Ganoderma
Tsugae
Cell Activity
Mycelium
and Serum Shen-Jeu
Won',
Enhances Interferon
Mao-Tsun
Splenic Production
Lin2, * and Wu-Lung
Natural
Killer
in Mice
Wu3
'Department of Microbiology and 2Department of Physiology, National Cheng Kung University Medical College, Tainan, Taiwan 70101 3Deparmmentof Pharmacy , National Defense Medical Center, Taipei, Taiwan Received
October
30, 1991
Accepted
February
20, 1992
ABSTRACT Effects of the water-soluble extract of Ganoderma tsugae mycelium (GT), its alcohol insoluble subfraction (GTI), and its alcohol-soluble subfraction (GTS) on splenic natural killer (NK) cell activity and serum interferon (IFN) production were assessed in mice. Intraperitoneal administra tion of GT (4-200 mg/kg) or GTI (1-50 mg/kg), but not GTS, augmented the NK cytotoxic activity in a dose-dependent manner in C3H/HeN mice. This augmentation of splenic NK cytolytic activity was not mouse-strain-dependent. The serum IFN titers of mice were also elevated after i.p.-doses of GTI. The GTI-induced serum IFN was reduced by either IFN-(a + ,8) antiserum or IFN-y monoclonal anti body in vitro. The treatment with antiserum neutralizing IFN-(a + ,8) resulted in a 70% reduction of GTI-induced IFN, while monoclonal antibody against mouse IFN-y, moderately neutralized the GTI induced IFN (50%). These results demonstrated that both the splenic NK activity and serum IFN [IFN (a + ,8) and IFN-y] titers are elevated by Ganoderma tsugae mycelium extracts in mice. Keywords: Ganoderma tsugae mycelium, Natural killer (splenic), Interferon production (serum), C3H/HeN mouse
The crude extracts of the fruit body of Ganoderma lucidum, a traditional Chinese herb, were demonstrated to inhibit the growth of Sarcoma 180 or fibrosarcoma (1, 2). The mycelium extract of Ganoderma lucidum was also shown to enhance interleukin-2 production in vitro (3). In addition, our previous studies showed that the alcohol-insoluble fraction of Ganoderma lucidum mycelium augmented the NK cell activity in mice (4). Recently, we also isolated a new compound, 3/3 hydroxy-26-oxo-5a -lanosta-8,24-dien-11-one, from the fruit body of Formosan Ganoderma lucidum which ex hibited potent inhibition of human KB and PLC/PRF/5 cells in vitro (5, 6). In the present study, we attempted to further assess the effects of extracts of Ganoderma tsugae mycelium, a species of Basidiomycetes, on splenic NK cytolytic activity and serum interferon production in mice.
MATERIALS AND METHODS Materials All drug solutions were prepared in pyrogen-free glassware, which was heated at 180°C for 5 hr before use. All solutions were steriled and non-pyrogenic; and as an additional precaution, they were passed through 0.22-,um Millipore bacterial filters. None of the extracts of Ganoderma tsugae used in this study induced gela tion in the limulus Amebocyte Lysate test (Associates of Cape Cod, Woods Hole, MA), so that any con tamination with endotoxin was below the level of 25 pg/ml. Mice Inbred C3H/HeN, C57BL/6, RIIIS/J, or C3H/HeJ mice were obtained from the Animal Center of the National Cheng Kung University (Tainan, Taiwan). Male or female 7 to 10-week-old mice were used in all experiments.
Preparation of extracts from mycelium of Ganoderma tsugae Mycelia (1000 g) was collected from malt extracted culture broth of Ganoderma tsugae (GT) and was washed twice with pyrogen-free water. The mycelia were extracted in water for 16 hr at 100°C and then fil tered through a Buchner funnel. The filtrate was con centrated by evaporation at 70°C under reduced pres sure. The dry powder (10 g) of the water-soluble ex tract of GT mycelium was immersed in 1000 ml 95% ethanol for 24 hr to fractionate the active substance. After alcohol extraction, the solution was centrifuged at 5000 rpm for 30 min to separate the alcohol-soluble fraction (GTS) and the precipitate, which was the alcohol-insoluble fraction (GTI). The resultant super natant was filtered through a Millipore filter (0.45,um) to remove the fine particles and then evaporated at 40°C under reduced pressure. Media Cell preparations were accomplished in RPMI 1640 (Gibco, Grand Island, NY) with 25 mM HEPES buffer (Gibco) plus 10% heat-inactivated fetal bovine serum (FBS, Gibco). The NK cytotoxic assays were carried out in Dulbecco's modified Eagle's medium (DMEM, Gibco) with 25 mM HEPES buffer, 10% FBS and the medium supplemented with 2 mM glutamine, 100 IU/ml penicillin G and 100 ,ug/ml streptomycin. Inter feron assays were made in Eagle's minimum essential medium (MEM, Gibco) supplemented with 5% new born calf serum (NCS, Gibco), L-glutamine and anti biotics. Cells The NK-sensitive cell line YAC-1, a Moloney virus induced T lymphoma, was grown as stationary suspen sion cultures in RPMI 1640 with 10% FBS, 100IU/ml penicillin, 100 ,ug/ml streptomycin, and 2 MM L-gluta mine. Preparation of spleen cells All spleens were aseptically removed and teased on a steel mesh immersed in chilled RPMI-1640 in a plastic dish. The cells that passed through the mesh were washed twice with RPMI-1640. The erythrocytes were lysed with double distilled water, while the remaining cells were suspended in DMEM with 10% FBS. Cytotoxicity assay The standard 4-hr 51Cr-release assay was performed as described previously (7). Briefly, the target cells were labelled with 100 pCi 51Cr (Amersham Interna tional plc, Amersham, U.K.) for 1 hr in 0.2 ml DMEM
containing 10% FBS. After washing three times in RPMI, effector cells at various concentrations were in cubated with 1 X 104 51Cr-labeled target cells in a 96 well, round-bottom microplate (Corning Laboratory Sciences Co.). After incubation, the plates were centri fuged at 800 X g for 10 min, and the radioactivity in 0.1 ml of the supernatant was measured in a gamma coun ter (Compu Gamma 1282, LKB, Sweden). The percent age of specific lysis was calculated by the following for mula:
where test cpm are the cpm released in the presence of effector cells, spontaneous cpm are the those cpm re leased from target cells added to culture medium alone, and maximal cpm are those cpm released from target cells, which was obtained by adding 0.2% SDS. All groups are tested in triplicate. The standard deviation was usually less than 3%. Spontaneous release is less than 12% of the maximal release. Interferon assay Interferon activity titers were assayed by the cyto pathic effect inhibition microassay method with mouse L cells and vesicular stomatitis virus (VSV, Indiana strain) as the challenge virus (4). The highest dilution of the titrated sample causing at least 50% protection was considered to be the endpoint. Titers were express ed in international reference units, as calibrated by the National Institutes of Health mouse reference IFN (G 002-904-511; NIH, Bethesda) and an internal IFN stan dard was used as reference units. Neutralization assays Rabbit antibody to mouse IFN-(a + 8) and mono clonal immunoglobulin to mouse IFN y were purchased from Lee Bio Molecular Research (San Diego, CA, USA). One-tenth milliliter of GTI-induced IFN was in cubated with 0.1 ml of 1 : 50 final dilution of anti-mouse IFN-(a + ,8) or anti-mouse IFN y for 1 hr at 37°C prior to assay on the L cells, and then the residual IFN activ ity was determined as described above. Statistical analysis Statistical significance was determined by normalizing the values of each assay and using the paired Student's t-test.
RESULTS Effect of Ganoderma tsugae mycelium extracts on sple nic NK cytotoxic activity in vivo Table 1 summarizes the effects of GT on splenic NK cytotoxic activity in C3H/HeN mice. The NK cytolytic activity was tested against YAC-1 target cells by the 4-hr 51Cr release assay 24 hr after an i.p. injection of GT. Intraperitoneal administration of 4 mg/kg of GT significantly augmented the mouse splenic NK activity at the E : T ratio of 40: 1, 80: 1, and 160: 1 (Table 1). GT over a dose range of 4 200 mg/kg produced a dose-related enhancement in the splenic NK activity in C3H/HeN mice (Table 1). Over a dose range of 1 50 mg/kg, i.p. injection of GTI, but not GTS, also re
Table 1. Dose-response effect of GT on splenic NK cytotoxic activity in C3H/HeN micea
Table 3.
suited in a dose-dependent augmentation of the splenic NK activity (Table 2). At the i.p.-dose of 2.5 mg/kg of GTI, the splenic NK activity started to increase at 15 hr after injection (Table 3). The NK activity reached its peak level at 24 72 hr and returned to its normal level by 120 hr. On the other hand, the similar augmenting effect on the splenic NK activity was also observed in the other mouse strains after an i.p.-dose of 2.5 mg/kg of GTI (Table 4). In particular, GTI enhanced splenic NK activity in LPS-resistant C3H/HeJ mice. Effects of GTI on serum IFN production Figure 1 shows the effects of GTI on serum IFN titers in mice. Intraperitoneal administration of 0.1 mg/kg of GTI had no effect on the serum level of IFN.
Table 2. Dose-response effect of GTI on splenic NK cytotoxic activity in C3H/HeN micea
Kinetic augmentation of splenic NK cytotoxic activity in C3H/HeN by GTIa
Intraperitoneal administration of 1 mg/kg, 10 mg/kg, or 50 mg/kg of GTI significantly elevated the serum levels of IFN in mice. At an i.p.-dose of 2.5 mg/kg of GTI, the IFN titer started to rise at 15 hr, reached its peak level at 24 48 hr, and returned to its control level at about 96 hr (Fig. 2). Table 5 summarizes the effects of mouse IFN-(a + ,8) antibody of IFN y antibody on the GTI-induced serum IFN. It was found that the GTI-induced serum IFN was reduced by either IFN-(a + 8) antiserum or IFN y monoclonal antibody in vitro. It can be seen that the treatment with antiserum neutralizing IFN-(a + f8) re sulted in a marked reduction of 70% of GTI-induced IFN. In contrast, monoclonal antibody against mouse IFN-y moderately neutralized the GTI-induced IFN (50%).
Table
4.
Effect
of GTI
on splenic
NK
Fig. 1. Effect of various doses of GTI on IFN induction in C3H/HeN mice. Titers represented IFN activities in the mouse sera obtained at 24 hr after i.p.-injection of GTI. All values of IFN titers in the mouse sera are the means ± S.E.M. of three separate experiments.
DISCUSSION The present study showed that the water-soluble ex tract of Ganoderma tsugae mycelium (GT) or its sub fraction, alcohol-insoluble fraction (GTI), augmented the splenic NK cytotoxic activity in mice. Such a biologi cal property is not demonstrated by the alcohol-soluble fraction (GTS). The kinetic study in mice revealed that the NK activity began to rise at 15 hr after an i.p.-dose of 2.5 mg/kg of GTI, reached its peak at 24-72 hr, and returned to the normal level by 120 hr. The results also showed that the GTI-induced NK activity enhancement was not mouse-strain-specific, since it has been demon strated in 4 different strains of mice. One may ask about the possibility that the biological effect of GTI may be due to LPS contamination. However, the cur rent results showed that the GT or GTI used in the
cytotoxic
activity
in different
mouse
strains'
Fig. 2. Kinetics of serum IFN induction after the injection of GTI at 2.5 mg/kg, i.p. in C3H/HeN mice. All values of IFN titers in the mouse sera are the means ± S.E.M. of three separate ex periments.
Table
5.
Characterization
of GTI-induced
present study did not induce gelation in the LAL test. Furthermore, GTI was also shown to augment the NK activity in LPS-non-responder C3H/HeJ mice. There fore, it seems that this possibility can be ruled out from the present study. Additionally, our purification study shows that the GTI contains mainly proteins and car bohydrates (S.-J. Won et al., unpublished data). There fore, certain kinds of glycoproteins may be useful for producing NK cytotoxic these effects. Previous studies showed that augmentation of NK activity by a variety of agents is mediated by induction of IFN (7-10). IFN has also been demonstrated to aug ment the NK activity in vitro or in vivo (7-14). In deed, our present results showed that 15 hours after an i.p.-injection of GTI (1-50 mg/kg), the serum IFN titer started to elevate in mice. Like the time course of the splenic NK activity, the GTI-induced IFN elevation reached its pleateau at 24-48 hr. The appearance of elevating IFN levels coincided-well with augmented NK activity in GTI-treated mice. These observations lead us to think that the augmentation of the splenic NK activ ity in mice after administration of GTI may be due to the induced IFN elevation in the mice serum. Inter ferons have been categorized as IFN-a, IFN-/3, and IFN-y based on antigenic heterogeneity (15). In the present results, the serum IFN elevation induced by GTI was reduced by treatment of the serum with either anti-mouse IFN-(a + ,8) or IFN y . The results sug gested that the serum IFN production induced by GTI is a mixture of types IFN-a, IFN-fl, and IFN-y. Final ly, it should be mentioned that in the current results, there is no dose-dependency for the IFN production in
IFN
by
antibody
neutralizations
duced by GTI (as shown in Fig. 1). This indicates that the GTI dose range of 1 50 mg/kg is the optimal con centration for IFN production. Any doses below or above this dose range, i.e., 0.1 mg/kg or 250 mg/kg of GTI, would have no effect on IFN production. Fur thermore, although we did not study any positive con trol substance (or drugs) in this study, our previous ex periments have already provided evidence to demon strate that Ganoderma lucidum had similar effects (4 6). In summary, the present results show that intraperi toneal administration of GT or its subfraction GTI, but not GTS, induced a dose-related increase in the splenic NK activity in mice. In addition to elevating the splenic NK activity, administration of GTI produced an in crease in serum IFN titers. Acknowledgments The work reported here was suppported by grants from the National Science Council (Taipei, Taiwan, No. NSC 80-0420 B006-11).
REFERENCES 1
2
3
Cheng, H.H., Tung, Y.C. and Tung, T.C.: Effects of Ganoderma lucidum extracts on sarcoma-180 tumor growth in mice. Bull. Chinese Oncol. Soc. 3, 22-28 (1982) Lee, S.S., Chen, F.D., Chang, S.C., Wei, Y.H., Liu, I., Chen, C.F., Wei, R.D., Chen, K.Y. and Han, P.W.: In vivo antitumor effect of crude extracts from the mycelium of Ganoderma lucidum. J. Chinese Oncol. Soc. 5, 22-28 (1984) Chang, H.H., Hsieh, K.H., Tung, Y.C. and Tung, T.C.: Effect of Ganoderma lucidum extract on interleukin-2 pro
duction in mice. J. Chinese Oncol. Soc. 4, 13-22 (1988) 4 Won, S.J., Lee, S.S., Ke, Y.H. and Lin, M.T.: Enhancement of splenic NK cytotoxic activity by extracts of Ganoderma lucidum mycelium in mice. J. Biomed. Lab. Sci. 2, 201-213 (1989) 5 Lin, C.N., Tome, W.P. and Won, S.J.: A lanostanoid of Formosan Ganoderma lucidum. Phytochemistry 29, 673-675 (1990) 6 Lin, C.N., Tome, W.P. and Won, S.J.: Novel cytotoxic principles of Formosan Ganoderma lucidum. J. Nat. Prod. 54, 998-1002 (1991) 7 Djeu, J.Y., Heinbaugh, J.A., Holden, H.T. and Herberman, R.B.: Augmentation of mouse natural killer cell activity by interferon and interferon inducers. J. Immunol. 122, 175 187 (1979) 8 Aso, H., Suzuki, F., Yamaguchi, T., Hayashi, Y., Ebina, T. and Ishida, N.: Induction of interferon and activation of NK cells and macrophages in mice by oral administration of Ge 132, an organic germanium compound. Microbiol. Immunol. 29, 65-74 (1985) 9 Langford, M.P., Weigent, D.A., Georgiades, J.A., Johnson, H.M. and Stanton, G.J.: Antibody to staphylococcal entero toxin A induced human immune interferon. J. Immunol. 126, 1620 1623(1978)
10 Gidlund, M., Orn, A., Wigzell,H., Senik, A. and Gresser, I.: Enhanced NK cell activity in mice injected with interferon and interferon inducers. Nature 273, 759-761 (1978) 11 Oldham, R.K.: Biologicalsand biological response modifiers: Fourth modality of cancer treatment. Cancer Treat. Reports 68, 221-232 (1984) 12 Oldham, R.K., Thurman, G.B., Talmadge, J.E., Stevenson, H.C. and Foon, K.A.: Lymphokines, monoclonal antibodies, and other biological response modifiers in the treatment of cancer. Cancer 54, 2795-2806 (1984) 13 Oldham, R.K.: Biologicalsand biologicalresponse modifiers: Design of clinical trials. J. Biol. Resp. Modif. 4, 117-128 (1985) 14 Lotzova, E., Savary, C.A. and Stringfellow, D.A.: 5-Halo-6 phenyl pyrimidinones: new molecules with cancer therapeutic potential and interferon-inducingcapacity are strong inducers of murine natural killer cells. J. Immunol. 130, 965-969 (1983) 15 Baron, S., Dianzani, F. and Stanton, G.J.: General consid eration of the interferon system. In Texas Reports on Biology and Medicine 41, 1981-1982: The Interferon System: A Re view to 1982. Part 1, Edited by Baron, S., Dianzani, F. and Stanton, G.T., Vol. 41, p. 1 12, The University of Texas Medical Branch, Galveston (1982)
Tsugae
Cell Activity
Mycelium
and Serum Shen-Jeu
Won',
Enhances Interferon
Mao-Tsun
Splenic Production
Lin2, * and Wu-Lung
Natural
Killer
in Mice
Wu3
'Department of Microbiology and 2Department of Physiology, National Cheng Kung University Medical College, Tainan, Taiwan 70101 3Deparmmentof Pharmacy , National Defense Medical Center, Taipei, Taiwan Received
October
30, 1991
Accepted
February
20, 1992
ABSTRACT Effects of the water-soluble extract of Ganoderma tsugae mycelium (GT), its alcohol insoluble subfraction (GTI), and its alcohol-soluble subfraction (GTS) on splenic natural killer (NK) cell activity and serum interferon (IFN) production were assessed in mice. Intraperitoneal administra tion of GT (4-200 mg/kg) or GTI (1-50 mg/kg), but not GTS, augmented the NK cytotoxic activity in a dose-dependent manner in C3H/HeN mice. This augmentation of splenic NK cytolytic activity was not mouse-strain-dependent. The serum IFN titers of mice were also elevated after i.p.-doses of GTI. The GTI-induced serum IFN was reduced by either IFN-(a + ,8) antiserum or IFN-y monoclonal anti body in vitro. The treatment with antiserum neutralizing IFN-(a + ,8) resulted in a 70% reduction of GTI-induced IFN, while monoclonal antibody against mouse IFN-y, moderately neutralized the GTI induced IFN (50%). These results demonstrated that both the splenic NK activity and serum IFN [IFN (a + ,8) and IFN-y] titers are elevated by Ganoderma tsugae mycelium extracts in mice. Keywords: Ganoderma tsugae mycelium, Natural killer (splenic), Interferon production (serum), C3H/HeN mouse
The crude extracts of the fruit body of Ganoderma lucidum, a traditional Chinese herb, were demonstrated to inhibit the growth of Sarcoma 180 or fibrosarcoma (1, 2). The mycelium extract of Ganoderma lucidum was also shown to enhance interleukin-2 production in vitro (3). In addition, our previous studies showed that the alcohol-insoluble fraction of Ganoderma lucidum mycelium augmented the NK cell activity in mice (4). Recently, we also isolated a new compound, 3/3 hydroxy-26-oxo-5a -lanosta-8,24-dien-11-one, from the fruit body of Formosan Ganoderma lucidum which ex hibited potent inhibition of human KB and PLC/PRF/5 cells in vitro (5, 6). In the present study, we attempted to further assess the effects of extracts of Ganoderma tsugae mycelium, a species of Basidiomycetes, on splenic NK cytolytic activity and serum interferon production in mice.
MATERIALS AND METHODS Materials All drug solutions were prepared in pyrogen-free glassware, which was heated at 180°C for 5 hr before use. All solutions were steriled and non-pyrogenic; and as an additional precaution, they were passed through 0.22-,um Millipore bacterial filters. None of the extracts of Ganoderma tsugae used in this study induced gela tion in the limulus Amebocyte Lysate test (Associates of Cape Cod, Woods Hole, MA), so that any con tamination with endotoxin was below the level of 25 pg/ml. Mice Inbred C3H/HeN, C57BL/6, RIIIS/J, or C3H/HeJ mice were obtained from the Animal Center of the National Cheng Kung University (Tainan, Taiwan). Male or female 7 to 10-week-old mice were used in all experiments.
Preparation of extracts from mycelium of Ganoderma tsugae Mycelia (1000 g) was collected from malt extracted culture broth of Ganoderma tsugae (GT) and was washed twice with pyrogen-free water. The mycelia were extracted in water for 16 hr at 100°C and then fil tered through a Buchner funnel. The filtrate was con centrated by evaporation at 70°C under reduced pres sure. The dry powder (10 g) of the water-soluble ex tract of GT mycelium was immersed in 1000 ml 95% ethanol for 24 hr to fractionate the active substance. After alcohol extraction, the solution was centrifuged at 5000 rpm for 30 min to separate the alcohol-soluble fraction (GTS) and the precipitate, which was the alcohol-insoluble fraction (GTI). The resultant super natant was filtered through a Millipore filter (0.45,um) to remove the fine particles and then evaporated at 40°C under reduced pressure. Media Cell preparations were accomplished in RPMI 1640 (Gibco, Grand Island, NY) with 25 mM HEPES buffer (Gibco) plus 10% heat-inactivated fetal bovine serum (FBS, Gibco). The NK cytotoxic assays were carried out in Dulbecco's modified Eagle's medium (DMEM, Gibco) with 25 mM HEPES buffer, 10% FBS and the medium supplemented with 2 mM glutamine, 100 IU/ml penicillin G and 100 ,ug/ml streptomycin. Inter feron assays were made in Eagle's minimum essential medium (MEM, Gibco) supplemented with 5% new born calf serum (NCS, Gibco), L-glutamine and anti biotics. Cells The NK-sensitive cell line YAC-1, a Moloney virus induced T lymphoma, was grown as stationary suspen sion cultures in RPMI 1640 with 10% FBS, 100IU/ml penicillin, 100 ,ug/ml streptomycin, and 2 MM L-gluta mine. Preparation of spleen cells All spleens were aseptically removed and teased on a steel mesh immersed in chilled RPMI-1640 in a plastic dish. The cells that passed through the mesh were washed twice with RPMI-1640. The erythrocytes were lysed with double distilled water, while the remaining cells were suspended in DMEM with 10% FBS. Cytotoxicity assay The standard 4-hr 51Cr-release assay was performed as described previously (7). Briefly, the target cells were labelled with 100 pCi 51Cr (Amersham Interna tional plc, Amersham, U.K.) for 1 hr in 0.2 ml DMEM
containing 10% FBS. After washing three times in RPMI, effector cells at various concentrations were in cubated with 1 X 104 51Cr-labeled target cells in a 96 well, round-bottom microplate (Corning Laboratory Sciences Co.). After incubation, the plates were centri fuged at 800 X g for 10 min, and the radioactivity in 0.1 ml of the supernatant was measured in a gamma coun ter (Compu Gamma 1282, LKB, Sweden). The percent age of specific lysis was calculated by the following for mula:
where test cpm are the cpm released in the presence of effector cells, spontaneous cpm are the those cpm re leased from target cells added to culture medium alone, and maximal cpm are those cpm released from target cells, which was obtained by adding 0.2% SDS. All groups are tested in triplicate. The standard deviation was usually less than 3%. Spontaneous release is less than 12% of the maximal release. Interferon assay Interferon activity titers were assayed by the cyto pathic effect inhibition microassay method with mouse L cells and vesicular stomatitis virus (VSV, Indiana strain) as the challenge virus (4). The highest dilution of the titrated sample causing at least 50% protection was considered to be the endpoint. Titers were express ed in international reference units, as calibrated by the National Institutes of Health mouse reference IFN (G 002-904-511; NIH, Bethesda) and an internal IFN stan dard was used as reference units. Neutralization assays Rabbit antibody to mouse IFN-(a + 8) and mono clonal immunoglobulin to mouse IFN y were purchased from Lee Bio Molecular Research (San Diego, CA, USA). One-tenth milliliter of GTI-induced IFN was in cubated with 0.1 ml of 1 : 50 final dilution of anti-mouse IFN-(a + ,8) or anti-mouse IFN y for 1 hr at 37°C prior to assay on the L cells, and then the residual IFN activ ity was determined as described above. Statistical analysis Statistical significance was determined by normalizing the values of each assay and using the paired Student's t-test.
RESULTS Effect of Ganoderma tsugae mycelium extracts on sple nic NK cytotoxic activity in vivo Table 1 summarizes the effects of GT on splenic NK cytotoxic activity in C3H/HeN mice. The NK cytolytic activity was tested against YAC-1 target cells by the 4-hr 51Cr release assay 24 hr after an i.p. injection of GT. Intraperitoneal administration of 4 mg/kg of GT significantly augmented the mouse splenic NK activity at the E : T ratio of 40: 1, 80: 1, and 160: 1 (Table 1). GT over a dose range of 4 200 mg/kg produced a dose-related enhancement in the splenic NK activity in C3H/HeN mice (Table 1). Over a dose range of 1 50 mg/kg, i.p. injection of GTI, but not GTS, also re
Table 1. Dose-response effect of GT on splenic NK cytotoxic activity in C3H/HeN micea
Table 3.
suited in a dose-dependent augmentation of the splenic NK activity (Table 2). At the i.p.-dose of 2.5 mg/kg of GTI, the splenic NK activity started to increase at 15 hr after injection (Table 3). The NK activity reached its peak level at 24 72 hr and returned to its normal level by 120 hr. On the other hand, the similar augmenting effect on the splenic NK activity was also observed in the other mouse strains after an i.p.-dose of 2.5 mg/kg of GTI (Table 4). In particular, GTI enhanced splenic NK activity in LPS-resistant C3H/HeJ mice. Effects of GTI on serum IFN production Figure 1 shows the effects of GTI on serum IFN titers in mice. Intraperitoneal administration of 0.1 mg/kg of GTI had no effect on the serum level of IFN.
Table 2. Dose-response effect of GTI on splenic NK cytotoxic activity in C3H/HeN micea
Kinetic augmentation of splenic NK cytotoxic activity in C3H/HeN by GTIa
Intraperitoneal administration of 1 mg/kg, 10 mg/kg, or 50 mg/kg of GTI significantly elevated the serum levels of IFN in mice. At an i.p.-dose of 2.5 mg/kg of GTI, the IFN titer started to rise at 15 hr, reached its peak level at 24 48 hr, and returned to its control level at about 96 hr (Fig. 2). Table 5 summarizes the effects of mouse IFN-(a + ,8) antibody of IFN y antibody on the GTI-induced serum IFN. It was found that the GTI-induced serum IFN was reduced by either IFN-(a + 8) antiserum or IFN y monoclonal antibody in vitro. It can be seen that the treatment with antiserum neutralizing IFN-(a + f8) re sulted in a marked reduction of 70% of GTI-induced IFN. In contrast, monoclonal antibody against mouse IFN-y moderately neutralized the GTI-induced IFN (50%).
Table
4.
Effect
of GTI
on splenic
NK
Fig. 1. Effect of various doses of GTI on IFN induction in C3H/HeN mice. Titers represented IFN activities in the mouse sera obtained at 24 hr after i.p.-injection of GTI. All values of IFN titers in the mouse sera are the means ± S.E.M. of three separate experiments.
DISCUSSION The present study showed that the water-soluble ex tract of Ganoderma tsugae mycelium (GT) or its sub fraction, alcohol-insoluble fraction (GTI), augmented the splenic NK cytotoxic activity in mice. Such a biologi cal property is not demonstrated by the alcohol-soluble fraction (GTS). The kinetic study in mice revealed that the NK activity began to rise at 15 hr after an i.p.-dose of 2.5 mg/kg of GTI, reached its peak at 24-72 hr, and returned to the normal level by 120 hr. The results also showed that the GTI-induced NK activity enhancement was not mouse-strain-specific, since it has been demon strated in 4 different strains of mice. One may ask about the possibility that the biological effect of GTI may be due to LPS contamination. However, the cur rent results showed that the GT or GTI used in the
cytotoxic
activity
in different
mouse
strains'
Fig. 2. Kinetics of serum IFN induction after the injection of GTI at 2.5 mg/kg, i.p. in C3H/HeN mice. All values of IFN titers in the mouse sera are the means ± S.E.M. of three separate ex periments.
Table
5.
Characterization
of GTI-induced
present study did not induce gelation in the LAL test. Furthermore, GTI was also shown to augment the NK activity in LPS-non-responder C3H/HeJ mice. There fore, it seems that this possibility can be ruled out from the present study. Additionally, our purification study shows that the GTI contains mainly proteins and car bohydrates (S.-J. Won et al., unpublished data). There fore, certain kinds of glycoproteins may be useful for producing NK cytotoxic these effects. Previous studies showed that augmentation of NK activity by a variety of agents is mediated by induction of IFN (7-10). IFN has also been demonstrated to aug ment the NK activity in vitro or in vivo (7-14). In deed, our present results showed that 15 hours after an i.p.-injection of GTI (1-50 mg/kg), the serum IFN titer started to elevate in mice. Like the time course of the splenic NK activity, the GTI-induced IFN elevation reached its pleateau at 24-48 hr. The appearance of elevating IFN levels coincided-well with augmented NK activity in GTI-treated mice. These observations lead us to think that the augmentation of the splenic NK activ ity in mice after administration of GTI may be due to the induced IFN elevation in the mice serum. Inter ferons have been categorized as IFN-a, IFN-/3, and IFN-y based on antigenic heterogeneity (15). In the present results, the serum IFN elevation induced by GTI was reduced by treatment of the serum with either anti-mouse IFN-(a + ,8) or IFN y . The results sug gested that the serum IFN production induced by GTI is a mixture of types IFN-a, IFN-fl, and IFN-y. Final ly, it should be mentioned that in the current results, there is no dose-dependency for the IFN production in
IFN
by
antibody
neutralizations
duced by GTI (as shown in Fig. 1). This indicates that the GTI dose range of 1 50 mg/kg is the optimal con centration for IFN production. Any doses below or above this dose range, i.e., 0.1 mg/kg or 250 mg/kg of GTI, would have no effect on IFN production. Fur thermore, although we did not study any positive con trol substance (or drugs) in this study, our previous ex periments have already provided evidence to demon strate that Ganoderma lucidum had similar effects (4 6). In summary, the present results show that intraperi toneal administration of GT or its subfraction GTI, but not GTS, induced a dose-related increase in the splenic NK activity in mice. In addition to elevating the splenic NK activity, administration of GTI produced an in crease in serum IFN titers. Acknowledgments The work reported here was suppported by grants from the National Science Council (Taipei, Taiwan, No. NSC 80-0420 B006-11).
REFERENCES 1
2
3
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