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Protective effects of a protein-bound polysaccharide, PSK, on Candida albicans infection in mice via tumor necrosis factor-α induction
International Immunopharmacology 1 Ž2001. 1797–1811 www.elsevier.comrlocaterintimp
Protective effects of a protein-bound polysaccharide, PSK, on Candida albicans infection in mice via tumor necrosis factor-a induction Yoshio Ohmura, Kenichi Matsunaga) , Isamu Motokawa, Katsuo Sakurai, Takao Ando Biomedical Research Laboratory, Kureha Chemical Industry, Co. Ltd. 3-26-2, Hyakunin-cho, Shinjuku-ku, Tokyo, 169-8503, Japan Received 8 February 2001; accepted 13 May 2001
Abstract We investigated the protective mechanism of a protein-bound polysaccharide, PSK, against lethal infection with Candida albicans Ž C. albicans . in mice. Ž1. In BALBrc mice inoculated intravenously with C. albicans, the intraperitoneal Žip. administration of PSK increased survival rates and prolonged the survival period depending on the time of administration, the dosage, and the size of fungal inoculum; the maximal effect was obtained when PSK 250 mgrkg was ip administered to mice 24 h before inoculation of 1 = 10 6 C. albicans Ž30 days survivors showed 60% and the mean survival period of mice with fatal infection increased 209%.. Ž2. The protective effect of PSK was significantly decreased in mice treated with cyclophosphamide or carrageenan, or in mice treated previously with anti-tumor necrosis factor-a ŽTNF-a . antibody. Ž3. The administration of PSK significantly enhanced the expression of TNF-a gene in spleen and increased leukocyte functions from 6 h to 1 day after inoculation. Ž4. When the PSK fraction subjected to hydrolysis with b 1–3 glucanase or hydrazine was used instead of PSK, the anti-fungal activities were significantly decreased. These findings suggested that the protective effect of PSK on lethal C. albicans infection in mice was mainly produced via TNF-a functions, and that b 1–3 glucan and protein moiety in PSK molecule were involved in the expression of the activities. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Anti-infectious effects of BRM; Induction of tumor necrosis factor-a ; Candida albicans; Protein-bound polysaccharide
1. Introduction AbbreÕiations: BRM, biological response modifier; C. albicans, Candida albicans; IL-1 a , interleukin-1 a ; IL-2, interleukin2; IL-6, interleukin-6; IFN-g , interferon-g ; ip, intraperitoneally; iv, intravenously; PEC, peritoneal exudate cells; QOL, quality of life; TNF-a , tumor necrosis factor-a ) Corresponding author. Tel.: q81-3-3362-7326; fax: q81-33362-8523. E-mail address: [email protected] ŽK. Matsunaga..
Despite recent advances in anti-fungal chemotherapy and diagnosis, the increase of high-risk patients and the appearance of drug-resistant fungi have resulted in an increase in the incidence of deep-seated mycosis w1x. In many cases, deep-seated
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 1 0 4 - 7
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mycosis occurs in individuals with decreased resistance due to underlying diseases such as cancer, or due to immunosuppressive therapies w2x. It is important to maintain and improve biophylaxis in order to prevent the onset or progression of deep-seated mycosis for the management of quality of life ŽQOL. in such patients. Certain biological response modifiers ŽBRMs. have been reported to produce not only anti-cancer effects but also anti-infectious effects by modifying responses of the host to cancer cells or pathogenic microorganisms w3,4x. PSK, a protein-bound polysaccharide derived from a basidiomycete is a representative BRM exhibiting a variety of biological activities such as immunoregulation and antioxidant activity w5x, while in randomized controlled trials, the concomitant chemotherapy prolongs postoperative survival of patients with gastric cancer w6x and colorectal cancer w7x. PSK has a mean molecular weight of 9.4 = 10 4 and is composed of protein and glucan having b 1–4 bond in the main chain and b 1–3 and b 1–6 bond in the side chain, that binds to a protein moiety through O- or N-glycoside bonds w8x. Although the protective effect of PSK against infection with pathogenic microorganisms such as virus, bacteria, and fungi has been reported in experimental animals as well as immunosuppressed patients w9– 15x, the detailed mechanism of its action remains to be elucidated. The present study reports that PSK is effective in the protection against lethal systemic infections in mice with Candida albicans Ž C. albicans . through enhanced formation of tumor necrosis factor-a ŽTNF-a ..
2. Materials and methods 2.1. Animals Specific pathogen-free female BALBrc mice, BALBrc nurnu mice and BALBrc nurqmice were purchased from CLEA Japan ŽTokyo., and C57BLr6 bgrbg mice and C57BL bgrqmice from Japan Charles River ŽAtsugi, Kanagawa.. After acclimatization, they were used in experiments at 8 weeks of age. They had free access to food CE-2 ŽOriental Yeast, Tokyo. and sterilized tap water.
Animals were kept at temperature of 24 " 2 8C, a humidity of 55 " 10%, and a luminous intensity of approximately 5 lx with a lighting cycle from 8:00 AM to 8:00 PM under luminary air flow. Only staff caring for the animals and people involved in the experiments entered the breeding room. 2.2. Inoculation of C. albicans C. albicans strain IFO1060 maintained in our laboratory was used: it was inoculated in Sabouraud culture media supplemented with 2% glucose ŽpH 5.6., followed by shaking the culture at 25 8C for 18 h. After incubation, fungal cells were washed three times with endotoxin-free, sterile phosphate-buffered saline ŽPBS., and suspended in PBS to an appropriate concentration. An aliquot of the suspension was injected into the caudal vein of mouse. 2.3. Reagents and antibodies Carrageenan ŽCA. and aminoguanidine were purchased from Sigma ŽSt. Louis, MO, USA., cyclophosphamide ŽCY. was from Shionogi ŽOsaka., amphotericin B was from Bristol–Myers–Squibb ŽNew York, USA., carboxymethylated b 1–3 glucan ŽCM-curdlan. of Alcaligenes faecalis Õar. myxogenes origin with number-average degree of carboxymethyl groups substituted per anhydroglucose unit 0.47 was from Wako ŽOsaka, Japan., recombinant mouse TNF-a with endotoxin content less than 2 EUrm g protein was from Endogen ŽWoburn, MA, USA.; anti-mouse TNF-a antibody ŽAb., anti-mouse interleukin-1 a ŽIL-1 a . Ab, anti-mouse interleukin-2 ŽIL-2. Ab, anti-mouse interleukin-6 ŽIL-6. Ab, and anti-mouse interferon-g ŽIFN-g . Ab were from R & D System ŽMinneapolis, MN, USA.. These reagents or antibodies were dissolved in PBS or sterile distilled water, and injected to animals. Aminoguanidine was injected ip at a dose of 300 mgrkg 2 days before inoculation, CM-curdlan was injected ip at a dose of 100 mgrkg 1 day before inoculation and recombinant mouse TNF-a was injected iv at a dose of 10 4 Ur mouse 1 h before inoculation, following the protocols of Vasquez-Torres et al. w16x, Sasaki et al. w17x and Havell w18x, respectively.
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2.4. PSK and its fractions PSK ŽKureha Chemical Industry, Tokyo. was dissolved in PBS for use in the experiments. To estimate the structure responsible for the biological activity of PSK Ž62% sugar and 38% protein., the following fractions were prepared. For the preparation of fractions with a relatively low protein content, the peptide moiety of PSK was decomposed by hydrazine following the method of Takasaki et al. w19x. Briefly, PSK was dissolved in anhydrous hydrazine and, heated in a sealed tube at 100 8C for 24 h. Then, hydrazine was removed in a vacuum desiccator. The resulting exsiccates were dissolved in water and subjected to purification by a combination of gel filtration ŽSephadex G-25, Pharmacia Biotech, Uppsala, Sweden. and diethylaminoethyl ŽDEAE. cellulose ion exchange chromatography ŽToyopearl 650S, Toyo, Tokyo.. Thus, a hydrazine-treated PSK ŽPSK-H, 94% sugar and 6% protein. was obtained. Next, for the preparation of fractions with less sugar content, polysaccharide moiety of PSK was decomposed by periodate oxidation. Briefly, PSK was incubated in 0.04 M metaperchloric acid at 4 8C for 14 days. After the reaction was stopped by adding ethylene glycol, sodium borohybride was added and the mixture was allowed to stand at 25 8C overnight, then dialyzed against distilled water for 48 h. Sulfuric acid was added to a concentration of 0.1 N, and the mixture was left at 25 8C for 24 h. After neutralization with sodium hydroxide, the reaction mixture was subjected to gel filtration on Sephadex G-25 to remove low-molecular-weight substances. The resulting fraction PSK-S had a sugar–protein ratio of 22:78. This fraction was further applied to a column of Cuq-coordinated Chelating Sepharose FF ŽPharmacia Biotech., and fractions SM1 Ž55% sugar and 45% protein., SM2 Ž12% sugar and 88% protein. and SM3 Ž7% sugar and 93% protein. were obtained as eluates. Enzymolysis products were prepared by treating these fractions with Arthrobacter luteus-derived b 1–3 glucanase Žprepared in our laboratory by the purification of Zymolyase w purchased from Seikagaku Kogyo, Tokyo. or with Tritirachium album-derived proteinase K w ŽMerck KgaA, Darmstadt, Ger-
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many. for 5 h at 37 or 65 8C, respectively, and then subjected to gel filtration on Sephadex G-25 to remove low-molecular-weight substances. 2.5. Determination of cytokines Plasma levels of TNF-a , IL-2, IL-6 and IFN-g were measured using a commercially available ELISA kit ŽR & D System.. 2.6. Expression of cytokine genes The expression of cytokine genes was measured by the reverse transcriptase-PCR technique. Mice were sacrificed, and their blood, spleen, liver and kidney were removed and immediately frozen in liquid nitrogen. These samples were stored at y80 8C until RNA preparation. After the addition of an ISOGEN w solution ŽNippon Gene, Tokyo., the frozen samples were homogenized, and total RNA was extracted according to the method of Chromczynski and Sacchi w20x. The total RNA was mixed with a buffer solution containing 20 mM Tris–HCl ŽpH 8.4., 50 mM KCl, 2.5 mM MgCl 2 , 1 mM each of dATP, dCTP, dGTP and dTTP, 0.5 m g of oligoŽdT.12 – 18 , and 200 U of MLV reverse transcriptase ŽGibco BRL, Gaithersburg, MD, USA., and reverse transcription was allowed to proceed at 42 8C for 50 min. The reaction was stopped by heating at 70 8C for 15 min, followed by cooling on ice. Subsequently, the reaction mixture was mixed with a buffer solution containing 10 mM Tris–HCl ŽpH 9.0., 50 mM KCl, 1.5 mM MgCl 2 , 200 m M each of dATP, dCTP, dGTP and dTTP, 5 pM each of 5X and 3X oligomers, and Taq polymerase ŽPromega, Madison, WI, USA.. The reaction mixture was subjected to PCR amplification on a Perkin-Elmer PCR Thermal Cycler TP-2000 ŽPerkin Elmer, Foster City, CA, USA.. In the case of b-actin, heating at 94 8C for 1 min and 25 cycles of amplification and, in the case of TNF-a , heating at 94 8C for 4 min and 35 cycles of amplification were performed, followed by annealing for 1 min. PCR products were electrophoretically separated on 2% agarose gels containing ethidium bromide, and measured semiquantitatively using a Bio-Rad Multi-Analyst System. Total RNA, derived from concanavalin-A-stimulated mouse splenic cells, was used as a positive control; and b-actin
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total RNA as an internal standard for the measurement. The following primers were used: b-actin wsensex 5X-ATG GAT GAC GATATCGCTG, wantisensex 5X-GATTCCATA CCCA GGAA GG; TNF-a wsensex 5X-GATCTCAAAGAC AACCAACTAGTG wantisensex 5X-CTCGAGCT GGAAGACT CCTCCCAG. 2.7. Phagocytic and killing actiÕity of peritoneal exudate cells (PEC) The activities of PEC were measured following the method of Netea et al. w21x as modified. Briefly, 1 ml of 10% proteose peptone ŽDifco Lab., Detroit, MI, USA. was injected into the peritoneal cavity of healthy or fungus-inoculated mice, and PEC were collected 4 h later. The cells were washed with endotoxin-free RPMI1640 medium, and PEC were adjusted to 5 = 10 5 cellsrml with the medium. In a silicon-pretreated culture tube, the PEC suspension 1 ml and a C. albicans suspension 5 = 10 4 CFU 1 ml were mixed to a ratio of 10:1, and the mixture was cultured in an incubator at 37 8C in an atmosphere of 5% CO 2 . For the evaluation of phagocytic activity, the PEC were recovered 5 min after the start of culture and were washed with PBS twice. After it was ascertained microscopically that PEC had phagocytized fungi and that no fungi were adherent to them, they were subjected to lysis by the addition of a 0.01% aqueous solution of bovine serum albumin ŽSigma.. Serial 10-fold dilutions of the lysate in sterile PBS were made, and an aliquot of each dilution was spread on Sabouraud agar medium. After 24 h of culture at 37 8C, colonies of C. albicans were counted. For the evaluation of killing activity, PEC were recovered 15 min after the start of culture. The recovered PEC were washed with RPMI1640 medium twice, and cultured at 37 8C for 1 h in an incubator in an atmosphere of 5% CO 2 . Similar to the evaluation of phagocytic activity, serial dilutions of the cell lysate were cultured on the medium, and the colonies formed were counted. 2.8. Superoxide production by PEC A suspension of 2 = 10 5 PEC and 50 m M CLAphenyl ŽTokyo Kasei, Tokyo. were dispensed in a
tube, and the volume of the mixture was adjusted to 175 m l with Hank’s Balanced Salt Solution containing 0.25% human albumin. Twenty-five microliters of 400 ngrml of phorbol 12-myristate 13-acetate ŽPMA, Sigma. was added, the reaction was allowed to proceed at 20 8C, and then the fluorescence emitted was sequentially measured on a Luminescence Meter ŽAloka, Tokyo.. 2.9. Determination of C. albicans-deriÕed polysaccharide Plasma levels of b-glucan, a cell wall component of C. albicans, were determined following the method of Obayashi et al. w22x by the use of a commercially available kit ŽSeikagaku Kogyo.. 2.10. Determination of endotoxin The endotoxin content of samples for activity evaluation, reagents, or culture media was determined colorimetrically using a commercially available kit ŽSeikagaku Kogyo.. 2.11. Statistical analysis Values are shown as mean " standard deviation. Test of significance was performed using a log rank test and Student’s t-test, and values of P - 0.05 were considered significant.
3. Results 3.1. Dependency of anti-infectious effects of PSK on time of administration, dosage, and size of inoculum To clarify the influence of time of administration on the anti-infectious effects of PSK, we gave healthy BALBrc mice an intraperitoneal Žip. injection of 50 mgrkg of PSK 5, 3 or 1 day or immediately before the intravenous Živ. inoculation of 2 = 10 6 C. albicans, and observed them for survival periods. Compared with a 30-day survival rate of 0% and a mean survival period of 5.7 " 1.9 days for control group A, those for the PSK-administered group ranged from 7% to 47% and from 7.1 to 8.6 days, respectively ŽFig. 1-Ž1... Of the PSK-administered groups,
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Fig. 1. Protective effect of PSK on lethal C. albicans infection in mice: dependency on time of administration and dosage. BALBrc mice Ž n s 15. were iv inoculated with 2.0 = 10 6 C. albicans and were observed for survival periods. Ž1. PSK 50 mgrkg was ip administered 5 days Žgroup B., 3 days Žgroup C., 1 day Žgroup D., or immediately before Žgroup E. inoculation. A control group Žgroup A. was ip given 0.2 ml of saline 1 day before inoculation. Statistically significant at ) P - 0.01 and ) ) P - 0.05 Žvs. group A. in log rank test. Ž2. One day before inoculation, 10 mgrkg Žgroup G., 50 mgrkg Žgroup H., or 250 mgrkg Žgroup I. of PSK was administered ip. Instead of PSK, 0.2 ml of saline was administered ip to a control group Žgroup F. on the same day of PSK administration. Statistically significant at ) P - 0.01 Žvs. group F. in log rank test.
group D, to which PSK had been administered 1 day before inoculation, had the greatest survival benefit: the 30-day survival rate was 47%, and the mean survival period 8.6 " 1.0 days Ž151% that of the control group.. The 30-day survival rates and mean survival periods for groups C and B Žreceiving PSK 3 and 5 days before inoculation, respectively. were 20% and 8.1 " 1.9 days, and 7% and 7.1 " 1.4 days, respectively, suggesting a tendency that the shorter the interval between PSK administration and inoculation, the greater the protective effect. However, the 30-day survival rate and mean survival period for group E receiving PSK immediately before inoculation were 7% and 7.2 " 1.5 days, respectively, indicating a decrease in the protective effect compared with the other PSK-administered groups. The administration of PSK 1 day after inoculation had no significant effect on the rate or period of survival Ždata not shown.. Subsequently, to clarify the dose-dependency of the PSK effect, we gave healthy BALBrc mice an ip injection of 10, 50 or 250 mgrkg of PSK 1 day before the iv inoculation of 2.2 = 10 6 C. albicans, and observed the post-inoculation survival periods. Compared with the 30-day survival rate of 7% for
control group F, those for PSK-administered groups G, H and I Žreceiving 10, 50 or 250 mgrkg of PSK, respectively. were 13%, 40% and 53%, respectively, indicating an increase in the rate of survival depending on the PSK dose ŽFig. 1-Ž2... Furthermore, compared with the mean survival period of 5.2 " 0.9 days for control group F, those for PSK-administered groups G, H and I were prolonged to 6.5 " 1.0, 8.6 " 1.3, and 10.4 " 1.5 days, respectively, depending on the dose. In order to investigate the influence of the inoculum size on the PSK effect, we inoculated various amounts of C. albicans iv into healthy BALBrc mice 1 day after an ip injection of 250 mgrkg of PSK, and observed them for survival periods. As listed in Table 1, when 1.0 = 10 6 , 5.0 = 10 6 or 3.0 = 10 7 C. albicans was inoculated, the 30-day survival rate for the control group was 13%, 0% or 0%, respectively, and the mean survival period was 5.3 " 0.5, 3.9 " 0.8 or 2.9 " 0.6 days, respectively, suggesting that as the inoculum size increased, the survival rate decreased, and the mean survival period shortened. The 30-day survival rates for the groups receiving a PSK injection 1 day before inoculation were 60%, 27% and 7%, respectively, and the mean
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Table 1 Dependency of the protective effects of PSK on the inoculum size Inoculum size
Treatment
Mean survival days
30-day survivors
1=10 6
Saline PSK Saline PSK Saline PSK
5.3"0.5 Ž100%. 11.1"1.9 Ž209%. 3.9"0.8 Ž100%. 6.7"1.9 Ž172%. 2.9"0.6 Ž100%. 4.4"0.9 Ž152%.
2r15 Ž13%. 9r15 Ž60%. ) 0r15 Ž0%. 4r15 Ž27%. ) 0r15 Ž0%. 1r15 Ž7%. )
5=10 6 3=10 7
BALBrc mice Ž ns15. were given PSK 250 mgrkg or saline 0.2 ml. Twenty-four hours later, they were iv inoculated with 1=10 6 , 5=10 6 , or 3=10 7 C. albicans, and were observed for subsequent survival periods. ) Statistically significant at P - 0.01 ŽPSK group vs. saline group..
survival periods were 11.1 " 1.9 Ž209% of that for the control group., 6.7 " 1.9 Ž172%. and 4.4 " 0.9 Ž152%. days, respectively, suggesting that as the inoculum size decreased, PSK became more effective. In addition, to compare the anti-infectious effects of PSK with those of other anti-fungal drugs or reagents, we gave healthy BALBrc mice an injection of amphotericin B, CM-curdlan or recombinant mouse TNF-a 1 day or 1 h before the iv inoculation of 2.5 = 10 6 C. albicans, and observed them for survival periods. As listed in Table 2, mean survival period for the group receiving amphotericin B 1 h before inoculation or the group receiving PSK 1 day before inoculation was significantly prolonged, when compared with that of the group receiving saline. However, neither injection of CM-curdlan 1 day before inoculation nor recombinant mouse TNF-a 1 h before inoculation had any significant effect on mean survival period in those mice. These findings suggest that the anti-infectious effect of PSK depends on the time of administration, the dose administered, and the size of fungal inoculum. 3.2. ProtectiÕe effect of PSK in mice with leukopenia or phagocytes-dysfunction, or in mice with congenital immunodeficiency We examined the influence of PSK administration on the rate and period of survival after the inoculation in mice treated with CY or CA, or in mice with congenital immunodeficiency.
The treatment of CY to healthy BALBrc mice caused peripheral leukocyte counts to decrease, with a nadir at 4 days, and slow recovery. Pretreatment of mice with PSK did not significantly influence peripheral leukocyte counts Ždata not shown.. Under these conditions, we inoculated C. albicans iv into mice 4 days after an ip treatment with CY 100 or 300 mgrkg, and observed them for survival periods. When 2.1 = 10 6 C. albicans was inoculated into mice, all the mice died within 5 days, with or without PSK administration Ždata not shown.. However, when the inoculum size was decreased to 4.8 = 10 5 C. albicans, the mean survival period for group B receiving PSK 1 day before inoculation was prolonged to 182% ŽFig. 2-Ž1.. of that for control group A after treatment with CY 100 mgrkg, and to 150% of that for the group A after treatment with CY 300 mgrkg, suggesting a tendency that the PSK effect was weaker in the group treated with higher dose of CY ŽFig. 2-Ž1. and -Ž2... Then, to examine the PSK effect in mice with phagocyte system dysfunction, we inoculated 2.1 = 10 6 C. albicans iv into BALBrc mice 4 days after an ip treatment with CA 100 or 300 mgrkg. PSK administration had no significant effect on survival periods in those mice, irrespective of CA doses ŽFig. 2-Ž3. and -Ž4... Furthermore, to examine the PSK effect in mice with congenital thymus defect, we inoculated 4.8 = 10 5 C. albicans iv into BALBrc nurnu mice. As a result, compared with that of BALBrc nurqmice, Table 2 Comparison of anti-infectious effects of PSK with those of antifungal drug or reagents Treatments
Mean survival days
Ž1. Saline 0.2 ml, ip, day y1 5.0"0.6 Ž100%. Ž2. Amphotericin B 13.4"3.1 Ž268%. 0.25 mgrkg, iv, day 0 Ž3. CM-curdlan 100 5.2"0.9 Ž104%. mgrkg, ip, day y1 Ž4. Recombinant mouse TNF-a 6.7"1.2 Ž134%. 1=10 4 Urmouse, iv, day 0 Ž5. PSK 250 mgrkg, ip, 9.3"1.1 Ž186%. day y1
30-days survivors 0r8 5r8 ) 0r8 1r8 3r8 )
BALBrc mice Ž ns8. were given anti-fungal drug or reagents 1 day or 1 h before inoculation with 2.5=10 6 C. albicans on day 0, and were observed subsequent survival periods. ) Statistically significant at P - 0.01 Žvs. saline group..
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Fig. 2. Influence of treatment with CY or CA on the protective effect of PSK. BALBrc mice Ž n s 15. were iv inoculated with 4.8 = 10 5 C. albicans and were observed for survival periods. PSK 250 mgrkg was ip administered 1 day before inoculation Žgroup B.. Instead of PSK, 0.2 ml of saline was ip administered to a control group Žgroup A. on the same day of PSK administration. CY 100 mgrkg Ž1., 300 mgrkg Ž2., CA 100 mgrkg Ž3. or 300 mgrkg Ž4. was ip injected 4 days before inoculation. Statistically significant at ) P - 0.01 Žvs. group A. in log rank test.
the PSK effect on the survival period decreased in BALBrc nurnu mice 10 days after inoculation and later, with all the mice dying of infection within 17 days ŽFig. 3-Ž1. and -Ž2... However, regarding C57BLr6 bgrbg mice with congenital defect of natural killer cell activity, PSK significantly improved the rate and period of their survival ŽFig. 3-Ž3. and -Ž4... These findings suggested that the PSK effect was mainly mediated by the function of the phagocytic system, although there was a possible involvement of the T cell system during the later stage of infection. 3.3. Influence of treatment with anti-cytokine Ab or nitrogen monoxide (NO) inhibitor on the PSK effect To identify the effector molecule responsible for the expression of the PSK effect, we investigated the
influence of treatment with anti-cytokine Ab or an NO inhibitor on the rate and period of survival after inoculation in saline-treated mice and PSK-administered mice. When 2.2 = 10 6 C. albicans was iv inoculated into BALBrc mice 2 days after treatment with antiTNF-a Ab, the mean survival period was significantly shorter than in mice treated with immunoglobulin ŽIg. with similar timing ŽTable 3.. In the anti-TNF-a Ab-treated mice, PSK had no effects on the 30-days survival and mean survival periods. However, in mice treated with anti-IL-6 Ab, anti-IL1 a Ab, anti-IFN-g Ab, or anti-IL-2 Ab, or aminoguanidine, the treatments did not significantly influence the rate or mean period of survival, and PSK significantly prolonged the period of survival of the mice.
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Fig. 3. Anti-infectious effect of PSK in mice with congenital immunodeficiency. Groups of 15 BALBrc nurqmice Ž1., BALBrc nurnu mice Ž2., C57BLr6 bgrqmice Ž3., or C57BLr6 bgrbg mice Ž4. were iv inoculated with 2.1 = 10 6 C. albicans and were observed for survival periods. One day before inoculation, 0.2 ml of saline Žgroup A. or 250 mgrkg of PSK Žgroup B. was administered ip. Statistically significant at ) P - 0.01 Žvs. group A. in log rank test.
These findings suggest that the main effector molecule involved in the PSK effect is possibly TNF-a . 3.4. Influence of PSK on the leÕel of TNF-a in plasma, expression of TNF-a gene in spleen and peripheral leukocyte functions in fungus-inoculated mice To elucidate the mechanism of PSK action, we inoculated 2.0 = 10 6 C. albicans iv into healthy BALBrc mice, and examined time-dependent changes in the level of TNF-a in plasma, the expression of TNF-a gene in spleen, and leukocyte functions in PSK administered mice. The expression of TNF-a gene in the spleen of control mice was slightly induced 6 h after inoculation, and thereafter the level of expression increased with the progression of infection. However, in the
group receiving PSK, the expression was markedly induced 6 h after inoculation, gradually decreased thereafter and, at 5 days and hereafter returned almost to the level of healthy control mice ŽFig. 4-Ž1. and -Ž3... Plasma levels of TNF-a showed a similar tendency ŽFig. 4-Ž2... Furthermore, in vitro phagocytic ability, killing activity, and superoxide-productivity in PEC of mice 24 h after inoculation were significantly enhanced in fungus-inoculated group receiving PSK ŽFig. 5-Ž1., -Ž2. and -Ž3.., although there were no significant differences in PEC recovery and their cell population Ž88–95% polymorphonuclear cells and 5–12% monocytes or macrophages. among groups tested. These findings suggest that the protective effect of PSK is produced by the enhanced formation of TNF-a , followed by the resultant increase in leukocyte anti-fungal activity.
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Table 3 Effects of treatments with anti-cytokine Ab or NO inhibitor on the protective effects of PSK Treatments Ig Anti-TNF-a Ab Anti-IL-1 a Ab Anti-IL-2 Ab Anti-IL-6 Ab Anti-IFN-g Ab Aminoguanidine
Saline PSK Saline PSK Saline PSK Saline PSK Saline PSK Saline PSK Saline PSK
Mean survival days
30-day survivors
5.1"1.2 Ž100%. 9.8"1.9 Ž192%. 3.0"0.8 Ž100%. 3.6"1.1 Ž120%. 4.1"1.1 Ž100%. 6.7"1.4 Ž163%. 5.1"1.0 Ž100%. 8.0"1.4 Ž157%. 4.3"1.0 Ž100%. 5.5"1.1 Ž145%. 4.4"0.9 Ž100%. 6.8"1.9 Ž155%. 4.9"0.8 Ž100%. 8.4"1.1 Ž171%.
0r8 3r8 ) 0r8 ) ) 0r8 NS a 0r8 1r8 ) 0r8 3r8 ) 0r8 0r8 ) 0r8 3r8 ) 0r8 3r8 )
BALBrc mice Ž ns8. were ip administered anti-cytokine Ab 1 mg or aminoguanidine 300 mgrkg on day y2, and PSK 250 mgrkg or saline 0.2 ml on day y1. They were iv inoculated with C. albicans on day 0, and were observed for subsequent survival periods. a NS, not significant Žvs. saline group.. ) Statistically significant at P - 0.01 Žsaline group vs. PSK group.. )) Statistically significant at P - 0.01 ŽIg-saline group vs. Anti-TNF-a-Ab-saline group..
3.5. Estimated actiÕe structure of PSK To estimate the structure responsible for the PSK effect, we prepared PSK fractions with less protein or sugar contents, and examined their TNF-a-inducing activity and anti-infectious activity. We administered an ip injection of the fractions 100 mgrkg Žsuboptimal dose of PSK. to healthy BALBrc mice, and examined the expression of TNF-a gene in the spleen 6 h after inoculation, the mean post-inoculation survival period, and the 30-day survival rate. As shown in Fig. 6, the activities of fraction PSK-H, which was prepared by removing most of the peptide portion with hydrazine, decreased below that of PSK. In contrast, the activities of fraction PSK-S, which was prepared by cleaving the 1–6 bond in polysaccharide portion with metaperchloric acid, were higher than those of PSK. When fraction PSK-S was fractionated into three fractions by metal ŽCu.-chelate-chromatography,
Fig. 4. Influence of PSK administration on level of TNF-a in plasma and expression of TNF-a gene in spleen of fungus-inoculated mice. BALBrc mice Ž ns 5. were iv inoculated with 2.1=10 6 C. albicans, and plasma and spleen specimens were collected in a time-dependent manner to measure Ž1. the level of fungus-derived b-glucan in plasma, Ž2. the level of TNF-a in plasma, and Ž3. the expression of TNF-a gene in spleen. One day before inoculation, 250 mgrkg of PSK ŽD. or 0.2 ml of saline ŽC. was administered ip. As a control, non-inoculated mice received 250 mgrkg of PSK ŽB. or 0.2 ml of saline ŽA. ip at the same time as the inoculated mice. Statistically significant at ) P - 0.01 and )) P - 0.05 Žvs. group A. in Student’s t-test.
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fraction SM2 exhibited the greatest activities reaching about 160% of PSK activities. The treatment of SM2 with b 1–3 glucanase or proteinase K caused a decrease in the activities. These findings suggest that the main structure involved in TNF-a-inducing activity and anti-infectious activity in the PSK molecule is possibly b 1–3 glucan and protein.
4. Discussion
Fig. 5. Influence of PSK administration on the activities of PEC in healthy mice and fungus-inoculated mice. BALBrc mice Ž ns8. were iv inoculated with 2.0=10 6 C. albicans. One day later, 1 ml of 10% proteose peptone was injected ip, and PEC were collected 4 h later. Ž1. In vitro phagocytic activity of PEC after 5 min of coculture with C. albicans; Ž2. in vitro killing activity of PEC after 1 h of coculture with C. albicans; Ž3. the production of superoxide 30 min after addition of PMA. The details are described in Materials and methods. Group C, healthy control mice given saline Žwithout inoculation.; Group P, healthy mice given PSK Žwithout inoculation.; group IC, fungus-inoculated mice given saline 1 day before inoculation; group IP, fungus-inoculated mice given PSK 1 day before inoculation. Statistically significant at ) P - 0.01 and ) ) P - 0.05 in Student’s t-test.
The present study showed that PSK exerted a protective effect against lethal infection with C. albicans, depending on the time of administration, dosage, and size of fungal inoculum. The ip administration of 250 mgrkg of PSK 1 day before the iv inoculation of 1 = 10 6 C. albicans was most effective: 60% of the mice escaped fatal infection, while the mice with fatal infection had a 209% prolonged survival ŽTable 1.. In murine systemic candidiasis models, the conditions to produce anti-infectious effects of BRM vary with their action mechanisms w23,24x. Such conditions of PSK resembled those of bacterial preparations w25,26x, unlike those of certain Chinese herb medicines w24,27x whose administration after inoculation is effective, recombinant colony stimulating factor w28x and b 1–3 glucan of Saccharomyces origin w29x requiring repeated administration before inoculation, a low-molecular-weight compound of microbial metabolite origin w30x, b 1–3 glucan of mushroom origin w23x and chitin and chitosan preparations w31x, which require repeated preand post-inoculation administration. Abe et al. w24x classified the functions of anti-fungal BRM into neutrophil activators and macrophage activators; the former is effective in prolonging survival periods when administered before inoculation, and the latter is effective when administered after inoculation. In addition, H-Young et al. w23x postulated that BRM activating phagocytic cells exert effects in the early stage of infection, and those enhancing cell-mediated immunity produce effects during the later stages of infection. Since the findings in the present study showed that the PSK effect reduced in mice in which the leukocyte counts had been reduced by treatment with a high dose of CY, or diminished in mice in
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Fig. 6. Influence of PSK fractions on the expression of TNF-a gene in spleen and anti-infectious activity. BALBrc mice Ž n s 13. were iv inoculated with 2.1 = 10 6 C. albicans. Six hours later, five mice were sacrificed, and their spleens were tested for the expression of the TNF-a gene. The remaining eight mice were observed for survival periods. PSK or its fractions were administered ip in a dose of 100 mgrkg 1 day before inoculation. The details of procedures for the preparation of the fractions are described in Materials and methods. Statistically significant at †1 P - 0.01 Žvs. Control group., † 2 P - 0.01 and † 2 P - 0.05 Žvs. PSK group., †3 P - 0.01 and †3 P - 0.05 Žvs. PSK-S group. and †4P - 0.01 Žvs. M2 group. in Student’s t-test or log rank test.
which the phagocytic function had been impaired by CA treatment ŽFig. 2., it was suggested that PSK mainly falls into a category of BRM that enhanced leukocyte function and had a protective activity in the early stage of infection. However, in T cell-deficient nude mice, PSK was not effective in prolonging survival 10 days after inoculation and later ŽFig.
3., suggesting a possible involvement of T cell function in the production of PSK effects in the later stages of infection. Among effector molecules that host cells produce in response to infecting pathogens, TNF-a is an important multifunctional cytokine during onset and progression of systemic candidiasis as evidenced by
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Marodi et al. w32x, Steinshamn and Waage w33x, Blasi et al. w34x, Louie et al. w35x, Ashman and Papadimitriou w36x, Steinshamn et al. w37x and Netea et al. w21x. During the early stage of infection, TNF-a , which is produced in the body by the stimulation with mannan or mannoprotein of C. albicans cell wall components w38x, acts on cells such as macrophages and polymorphonuclear cells to enhance their anti-fungal functions. However, during the later stages of infection, as the fungus rapidly proliferates, the level of TNF-a in the body increases, affecting negative effects on the cytokine network in the host defense system, often resulting in end-organ failure or septic shock w39x. The observations in the present study that anti-fungal activities of PEC in control mice were enhanced 1 day after inoculation ŽFig. 5., as TNF-a was induced in plasma and spleen ŽFig. 4., and that blocking TNF-a functions by pretreatment with specific Ab shortened survival periods of control mice that had been iv inoculated ŽTable 3., also demonstrated the physiological role of this cytokine in the host defense mechanism. That TNF-a is a candidate molecule responsible for the expression of the protective effect of PSK on C. albicans infection can be inferred from Ži. the disappearance of the effect by treatment with antiTNF-a Ab ŽTable 3., Žii. a marked induction of TNF-a gene in spleen 6 h after inoculation ŽFig. 4., and Žiii. a significant enhancement of PEC anti-fungal activities 1 day after inoculation ŽFig. 5.. Since TNF-a production requires a two-stage activation; priming and triggering w40x, combination of priming by PSK and triggering 24 h later is considered the optimum condition for the production of the effect in the present study, which was different from that of BRM of different origin streptococcal preparation OK-432 in its combination sequence; priming by the fungus followed by triggering by the BRM 48 h later w27x. It is suggested that PSK enhances leukocyte functions through increased TNF-a induction during the early stage of infection, which inhibits the proliferation of systemically disseminated C. albicans, resulting in improved survival rates and prolonged survival periods. However, PSK administration during the later stages of infection had little influence on TNF-a production Ždata not shown.. These findings suggested that PSK does not significantly influ-
ence leukocyte functions, survival rates or survival periods in the host in which the C. albicans dissemination has already caused on overproduction of TNF-a . The involvement of the cytokine in the expression of the biological effects of PSK was also reported by Ishihara et al. w41x that pulmonary metastasis is suppressed by PSK in mice with Lewis lung cancer in a TNF-a-dependent manner. TNF-a induces the production of cytokines that could participate in the host response to infecting pathogens, such as IL-6 and IL-1 w42x. However, induction of IL-6 and IL-1 does not appear to correlate directly with the PSK effect in the present study, since PSK injection significantly prolonged the mean survival periods of anti-IL-6 mAb-treated or anti-IL1 a mAb-treated mice, although the treatment partially reduced the PSK effect ŽTable 3.. Further, the PSK effect on plasma level of IL-6 and gene expression of IL-6 in spleen of mice after inoculation was not so marked as in case of TNF-a Ždata not shown.. These mechanisms are under investigations. Fractionation studies suggested the involvement of both b 1–3 glucan and hydrazine-sensitive proteins in the expression of PSK activities ŽFig. 6.. The b 1–3 glucan with a unique triple-helix structure exerts its various biological activities via the receptors of host cells such as macrophages w43x; the metallophilic portion of the protein moiety of PSK molecules has histidine, cysteine or tryptophan residue possibly on the molecular surface in the formation of complex with a transitional metal ion. The parameter of Levitt w44x regarding SM2 fraction in Fig. 6 revealed that the protein was hydrophobic Ždata not shown.. It was deduced that the b 1–3 glucan moieties in PSK molecule bind the receptors on host cells such as macrophages, followed by the metallophilic proteins in PSK molecule to induce intracellular signal transduction for TNF-a priming. The estimated structure for the exertion of various biological activities of PSK has been investigated with different assay systems; Matsunaga et al. w45x reported that protein with hydrophobicity has an important role to bind directly with human transforming growth factor-b and to modulate its activity; Nakamura and Matsunaga w46x reported proteins to have a role in capturing active oxygen and recovering natural killer cell activities in spleen cells of tumor-bearing rats; Ishihara et al. w47x reported that
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b-glucan and proteins had in vitro chemokine-inducing activity in human alveolar macrophages. Detailed investigation on SM2 fraction is now progressing to clarify fine structures in PSK molecule responsible for expression of biological activities. PSK exerts its immunomodulative effects by oral route in experimental animals w48x as well as cancer patients w49x. According to the studies of in vivo fate of radioisotope-labeled PSK, radioactive substances with molecular weight equivalent to PSK w50x or radioactive substances with antigenic epitopes of b 1–3 glucan portion in PSK w51x appear in blood from 4 to 24 h after oral administration to healthy rats, suggesting the absorption of PSK in the original form from digestive tract. In our preliminary experiments, oral administration of PSK significantly reduced the viable fungal counts in kidneys and prolonged the survival period, when lethal or sublethal amounts of C. albicans was iv inoculated to mice bearing syngeneic tumor w52x. However, the expression of the effect of PSK per os appears to be slightly different from that of ip injectionw53x, possibly due to the different pharmacokinetics of PSK-derived b glucan–protein complex in the body. Novel anti-infectious BRMs such as cytokine inducers and vaccines have been designed and developed w4,54,55x. The finding in the present study will provide not only the protective mechanism of BRM on systemic candidiasis but also information useful for their development or applications.
References w1x Jarvis WR. Epidemiology of nosocomial fungal infections, with emphasis to Candida species. Clin Infect Dis 1995; 20:1526–30. w2x Romani L, Howard DH. Mechanisms of resistance to fungal infections. Curr Opin Immunol 1995;7:517–23. w3x Miller LL, Nagler CH. Colony-stimulating factors: clinical applications. In: DeVita VT, Hellman S, Rosenberg SA, editors. Biologic Therapy of Cancer. 2nd edn. USA: J.B. Lippincott 1995:141–82. w4x Kullberg BJ. Trends in immunotherapy of fungal infections. Eur J Clin Microbiol Infect Dis 1997;16:51–5. w5x Kobayashi H, Matsunaga K, Oguchi Y. Anti-metastatic effects of PSK ŽKrestin., a protein-bound polysaccharide obtained from basidiomycetes, an overview. Cancer Epidemiol, Biomarkers Prev 1995;4:275–81.
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w6x Nakazato H, Koike A, Saji S, Ogawa N, Sakamoto J. Efficacy of immunochemotherapy as adjuvant treatment after curative resection of gastric cancer. Lancet 1994;343:1122–6. w7x Mitomi T, Tsuchiya S, Iijima N, Aso K, Nishiyama K, et al. Randomized controlled study on adjuvant immunochemotherapy with PSK in curatively resected colorectal cancer. Dis Colon Rectum 1992;35:123–30. w8x Tsukagoshi S, Hashimoto Y, Fujii G, Kobayashi H, Nomoto K, et al. Krestin ŽPSK.. Cancer Treat Rev 1984;11:131–55. w9x Uetsuka A, Satoh S, Ohno Y. Protective effect of PSK, a protein-bound polysaccharide preparation against candidiasis in tumor-bearing mice. In: Escobar MR, Friedman H, editors. Macrophages and Lymphocytes, Part B. New York: Plenum 1980:21–31. w10x Mayer P, Drews J. The effect of a protein-bound polysaccharide from Coriolus Õersicolor on immunological parameters and experimental infection in mice. Infection 1980;8:13–20. w11x Ebihara K, Minamishima Y. Protective effect of biological response modifiers on murine cytomegalovirus infection. J Virol 1984;51:117–22. w12x Ando T, Motokawa I, Sakurai K, Ohmura Y, Fujii T, et al. Influence of PSK ŽKrestin. on resistance to infection of Pseudomonas aeruginosa in tumor-bearing mice. Cancer Chemother Pharmacol 1987;20:198–202. w13x Goya T, Torisu M, Uchiyama A, Hayashi Y, Miyata T, et al. Immunotherapy of patients with multiple organ failure ŽMOF.. In: Torisu M, Yoshida T, editors. New horizons of Tumor Immunotherapy. Ireland: Elsevier 1989:583–95. w14x Kawana T. Effect of PSK on recurrent genital herpes. Chemotherapy 1990;38:827–31. w15x Monnma Y, Kawana T, Shimizu F. In vitro inactivation of herpes simplex virus by a biological response modifier, PSK. Antiviral Res 1997;35:131–8. w16x Vazquez-Torres A, Jones-Carlson J, Balish E. Nitric oxide production does not directly increase macrophage candicidal activity. Infect Immun 1995;63:1142–4. w17x Sasaki T, Abiko N, Nitta K, Takasuka N, Sugino Y. Antitumor activity of carboxymethylglucans obtained by carboxymethylation of b Ž1–3.-D-glucan from Alcaligenes faecalis Õar. myxogenes IFO 13140. Eur J Cancer 1979;15: 211–5. w18x Havell EA. Evidence that tumor necrosis factor has an important role in antibacterial resistance. J Immunol 1989; 143:2894–9. w19x Takasaki S, Mizouchi T, Kobata A. Hydrazinolysis of asparagines-linked sugar chain to produce free oligosaccharide. In: Ginsburg V, editor. Methods in Enzymology, vol. 83, New York: Academic Press 1982:263–8. w20x Chromczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–9. w21x Netea MG, van Tits LJH, Curfs JHAJ, Amiot F, Meis JF, et al. Increased susceptibility of TNF-a lymphotoxin-a double knockout mice to systemic candidiasis through impaired recruitment of neutrophils and phagocytosis of Candida albicans. J Immunol 1999;163:1498–505. w22x Obayashi T, Yoshida M, Mori T, Goto H, Yasuoka A, et al.
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albicans infection: evidence for an important role of TNF receptor p55 in antifungal defense. J Immunol 1996;157: 2155–9. Vecchiarelli A, Puliti M, Torosantucci A, Cassone A, Bistoni F. In vitro production of tumor necrosis factor by murine splenic macrophages stimulated with mannoprotein constituents of Candida albicans cell wall. Cell Immunol 1991; 134:65–76. Walley KR, Lukacs NW, Standifoerd TJ, Strieter RM, Kunkel SL. Balance of inflammatory cytokines related to severity and mortality of murine sepsis. Infect Immun 1996;64: 4733–8. Satoh M, Shimada Y, Inagawa H, Minagawa H, Kajikawa T, et al. Priming effect of interferons and interleukin 2 on endogenous production of tumor necrosis factor in mice. Jpn J Cancer Res 1986;77:342–4. Ishihara Y, Iijima H, Matsunaga K. Contribution of cytokines on suppression of lung metastasis. Biotherapy 1998;11: 267–75. Jabions DM, Mule JJ, McIntosh JK, Sehgal PB, May LT, et al. Interleukin-6rinterferon-b 2 as a circulating hormone: induction by cytokine administration in man. J Immunol 1989; 142:1542–7. Xia Y, Vetvicka V, Yan J, Hanikyrova M, Mayadas T, et al. The b -glucan binding lectin site of mouse CR3 ŽCD11brCD18. and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells. J Immunol 1999;162:2281–90. Levitt M. A simplified representation of protein conformations for rapid simulation of protein folding. J Mol Biol 1976;104:59–107. Matsunaga K, Hosokawa A, Oohara M, Sugita N, Harada M, et al. Direct action of a protein-bound polysaccharide, PSK, on transforming growth factor-b . Immunopharmacology 1998;40:219–30. Nakamura K, Matsunaga K. Susceptibility of natural killer ŽNK. cells to reactive oxygen species ŽROS. and their restoration by the mimics of superoxide dismutase ŽSOD.. Cancer Biother Radiopharm 1998;13:275–90. Ishihara Y, Matsunaga K, Kagawa J. C–C and C–X–C chemokine synthesis and release via receptors by human alveolar macrophages treated with three types of stimulants. In Vitro Mol Toxicol 1999;12:213–23. Harada M, Matsunaga K, Oguchi Y, Iijima H, Tamada K, et al. Oral administration of PSK can improve the impaired anti-tumor CD4q T-cell response in gut-associated lymphoid tissue ŽGALT. of specific pathogen-free mice. Int J Cancer 1997;70:362–72. Torisu M, Hayashi Y, Ishimitsu T, Iwasaki K, Katano M, et al. Significant prolongation of disease-free period gained by oral polysaccharide K ŽPSK. administration after curative surgical operation of colorectal cancer. Cancer Immunol Immunother 1990;31:261–8. Ikuzawa M, Matsunaga K, Nishiyama S, Nakajima S, Kobayashi Y, et al. Fate and distribution of an antitumor protein-bound polysaccharide PSK ŽKrestin.. Int J Immunopharmacol 1988;10:415–23. Endoh H, Matsunaga K, Yoshikumi C, Kawai Y, Suzuki T,
Y. Ohmura et al.r International Immunopharmacology 1 (2001) 1797–1811 et al. Production of antiserum against antitumor protein-bound polysaccharide preparation, PSK ŽKrestin. and its pharmacologyical application. Int J Immunopharmacol 1988;10:103–9. w52x Y. Ohmura, K. Matsunaga, I. Motokawa, K. Sakurai, T. Ando, Protective effects of a protein-bound polysaccharide, PSK, on Candida albicans infection in mice bearing syngeneic tumor 2001, in submission. w53x Nomoto K, Yoshikumi C, Matsunaga K, Fujii T, Takeya K. Restoration of antibody-forming capacities by PS-K in tumor-bearing mice. Jpn J Cancer Res 1975;66:365–74.
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w54x Li SP, Lee S, Domer JE. Alteration in frequency of interleukin-2 ŽIL-2.-, gamma interferon-, or IL-4-secreting splenocytes induced by Candida albicans mannan andror monophosphoryl lipid A. Infect Immun 1998;66:1392–9. w55x Cardenas-Freytag L, Cheng E, Mayeux P, Domer J, Clements JD. Effectiveness of a vaccine composed of heat-killed Candida albicans and a novel adjuvant, LT ŽR192G., against systemic candidiasis. Infect Immun 1999;67:826–33.
Protective effects of a protein-bound polysaccharide, PSK, on Candida albicans infection in mice via tumor necrosis factor-a induction Yoshio Ohmura, Kenichi Matsunaga) , Isamu Motokawa, Katsuo Sakurai, Takao Ando Biomedical Research Laboratory, Kureha Chemical Industry, Co. Ltd. 3-26-2, Hyakunin-cho, Shinjuku-ku, Tokyo, 169-8503, Japan Received 8 February 2001; accepted 13 May 2001
Abstract We investigated the protective mechanism of a protein-bound polysaccharide, PSK, against lethal infection with Candida albicans Ž C. albicans . in mice. Ž1. In BALBrc mice inoculated intravenously with C. albicans, the intraperitoneal Žip. administration of PSK increased survival rates and prolonged the survival period depending on the time of administration, the dosage, and the size of fungal inoculum; the maximal effect was obtained when PSK 250 mgrkg was ip administered to mice 24 h before inoculation of 1 = 10 6 C. albicans Ž30 days survivors showed 60% and the mean survival period of mice with fatal infection increased 209%.. Ž2. The protective effect of PSK was significantly decreased in mice treated with cyclophosphamide or carrageenan, or in mice treated previously with anti-tumor necrosis factor-a ŽTNF-a . antibody. Ž3. The administration of PSK significantly enhanced the expression of TNF-a gene in spleen and increased leukocyte functions from 6 h to 1 day after inoculation. Ž4. When the PSK fraction subjected to hydrolysis with b 1–3 glucanase or hydrazine was used instead of PSK, the anti-fungal activities were significantly decreased. These findings suggested that the protective effect of PSK on lethal C. albicans infection in mice was mainly produced via TNF-a functions, and that b 1–3 glucan and protein moiety in PSK molecule were involved in the expression of the activities. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Anti-infectious effects of BRM; Induction of tumor necrosis factor-a ; Candida albicans; Protein-bound polysaccharide
1. Introduction AbbreÕiations: BRM, biological response modifier; C. albicans, Candida albicans; IL-1 a , interleukin-1 a ; IL-2, interleukin2; IL-6, interleukin-6; IFN-g , interferon-g ; ip, intraperitoneally; iv, intravenously; PEC, peritoneal exudate cells; QOL, quality of life; TNF-a , tumor necrosis factor-a ) Corresponding author. Tel.: q81-3-3362-7326; fax: q81-33362-8523. E-mail address: [email protected] ŽK. Matsunaga..
Despite recent advances in anti-fungal chemotherapy and diagnosis, the increase of high-risk patients and the appearance of drug-resistant fungi have resulted in an increase in the incidence of deep-seated mycosis w1x. In many cases, deep-seated
1567-5769r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 Ž 0 1 . 0 0 1 0 4 - 7
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mycosis occurs in individuals with decreased resistance due to underlying diseases such as cancer, or due to immunosuppressive therapies w2x. It is important to maintain and improve biophylaxis in order to prevent the onset or progression of deep-seated mycosis for the management of quality of life ŽQOL. in such patients. Certain biological response modifiers ŽBRMs. have been reported to produce not only anti-cancer effects but also anti-infectious effects by modifying responses of the host to cancer cells or pathogenic microorganisms w3,4x. PSK, a protein-bound polysaccharide derived from a basidiomycete is a representative BRM exhibiting a variety of biological activities such as immunoregulation and antioxidant activity w5x, while in randomized controlled trials, the concomitant chemotherapy prolongs postoperative survival of patients with gastric cancer w6x and colorectal cancer w7x. PSK has a mean molecular weight of 9.4 = 10 4 and is composed of protein and glucan having b 1–4 bond in the main chain and b 1–3 and b 1–6 bond in the side chain, that binds to a protein moiety through O- or N-glycoside bonds w8x. Although the protective effect of PSK against infection with pathogenic microorganisms such as virus, bacteria, and fungi has been reported in experimental animals as well as immunosuppressed patients w9– 15x, the detailed mechanism of its action remains to be elucidated. The present study reports that PSK is effective in the protection against lethal systemic infections in mice with Candida albicans Ž C. albicans . through enhanced formation of tumor necrosis factor-a ŽTNF-a ..
2. Materials and methods 2.1. Animals Specific pathogen-free female BALBrc mice, BALBrc nurnu mice and BALBrc nurqmice were purchased from CLEA Japan ŽTokyo., and C57BLr6 bgrbg mice and C57BL bgrqmice from Japan Charles River ŽAtsugi, Kanagawa.. After acclimatization, they were used in experiments at 8 weeks of age. They had free access to food CE-2 ŽOriental Yeast, Tokyo. and sterilized tap water.
Animals were kept at temperature of 24 " 2 8C, a humidity of 55 " 10%, and a luminous intensity of approximately 5 lx with a lighting cycle from 8:00 AM to 8:00 PM under luminary air flow. Only staff caring for the animals and people involved in the experiments entered the breeding room. 2.2. Inoculation of C. albicans C. albicans strain IFO1060 maintained in our laboratory was used: it was inoculated in Sabouraud culture media supplemented with 2% glucose ŽpH 5.6., followed by shaking the culture at 25 8C for 18 h. After incubation, fungal cells were washed three times with endotoxin-free, sterile phosphate-buffered saline ŽPBS., and suspended in PBS to an appropriate concentration. An aliquot of the suspension was injected into the caudal vein of mouse. 2.3. Reagents and antibodies Carrageenan ŽCA. and aminoguanidine were purchased from Sigma ŽSt. Louis, MO, USA., cyclophosphamide ŽCY. was from Shionogi ŽOsaka., amphotericin B was from Bristol–Myers–Squibb ŽNew York, USA., carboxymethylated b 1–3 glucan ŽCM-curdlan. of Alcaligenes faecalis Õar. myxogenes origin with number-average degree of carboxymethyl groups substituted per anhydroglucose unit 0.47 was from Wako ŽOsaka, Japan., recombinant mouse TNF-a with endotoxin content less than 2 EUrm g protein was from Endogen ŽWoburn, MA, USA.; anti-mouse TNF-a antibody ŽAb., anti-mouse interleukin-1 a ŽIL-1 a . Ab, anti-mouse interleukin-2 ŽIL-2. Ab, anti-mouse interleukin-6 ŽIL-6. Ab, and anti-mouse interferon-g ŽIFN-g . Ab were from R & D System ŽMinneapolis, MN, USA.. These reagents or antibodies were dissolved in PBS or sterile distilled water, and injected to animals. Aminoguanidine was injected ip at a dose of 300 mgrkg 2 days before inoculation, CM-curdlan was injected ip at a dose of 100 mgrkg 1 day before inoculation and recombinant mouse TNF-a was injected iv at a dose of 10 4 Ur mouse 1 h before inoculation, following the protocols of Vasquez-Torres et al. w16x, Sasaki et al. w17x and Havell w18x, respectively.
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2.4. PSK and its fractions PSK ŽKureha Chemical Industry, Tokyo. was dissolved in PBS for use in the experiments. To estimate the structure responsible for the biological activity of PSK Ž62% sugar and 38% protein., the following fractions were prepared. For the preparation of fractions with a relatively low protein content, the peptide moiety of PSK was decomposed by hydrazine following the method of Takasaki et al. w19x. Briefly, PSK was dissolved in anhydrous hydrazine and, heated in a sealed tube at 100 8C for 24 h. Then, hydrazine was removed in a vacuum desiccator. The resulting exsiccates were dissolved in water and subjected to purification by a combination of gel filtration ŽSephadex G-25, Pharmacia Biotech, Uppsala, Sweden. and diethylaminoethyl ŽDEAE. cellulose ion exchange chromatography ŽToyopearl 650S, Toyo, Tokyo.. Thus, a hydrazine-treated PSK ŽPSK-H, 94% sugar and 6% protein. was obtained. Next, for the preparation of fractions with less sugar content, polysaccharide moiety of PSK was decomposed by periodate oxidation. Briefly, PSK was incubated in 0.04 M metaperchloric acid at 4 8C for 14 days. After the reaction was stopped by adding ethylene glycol, sodium borohybride was added and the mixture was allowed to stand at 25 8C overnight, then dialyzed against distilled water for 48 h. Sulfuric acid was added to a concentration of 0.1 N, and the mixture was left at 25 8C for 24 h. After neutralization with sodium hydroxide, the reaction mixture was subjected to gel filtration on Sephadex G-25 to remove low-molecular-weight substances. The resulting fraction PSK-S had a sugar–protein ratio of 22:78. This fraction was further applied to a column of Cuq-coordinated Chelating Sepharose FF ŽPharmacia Biotech., and fractions SM1 Ž55% sugar and 45% protein., SM2 Ž12% sugar and 88% protein. and SM3 Ž7% sugar and 93% protein. were obtained as eluates. Enzymolysis products were prepared by treating these fractions with Arthrobacter luteus-derived b 1–3 glucanase Žprepared in our laboratory by the purification of Zymolyase w purchased from Seikagaku Kogyo, Tokyo. or with Tritirachium album-derived proteinase K w ŽMerck KgaA, Darmstadt, Ger-
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many. for 5 h at 37 or 65 8C, respectively, and then subjected to gel filtration on Sephadex G-25 to remove low-molecular-weight substances. 2.5. Determination of cytokines Plasma levels of TNF-a , IL-2, IL-6 and IFN-g were measured using a commercially available ELISA kit ŽR & D System.. 2.6. Expression of cytokine genes The expression of cytokine genes was measured by the reverse transcriptase-PCR technique. Mice were sacrificed, and their blood, spleen, liver and kidney were removed and immediately frozen in liquid nitrogen. These samples were stored at y80 8C until RNA preparation. After the addition of an ISOGEN w solution ŽNippon Gene, Tokyo., the frozen samples were homogenized, and total RNA was extracted according to the method of Chromczynski and Sacchi w20x. The total RNA was mixed with a buffer solution containing 20 mM Tris–HCl ŽpH 8.4., 50 mM KCl, 2.5 mM MgCl 2 , 1 mM each of dATP, dCTP, dGTP and dTTP, 0.5 m g of oligoŽdT.12 – 18 , and 200 U of MLV reverse transcriptase ŽGibco BRL, Gaithersburg, MD, USA., and reverse transcription was allowed to proceed at 42 8C for 50 min. The reaction was stopped by heating at 70 8C for 15 min, followed by cooling on ice. Subsequently, the reaction mixture was mixed with a buffer solution containing 10 mM Tris–HCl ŽpH 9.0., 50 mM KCl, 1.5 mM MgCl 2 , 200 m M each of dATP, dCTP, dGTP and dTTP, 5 pM each of 5X and 3X oligomers, and Taq polymerase ŽPromega, Madison, WI, USA.. The reaction mixture was subjected to PCR amplification on a Perkin-Elmer PCR Thermal Cycler TP-2000 ŽPerkin Elmer, Foster City, CA, USA.. In the case of b-actin, heating at 94 8C for 1 min and 25 cycles of amplification and, in the case of TNF-a , heating at 94 8C for 4 min and 35 cycles of amplification were performed, followed by annealing for 1 min. PCR products were electrophoretically separated on 2% agarose gels containing ethidium bromide, and measured semiquantitatively using a Bio-Rad Multi-Analyst System. Total RNA, derived from concanavalin-A-stimulated mouse splenic cells, was used as a positive control; and b-actin
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total RNA as an internal standard for the measurement. The following primers were used: b-actin wsensex 5X-ATG GAT GAC GATATCGCTG, wantisensex 5X-GATTCCATA CCCA GGAA GG; TNF-a wsensex 5X-GATCTCAAAGAC AACCAACTAGTG wantisensex 5X-CTCGAGCT GGAAGACT CCTCCCAG. 2.7. Phagocytic and killing actiÕity of peritoneal exudate cells (PEC) The activities of PEC were measured following the method of Netea et al. w21x as modified. Briefly, 1 ml of 10% proteose peptone ŽDifco Lab., Detroit, MI, USA. was injected into the peritoneal cavity of healthy or fungus-inoculated mice, and PEC were collected 4 h later. The cells were washed with endotoxin-free RPMI1640 medium, and PEC were adjusted to 5 = 10 5 cellsrml with the medium. In a silicon-pretreated culture tube, the PEC suspension 1 ml and a C. albicans suspension 5 = 10 4 CFU 1 ml were mixed to a ratio of 10:1, and the mixture was cultured in an incubator at 37 8C in an atmosphere of 5% CO 2 . For the evaluation of phagocytic activity, the PEC were recovered 5 min after the start of culture and were washed with PBS twice. After it was ascertained microscopically that PEC had phagocytized fungi and that no fungi were adherent to them, they were subjected to lysis by the addition of a 0.01% aqueous solution of bovine serum albumin ŽSigma.. Serial 10-fold dilutions of the lysate in sterile PBS were made, and an aliquot of each dilution was spread on Sabouraud agar medium. After 24 h of culture at 37 8C, colonies of C. albicans were counted. For the evaluation of killing activity, PEC were recovered 15 min after the start of culture. The recovered PEC were washed with RPMI1640 medium twice, and cultured at 37 8C for 1 h in an incubator in an atmosphere of 5% CO 2 . Similar to the evaluation of phagocytic activity, serial dilutions of the cell lysate were cultured on the medium, and the colonies formed were counted. 2.8. Superoxide production by PEC A suspension of 2 = 10 5 PEC and 50 m M CLAphenyl ŽTokyo Kasei, Tokyo. were dispensed in a
tube, and the volume of the mixture was adjusted to 175 m l with Hank’s Balanced Salt Solution containing 0.25% human albumin. Twenty-five microliters of 400 ngrml of phorbol 12-myristate 13-acetate ŽPMA, Sigma. was added, the reaction was allowed to proceed at 20 8C, and then the fluorescence emitted was sequentially measured on a Luminescence Meter ŽAloka, Tokyo.. 2.9. Determination of C. albicans-deriÕed polysaccharide Plasma levels of b-glucan, a cell wall component of C. albicans, were determined following the method of Obayashi et al. w22x by the use of a commercially available kit ŽSeikagaku Kogyo.. 2.10. Determination of endotoxin The endotoxin content of samples for activity evaluation, reagents, or culture media was determined colorimetrically using a commercially available kit ŽSeikagaku Kogyo.. 2.11. Statistical analysis Values are shown as mean " standard deviation. Test of significance was performed using a log rank test and Student’s t-test, and values of P - 0.05 were considered significant.
3. Results 3.1. Dependency of anti-infectious effects of PSK on time of administration, dosage, and size of inoculum To clarify the influence of time of administration on the anti-infectious effects of PSK, we gave healthy BALBrc mice an intraperitoneal Žip. injection of 50 mgrkg of PSK 5, 3 or 1 day or immediately before the intravenous Živ. inoculation of 2 = 10 6 C. albicans, and observed them for survival periods. Compared with a 30-day survival rate of 0% and a mean survival period of 5.7 " 1.9 days for control group A, those for the PSK-administered group ranged from 7% to 47% and from 7.1 to 8.6 days, respectively ŽFig. 1-Ž1... Of the PSK-administered groups,
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Fig. 1. Protective effect of PSK on lethal C. albicans infection in mice: dependency on time of administration and dosage. BALBrc mice Ž n s 15. were iv inoculated with 2.0 = 10 6 C. albicans and were observed for survival periods. Ž1. PSK 50 mgrkg was ip administered 5 days Žgroup B., 3 days Žgroup C., 1 day Žgroup D., or immediately before Žgroup E. inoculation. A control group Žgroup A. was ip given 0.2 ml of saline 1 day before inoculation. Statistically significant at ) P - 0.01 and ) ) P - 0.05 Žvs. group A. in log rank test. Ž2. One day before inoculation, 10 mgrkg Žgroup G., 50 mgrkg Žgroup H., or 250 mgrkg Žgroup I. of PSK was administered ip. Instead of PSK, 0.2 ml of saline was administered ip to a control group Žgroup F. on the same day of PSK administration. Statistically significant at ) P - 0.01 Žvs. group F. in log rank test.
group D, to which PSK had been administered 1 day before inoculation, had the greatest survival benefit: the 30-day survival rate was 47%, and the mean survival period 8.6 " 1.0 days Ž151% that of the control group.. The 30-day survival rates and mean survival periods for groups C and B Žreceiving PSK 3 and 5 days before inoculation, respectively. were 20% and 8.1 " 1.9 days, and 7% and 7.1 " 1.4 days, respectively, suggesting a tendency that the shorter the interval between PSK administration and inoculation, the greater the protective effect. However, the 30-day survival rate and mean survival period for group E receiving PSK immediately before inoculation were 7% and 7.2 " 1.5 days, respectively, indicating a decrease in the protective effect compared with the other PSK-administered groups. The administration of PSK 1 day after inoculation had no significant effect on the rate or period of survival Ždata not shown.. Subsequently, to clarify the dose-dependency of the PSK effect, we gave healthy BALBrc mice an ip injection of 10, 50 or 250 mgrkg of PSK 1 day before the iv inoculation of 2.2 = 10 6 C. albicans, and observed the post-inoculation survival periods. Compared with the 30-day survival rate of 7% for
control group F, those for PSK-administered groups G, H and I Žreceiving 10, 50 or 250 mgrkg of PSK, respectively. were 13%, 40% and 53%, respectively, indicating an increase in the rate of survival depending on the PSK dose ŽFig. 1-Ž2... Furthermore, compared with the mean survival period of 5.2 " 0.9 days for control group F, those for PSK-administered groups G, H and I were prolonged to 6.5 " 1.0, 8.6 " 1.3, and 10.4 " 1.5 days, respectively, depending on the dose. In order to investigate the influence of the inoculum size on the PSK effect, we inoculated various amounts of C. albicans iv into healthy BALBrc mice 1 day after an ip injection of 250 mgrkg of PSK, and observed them for survival periods. As listed in Table 1, when 1.0 = 10 6 , 5.0 = 10 6 or 3.0 = 10 7 C. albicans was inoculated, the 30-day survival rate for the control group was 13%, 0% or 0%, respectively, and the mean survival period was 5.3 " 0.5, 3.9 " 0.8 or 2.9 " 0.6 days, respectively, suggesting that as the inoculum size increased, the survival rate decreased, and the mean survival period shortened. The 30-day survival rates for the groups receiving a PSK injection 1 day before inoculation were 60%, 27% and 7%, respectively, and the mean
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Table 1 Dependency of the protective effects of PSK on the inoculum size Inoculum size
Treatment
Mean survival days
30-day survivors
1=10 6
Saline PSK Saline PSK Saline PSK
5.3"0.5 Ž100%. 11.1"1.9 Ž209%. 3.9"0.8 Ž100%. 6.7"1.9 Ž172%. 2.9"0.6 Ž100%. 4.4"0.9 Ž152%.
2r15 Ž13%. 9r15 Ž60%. ) 0r15 Ž0%. 4r15 Ž27%. ) 0r15 Ž0%. 1r15 Ž7%. )
5=10 6 3=10 7
BALBrc mice Ž ns15. were given PSK 250 mgrkg or saline 0.2 ml. Twenty-four hours later, they were iv inoculated with 1=10 6 , 5=10 6 , or 3=10 7 C. albicans, and were observed for subsequent survival periods. ) Statistically significant at P - 0.01 ŽPSK group vs. saline group..
survival periods were 11.1 " 1.9 Ž209% of that for the control group., 6.7 " 1.9 Ž172%. and 4.4 " 0.9 Ž152%. days, respectively, suggesting that as the inoculum size decreased, PSK became more effective. In addition, to compare the anti-infectious effects of PSK with those of other anti-fungal drugs or reagents, we gave healthy BALBrc mice an injection of amphotericin B, CM-curdlan or recombinant mouse TNF-a 1 day or 1 h before the iv inoculation of 2.5 = 10 6 C. albicans, and observed them for survival periods. As listed in Table 2, mean survival period for the group receiving amphotericin B 1 h before inoculation or the group receiving PSK 1 day before inoculation was significantly prolonged, when compared with that of the group receiving saline. However, neither injection of CM-curdlan 1 day before inoculation nor recombinant mouse TNF-a 1 h before inoculation had any significant effect on mean survival period in those mice. These findings suggest that the anti-infectious effect of PSK depends on the time of administration, the dose administered, and the size of fungal inoculum. 3.2. ProtectiÕe effect of PSK in mice with leukopenia or phagocytes-dysfunction, or in mice with congenital immunodeficiency We examined the influence of PSK administration on the rate and period of survival after the inoculation in mice treated with CY or CA, or in mice with congenital immunodeficiency.
The treatment of CY to healthy BALBrc mice caused peripheral leukocyte counts to decrease, with a nadir at 4 days, and slow recovery. Pretreatment of mice with PSK did not significantly influence peripheral leukocyte counts Ždata not shown.. Under these conditions, we inoculated C. albicans iv into mice 4 days after an ip treatment with CY 100 or 300 mgrkg, and observed them for survival periods. When 2.1 = 10 6 C. albicans was inoculated into mice, all the mice died within 5 days, with or without PSK administration Ždata not shown.. However, when the inoculum size was decreased to 4.8 = 10 5 C. albicans, the mean survival period for group B receiving PSK 1 day before inoculation was prolonged to 182% ŽFig. 2-Ž1.. of that for control group A after treatment with CY 100 mgrkg, and to 150% of that for the group A after treatment with CY 300 mgrkg, suggesting a tendency that the PSK effect was weaker in the group treated with higher dose of CY ŽFig. 2-Ž1. and -Ž2... Then, to examine the PSK effect in mice with phagocyte system dysfunction, we inoculated 2.1 = 10 6 C. albicans iv into BALBrc mice 4 days after an ip treatment with CA 100 or 300 mgrkg. PSK administration had no significant effect on survival periods in those mice, irrespective of CA doses ŽFig. 2-Ž3. and -Ž4... Furthermore, to examine the PSK effect in mice with congenital thymus defect, we inoculated 4.8 = 10 5 C. albicans iv into BALBrc nurnu mice. As a result, compared with that of BALBrc nurqmice, Table 2 Comparison of anti-infectious effects of PSK with those of antifungal drug or reagents Treatments
Mean survival days
Ž1. Saline 0.2 ml, ip, day y1 5.0"0.6 Ž100%. Ž2. Amphotericin B 13.4"3.1 Ž268%. 0.25 mgrkg, iv, day 0 Ž3. CM-curdlan 100 5.2"0.9 Ž104%. mgrkg, ip, day y1 Ž4. Recombinant mouse TNF-a 6.7"1.2 Ž134%. 1=10 4 Urmouse, iv, day 0 Ž5. PSK 250 mgrkg, ip, 9.3"1.1 Ž186%. day y1
30-days survivors 0r8 5r8 ) 0r8 1r8 3r8 )
BALBrc mice Ž ns8. were given anti-fungal drug or reagents 1 day or 1 h before inoculation with 2.5=10 6 C. albicans on day 0, and were observed subsequent survival periods. ) Statistically significant at P - 0.01 Žvs. saline group..
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Fig. 2. Influence of treatment with CY or CA on the protective effect of PSK. BALBrc mice Ž n s 15. were iv inoculated with 4.8 = 10 5 C. albicans and were observed for survival periods. PSK 250 mgrkg was ip administered 1 day before inoculation Žgroup B.. Instead of PSK, 0.2 ml of saline was ip administered to a control group Žgroup A. on the same day of PSK administration. CY 100 mgrkg Ž1., 300 mgrkg Ž2., CA 100 mgrkg Ž3. or 300 mgrkg Ž4. was ip injected 4 days before inoculation. Statistically significant at ) P - 0.01 Žvs. group A. in log rank test.
the PSK effect on the survival period decreased in BALBrc nurnu mice 10 days after inoculation and later, with all the mice dying of infection within 17 days ŽFig. 3-Ž1. and -Ž2... However, regarding C57BLr6 bgrbg mice with congenital defect of natural killer cell activity, PSK significantly improved the rate and period of their survival ŽFig. 3-Ž3. and -Ž4... These findings suggested that the PSK effect was mainly mediated by the function of the phagocytic system, although there was a possible involvement of the T cell system during the later stage of infection. 3.3. Influence of treatment with anti-cytokine Ab or nitrogen monoxide (NO) inhibitor on the PSK effect To identify the effector molecule responsible for the expression of the PSK effect, we investigated the
influence of treatment with anti-cytokine Ab or an NO inhibitor on the rate and period of survival after inoculation in saline-treated mice and PSK-administered mice. When 2.2 = 10 6 C. albicans was iv inoculated into BALBrc mice 2 days after treatment with antiTNF-a Ab, the mean survival period was significantly shorter than in mice treated with immunoglobulin ŽIg. with similar timing ŽTable 3.. In the anti-TNF-a Ab-treated mice, PSK had no effects on the 30-days survival and mean survival periods. However, in mice treated with anti-IL-6 Ab, anti-IL1 a Ab, anti-IFN-g Ab, or anti-IL-2 Ab, or aminoguanidine, the treatments did not significantly influence the rate or mean period of survival, and PSK significantly prolonged the period of survival of the mice.
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Fig. 3. Anti-infectious effect of PSK in mice with congenital immunodeficiency. Groups of 15 BALBrc nurqmice Ž1., BALBrc nurnu mice Ž2., C57BLr6 bgrqmice Ž3., or C57BLr6 bgrbg mice Ž4. were iv inoculated with 2.1 = 10 6 C. albicans and were observed for survival periods. One day before inoculation, 0.2 ml of saline Žgroup A. or 250 mgrkg of PSK Žgroup B. was administered ip. Statistically significant at ) P - 0.01 Žvs. group A. in log rank test.
These findings suggest that the main effector molecule involved in the PSK effect is possibly TNF-a . 3.4. Influence of PSK on the leÕel of TNF-a in plasma, expression of TNF-a gene in spleen and peripheral leukocyte functions in fungus-inoculated mice To elucidate the mechanism of PSK action, we inoculated 2.0 = 10 6 C. albicans iv into healthy BALBrc mice, and examined time-dependent changes in the level of TNF-a in plasma, the expression of TNF-a gene in spleen, and leukocyte functions in PSK administered mice. The expression of TNF-a gene in the spleen of control mice was slightly induced 6 h after inoculation, and thereafter the level of expression increased with the progression of infection. However, in the
group receiving PSK, the expression was markedly induced 6 h after inoculation, gradually decreased thereafter and, at 5 days and hereafter returned almost to the level of healthy control mice ŽFig. 4-Ž1. and -Ž3... Plasma levels of TNF-a showed a similar tendency ŽFig. 4-Ž2... Furthermore, in vitro phagocytic ability, killing activity, and superoxide-productivity in PEC of mice 24 h after inoculation were significantly enhanced in fungus-inoculated group receiving PSK ŽFig. 5-Ž1., -Ž2. and -Ž3.., although there were no significant differences in PEC recovery and their cell population Ž88–95% polymorphonuclear cells and 5–12% monocytes or macrophages. among groups tested. These findings suggest that the protective effect of PSK is produced by the enhanced formation of TNF-a , followed by the resultant increase in leukocyte anti-fungal activity.
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Table 3 Effects of treatments with anti-cytokine Ab or NO inhibitor on the protective effects of PSK Treatments Ig Anti-TNF-a Ab Anti-IL-1 a Ab Anti-IL-2 Ab Anti-IL-6 Ab Anti-IFN-g Ab Aminoguanidine
Saline PSK Saline PSK Saline PSK Saline PSK Saline PSK Saline PSK Saline PSK
Mean survival days
30-day survivors
5.1"1.2 Ž100%. 9.8"1.9 Ž192%. 3.0"0.8 Ž100%. 3.6"1.1 Ž120%. 4.1"1.1 Ž100%. 6.7"1.4 Ž163%. 5.1"1.0 Ž100%. 8.0"1.4 Ž157%. 4.3"1.0 Ž100%. 5.5"1.1 Ž145%. 4.4"0.9 Ž100%. 6.8"1.9 Ž155%. 4.9"0.8 Ž100%. 8.4"1.1 Ž171%.
0r8 3r8 ) 0r8 ) ) 0r8 NS a 0r8 1r8 ) 0r8 3r8 ) 0r8 0r8 ) 0r8 3r8 ) 0r8 3r8 )
BALBrc mice Ž ns8. were ip administered anti-cytokine Ab 1 mg or aminoguanidine 300 mgrkg on day y2, and PSK 250 mgrkg or saline 0.2 ml on day y1. They were iv inoculated with C. albicans on day 0, and were observed for subsequent survival periods. a NS, not significant Žvs. saline group.. ) Statistically significant at P - 0.01 Žsaline group vs. PSK group.. )) Statistically significant at P - 0.01 ŽIg-saline group vs. Anti-TNF-a-Ab-saline group..
3.5. Estimated actiÕe structure of PSK To estimate the structure responsible for the PSK effect, we prepared PSK fractions with less protein or sugar contents, and examined their TNF-a-inducing activity and anti-infectious activity. We administered an ip injection of the fractions 100 mgrkg Žsuboptimal dose of PSK. to healthy BALBrc mice, and examined the expression of TNF-a gene in the spleen 6 h after inoculation, the mean post-inoculation survival period, and the 30-day survival rate. As shown in Fig. 6, the activities of fraction PSK-H, which was prepared by removing most of the peptide portion with hydrazine, decreased below that of PSK. In contrast, the activities of fraction PSK-S, which was prepared by cleaving the 1–6 bond in polysaccharide portion with metaperchloric acid, were higher than those of PSK. When fraction PSK-S was fractionated into three fractions by metal ŽCu.-chelate-chromatography,
Fig. 4. Influence of PSK administration on level of TNF-a in plasma and expression of TNF-a gene in spleen of fungus-inoculated mice. BALBrc mice Ž ns 5. were iv inoculated with 2.1=10 6 C. albicans, and plasma and spleen specimens were collected in a time-dependent manner to measure Ž1. the level of fungus-derived b-glucan in plasma, Ž2. the level of TNF-a in plasma, and Ž3. the expression of TNF-a gene in spleen. One day before inoculation, 250 mgrkg of PSK ŽD. or 0.2 ml of saline ŽC. was administered ip. As a control, non-inoculated mice received 250 mgrkg of PSK ŽB. or 0.2 ml of saline ŽA. ip at the same time as the inoculated mice. Statistically significant at ) P - 0.01 and )) P - 0.05 Žvs. group A. in Student’s t-test.
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fraction SM2 exhibited the greatest activities reaching about 160% of PSK activities. The treatment of SM2 with b 1–3 glucanase or proteinase K caused a decrease in the activities. These findings suggest that the main structure involved in TNF-a-inducing activity and anti-infectious activity in the PSK molecule is possibly b 1–3 glucan and protein.
4. Discussion
Fig. 5. Influence of PSK administration on the activities of PEC in healthy mice and fungus-inoculated mice. BALBrc mice Ž ns8. were iv inoculated with 2.0=10 6 C. albicans. One day later, 1 ml of 10% proteose peptone was injected ip, and PEC were collected 4 h later. Ž1. In vitro phagocytic activity of PEC after 5 min of coculture with C. albicans; Ž2. in vitro killing activity of PEC after 1 h of coculture with C. albicans; Ž3. the production of superoxide 30 min after addition of PMA. The details are described in Materials and methods. Group C, healthy control mice given saline Žwithout inoculation.; Group P, healthy mice given PSK Žwithout inoculation.; group IC, fungus-inoculated mice given saline 1 day before inoculation; group IP, fungus-inoculated mice given PSK 1 day before inoculation. Statistically significant at ) P - 0.01 and ) ) P - 0.05 in Student’s t-test.
The present study showed that PSK exerted a protective effect against lethal infection with C. albicans, depending on the time of administration, dosage, and size of fungal inoculum. The ip administration of 250 mgrkg of PSK 1 day before the iv inoculation of 1 = 10 6 C. albicans was most effective: 60% of the mice escaped fatal infection, while the mice with fatal infection had a 209% prolonged survival ŽTable 1.. In murine systemic candidiasis models, the conditions to produce anti-infectious effects of BRM vary with their action mechanisms w23,24x. Such conditions of PSK resembled those of bacterial preparations w25,26x, unlike those of certain Chinese herb medicines w24,27x whose administration after inoculation is effective, recombinant colony stimulating factor w28x and b 1–3 glucan of Saccharomyces origin w29x requiring repeated administration before inoculation, a low-molecular-weight compound of microbial metabolite origin w30x, b 1–3 glucan of mushroom origin w23x and chitin and chitosan preparations w31x, which require repeated preand post-inoculation administration. Abe et al. w24x classified the functions of anti-fungal BRM into neutrophil activators and macrophage activators; the former is effective in prolonging survival periods when administered before inoculation, and the latter is effective when administered after inoculation. In addition, H-Young et al. w23x postulated that BRM activating phagocytic cells exert effects in the early stage of infection, and those enhancing cell-mediated immunity produce effects during the later stages of infection. Since the findings in the present study showed that the PSK effect reduced in mice in which the leukocyte counts had been reduced by treatment with a high dose of CY, or diminished in mice in
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Fig. 6. Influence of PSK fractions on the expression of TNF-a gene in spleen and anti-infectious activity. BALBrc mice Ž n s 13. were iv inoculated with 2.1 = 10 6 C. albicans. Six hours later, five mice were sacrificed, and their spleens were tested for the expression of the TNF-a gene. The remaining eight mice were observed for survival periods. PSK or its fractions were administered ip in a dose of 100 mgrkg 1 day before inoculation. The details of procedures for the preparation of the fractions are described in Materials and methods. Statistically significant at †1 P - 0.01 Žvs. Control group., † 2 P - 0.01 and † 2 P - 0.05 Žvs. PSK group., †3 P - 0.01 and †3 P - 0.05 Žvs. PSK-S group. and †4P - 0.01 Žvs. M2 group. in Student’s t-test or log rank test.
which the phagocytic function had been impaired by CA treatment ŽFig. 2., it was suggested that PSK mainly falls into a category of BRM that enhanced leukocyte function and had a protective activity in the early stage of infection. However, in T cell-deficient nude mice, PSK was not effective in prolonging survival 10 days after inoculation and later ŽFig.
3., suggesting a possible involvement of T cell function in the production of PSK effects in the later stages of infection. Among effector molecules that host cells produce in response to infecting pathogens, TNF-a is an important multifunctional cytokine during onset and progression of systemic candidiasis as evidenced by
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Marodi et al. w32x, Steinshamn and Waage w33x, Blasi et al. w34x, Louie et al. w35x, Ashman and Papadimitriou w36x, Steinshamn et al. w37x and Netea et al. w21x. During the early stage of infection, TNF-a , which is produced in the body by the stimulation with mannan or mannoprotein of C. albicans cell wall components w38x, acts on cells such as macrophages and polymorphonuclear cells to enhance their anti-fungal functions. However, during the later stages of infection, as the fungus rapidly proliferates, the level of TNF-a in the body increases, affecting negative effects on the cytokine network in the host defense system, often resulting in end-organ failure or septic shock w39x. The observations in the present study that anti-fungal activities of PEC in control mice were enhanced 1 day after inoculation ŽFig. 5., as TNF-a was induced in plasma and spleen ŽFig. 4., and that blocking TNF-a functions by pretreatment with specific Ab shortened survival periods of control mice that had been iv inoculated ŽTable 3., also demonstrated the physiological role of this cytokine in the host defense mechanism. That TNF-a is a candidate molecule responsible for the expression of the protective effect of PSK on C. albicans infection can be inferred from Ži. the disappearance of the effect by treatment with antiTNF-a Ab ŽTable 3., Žii. a marked induction of TNF-a gene in spleen 6 h after inoculation ŽFig. 4., and Žiii. a significant enhancement of PEC anti-fungal activities 1 day after inoculation ŽFig. 5.. Since TNF-a production requires a two-stage activation; priming and triggering w40x, combination of priming by PSK and triggering 24 h later is considered the optimum condition for the production of the effect in the present study, which was different from that of BRM of different origin streptococcal preparation OK-432 in its combination sequence; priming by the fungus followed by triggering by the BRM 48 h later w27x. It is suggested that PSK enhances leukocyte functions through increased TNF-a induction during the early stage of infection, which inhibits the proliferation of systemically disseminated C. albicans, resulting in improved survival rates and prolonged survival periods. However, PSK administration during the later stages of infection had little influence on TNF-a production Ždata not shown.. These findings suggested that PSK does not significantly influ-
ence leukocyte functions, survival rates or survival periods in the host in which the C. albicans dissemination has already caused on overproduction of TNF-a . The involvement of the cytokine in the expression of the biological effects of PSK was also reported by Ishihara et al. w41x that pulmonary metastasis is suppressed by PSK in mice with Lewis lung cancer in a TNF-a-dependent manner. TNF-a induces the production of cytokines that could participate in the host response to infecting pathogens, such as IL-6 and IL-1 w42x. However, induction of IL-6 and IL-1 does not appear to correlate directly with the PSK effect in the present study, since PSK injection significantly prolonged the mean survival periods of anti-IL-6 mAb-treated or anti-IL1 a mAb-treated mice, although the treatment partially reduced the PSK effect ŽTable 3.. Further, the PSK effect on plasma level of IL-6 and gene expression of IL-6 in spleen of mice after inoculation was not so marked as in case of TNF-a Ždata not shown.. These mechanisms are under investigations. Fractionation studies suggested the involvement of both b 1–3 glucan and hydrazine-sensitive proteins in the expression of PSK activities ŽFig. 6.. The b 1–3 glucan with a unique triple-helix structure exerts its various biological activities via the receptors of host cells such as macrophages w43x; the metallophilic portion of the protein moiety of PSK molecules has histidine, cysteine or tryptophan residue possibly on the molecular surface in the formation of complex with a transitional metal ion. The parameter of Levitt w44x regarding SM2 fraction in Fig. 6 revealed that the protein was hydrophobic Ždata not shown.. It was deduced that the b 1–3 glucan moieties in PSK molecule bind the receptors on host cells such as macrophages, followed by the metallophilic proteins in PSK molecule to induce intracellular signal transduction for TNF-a priming. The estimated structure for the exertion of various biological activities of PSK has been investigated with different assay systems; Matsunaga et al. w45x reported that protein with hydrophobicity has an important role to bind directly with human transforming growth factor-b and to modulate its activity; Nakamura and Matsunaga w46x reported proteins to have a role in capturing active oxygen and recovering natural killer cell activities in spleen cells of tumor-bearing rats; Ishihara et al. w47x reported that
Y. Ohmura et al.r International Immunopharmacology 1 (2001) 1797–1811
b-glucan and proteins had in vitro chemokine-inducing activity in human alveolar macrophages. Detailed investigation on SM2 fraction is now progressing to clarify fine structures in PSK molecule responsible for expression of biological activities. PSK exerts its immunomodulative effects by oral route in experimental animals w48x as well as cancer patients w49x. According to the studies of in vivo fate of radioisotope-labeled PSK, radioactive substances with molecular weight equivalent to PSK w50x or radioactive substances with antigenic epitopes of b 1–3 glucan portion in PSK w51x appear in blood from 4 to 24 h after oral administration to healthy rats, suggesting the absorption of PSK in the original form from digestive tract. In our preliminary experiments, oral administration of PSK significantly reduced the viable fungal counts in kidneys and prolonged the survival period, when lethal or sublethal amounts of C. albicans was iv inoculated to mice bearing syngeneic tumor w52x. However, the expression of the effect of PSK per os appears to be slightly different from that of ip injectionw53x, possibly due to the different pharmacokinetics of PSK-derived b glucan–protein complex in the body. Novel anti-infectious BRMs such as cytokine inducers and vaccines have been designed and developed w4,54,55x. The finding in the present study will provide not only the protective mechanism of BRM on systemic candidiasis but also information useful for their development or applications.
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