxidized-ZYM (100 !,g/ml) for 24 h. TNF-a concentrations in the culture supernatants of stimulated macrophage cultures were measured by ELISA. No detectable con -

centrations of TNF-a could he observed in the culture supernatant in the absence of ZYM or oxidized-ZYM (control culture ).

Sonication of the oxidized ZYM preparations was repeated several times and the resulting suspensions were defined as weak (50 Hz x 30 sec x 1), medium (50 Hz X 330 sec X 2),and strong (50 Hz X 30 sec X3). After treatment, suspensions were centrifuged to separate the sediment from any solubilized fractions. Figure 4 shows TNF-a synthesis induced by sediments after sonication. The TNF-a production was only slightly reduced even after stimulation with fractions obtained by strong sonication. In the case of H 20 2 synthesis, activity of parent ZYM to macrophages was not changed after sonication, but UB-ZYM induced an enhanced activity (Fig. 5). However, the activity of UBfH-ZYM was significantly reduced after prolonged sonication. In addition, carbohydrate (sup-3 ) was solubilized during extensive sonication. Sup-3 (7 % of UBfH-ZYM) showed a comparable inhibitory activity to laminarin in ZYM-mediated H 2 0 2 synthesis (Fig. 7). In contrast, sup-3 did not inhibit PMA-mediated H 2 0 2 synthesis (data not shown). These facts suggest that the active components responsible for H 20 2 synthesis and for TNF-a synthesis are not identical. The chemical nature of sup-3 was further analyzed by 'H-NMR (Fig. 1d). Figure 1 shows the partial ' H-NMR spectrum of sup-3. Referring to the spec-

Activity of Oxidized Zymosan

73

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2' 1500

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Fig.S. Effect of sonication of NaIO,-oxidized ZYM on H20 , synthesis of macrophag-

es. Conditions of sonication (weak, medium, srrooges) are described in Materials and

Methods. ZYM or oxidized ZYM (100 I'g/wll) were added to the macrophage culture and H2 0! production was monitored by the scopolctin method as described in Materials and Methods. Spontaneous release of H 2 0 ': p < 0.05.

2

was subtracted in each experiment

tra of zymolyase digests shown in Fig. la-c, sup-3 contains both 1,3-~- and 1,6-~-glucosidic linkages. The signal at 4.72 ppm strongly suggests the presence of a branched 1,3-~-glucosidic linkage. These facts suggested that 1,3-~/ 1,6-~-linked oligosaccharides were physically associated with large glucan components in UBIH-ZYM.

Discussion Zymosan (ZYM) is a particle prepared from Saccharomyces cerevisiae. It strongly activates the alternative pathway of complement in the presence of the serum component properdin (2). ZYM is prepared by repeated hot water extraction and tryptic degradation of the yeast cells. Mannan is a major antigenic substance in the cell wall of yeast and is present on the cell surface as "manno-protein" (4). The protein part of the manno-protein is sensitive to tryptic digestion. ZYM is a representative agonist for the functional ~-glucan receptor, however, ZYM is not a pure ~-glucan preparation and still contains proteins, peptides, and mannan, in addition to cell wall ~-glucan.

74

N. Ohno et a!. 6000 OZYM

E!I+loo

5000

~

.,

~+250

4000

0 .0 0

.fi 0 a."

3000

q: 2000

I

1000

0 Sup-l IIIB)

Sup- 2 IIiB/ H)

Fig. 6. Inhibitory effect of oligosaccharides (sup-l and -2) on Zymosan-inducible HzO z production by murine peritoneal macrophages. Adherent macrophages were

pretreated with sup-! or -2 (100 or 250 ~glml ) for 6 h at 37 °C, then cells were stimulared by ZYM (100 ~glml). Released H2 0, was estimated from the reduction of the fluorescence intensity of scopoletin *: p < 0.005, ** : p < 0.001,

In preliminary studies, we have used the NaCIO oxidation method to prepare derivatives of ZYM, and found that NaCIO treatment is strong enough to degrade almost all the bio-active components. The resulting particles showed only a weak activity, even though they were composed mainly of 1,3an 1,6-linked ~-glucans. We have already reported that synthetic particulate 1 ,3-~-glucans, such as curdlan and cross-linked laminarin, do not have significant biological activity (1). These facts and the data shown in this paper strongly support the claim that 1,3-~-linkage is not sufficient for biological activity. At present, it is difficult to clearly discriminate between the structure of I/B/H-ZYM and NaClO-oxidized ZYM. NaClO and Nal04 oxidation products have different recovery rates and immunogenicity in addition to divergent inflammatory and immunopharmacological activities. The remaining cell wall components in NaCIO-oxidized ZYM mainly consisted of 1 ,6-~- and 1,3-~­ glucans. We have already reported that the branched/terminal residue of the 6-branched 1,3 - ~-glucan was more sensitive to NaCIO oxidation (5). Analysis of the linkages sensitive to Nal04 oxidation and the differences in recovery rates between oxidation methods suggests that ZYM has a large amount of a highly branched 1,3- and 1,6-~-linkages. In addition, it is also well known that a 1,6-f)-glucan, islandican, can not be dissolved in neutral aqueous solu-

Activity of Oxidized Zymosan

75

2000 1BOO 1600 ~ 1400

S

c 1200 0

", 1000 0

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£: 600 400

••

200 0 ZYM50

ZYM50

ZYM50

Lam 100

Lam 250

ZYM50 sup-3 200

ZYM50 sup- 3500

ZYM50 sup-3 1000

Fig. 7. Inhibition of H20 1 synthesis of ZYM-stimulatcd murine peritoneal macro-

phages by ultrasonic solubilized fraction sup3 ofIlBIH -ZYM . The ZYM co ncentration in this experiment was 50 ~glml. Co ncentrations (flglml ) of th e solubilized fractions are shown in the figure. *: p < 0.05, **: p < 0.01.

tion. It might be a plausible story that the water solubility of polysaccharides, such as 1,3-~- and 1,6-~-glucans, makes a physical barrier to water soluble chemicals. One important finding in this paper was that the size of the particles, strongly related to the duration of the ultrasonic disruption, significantly affected H 2 0, synthesis (Fig. 5) but not TNF-a synthesis (Fig. 4). This suggests that structure(s) required to induce each activity are related but not identical. This is supported by the fact that the solubilized fragments after IIBIHtreatment were good antagonists, not agonists, for H,O, synthesis. Antagonist activity was only found in the IIBIH-ZYM fraction, suggesting the absence of non-specific release. The activity shown by the fragments released during sonication might be mediated by a similar structure, since a major glucan fragment solubilized by IIBIH-treatment or by sonication contained 1,3~-linkages. Further, 1,3-~-glucan is usually stahilized by forming a triple helix conformation in aqueous solution, strongly supporting that 1 , 3-~-glucan fragments are solubilized by these treatments. As mentioned in the Introduction, ZYM is a representative inflammatory substance widely used in basic research. However, the molecular mechanism of ZYM-mediatcd biological actions is unclear as well as the function of the

76

N.Ohnoetal.

~-glucan receptor(s). The characteristic physicochemical properties of ~­ glucans including limited solubility in water and helix formation increase the difficulty in its study. The incidence of deep-seated mycosis has increased significantly with the expansion of the population of aged people, of intensive medical treatment, and of immunocompromised patients (7, 8). The data shown in this paper strongly suggest that even after complete recovery from the fungal infection, cell wall ~-glucan may be deposited in organs for long periods of time and might induce chronic inflammation. A detailed examination of recovering patients and further studies of ~-glucan are needed.

Acknowledgements. This study was supported by a Grant-in-Aid for Scientific Research (C, 10672059) from the Ministry of Education, Science, Sports and Culture.

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Corresponding author: Prof. Dr. Toshiro Yadomae, School of Pharmacy, Tokyo Univer-

sity of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan