B-Cell Mitogenic Activity of Slime Products Produced from Slime-Forming, Encapsulated Lsctococcus lactls ssp. cremorls HARUKI KlTAZAWA,1 TAKAHIRO YAMAGUCHI,2 and TAKATOSHIITOH 1 Department of Animal Science Faculty of Agriculture, Tohoku University Aobaku, Sendal, 981, Japan ABSTRACT
[3H]TdR = tritiated thymidine, LPS = lipopolysaccharide. Mff = macrophage, SI = stimulation index, T f = fraction enriched with T cells, WCP = whole celllyophylized preparation.
The mitogenic activities of whole cell lyophylized preparations, cell-wall components, and slime products obtained from Lactococcus lactis ssp. cremoris KVS20 were examined on murine spleen cells. Whole cell lyophylized preparations and slime products significantly (P < .05) stimulated mitogenic responses of the cells. The highest activity was induced by slime products in which the optimal concentration was 116 Jlglml. The significant (P < .05) increase of mitogenic activity induced by slime products occurred at 24 h, and the peak response was obtained 48 h after the stimulation. The activity was much higher in the fraction enriched with B cells than in the fraction enriched with T cells. In addition, slime products induced mitogenic activity to spleen cells of athymic nulnu mice. The chemical analysis of lipopolysaccharide and the minimal concentration for mitogenic response eliminated the possibility that the activity of slime products may be due to the contamination of lipopolysaccharide. The data demonstrate that slime products are a potent B-cell-dependent mitogen. (Key words: Lactococcus lacns ssp. cremoris, slime products, mitogenic activity, B-cell-dependent mitogen)
INTRODUCTION
The immunobiological effects with dairy lactic acid bacteria and their products have been an area of great interest (4, 5). In previous studies (12, 13), we reported that Scandinavian ropy sour milk, viili, and one of the starter cultures--the slime-forming, encapsulated Lactococcus lactis ssp. cremoris (designated L. lachs ssp. cremoris KVS20}-exhibited antitumoral activity against sarcoma180 in vivo. In addition, an intraperitoneal injection of L. lactis ssp. crenwris KVS20 and the slime products (CSP) induced cytotoxic macrophage (Mff) in a short-term study (10. 11). However, CSP failed to render thioglycollate-induced MIlS cytotoxic by the stimulation in vitro unlike L. lactis ssp. cremoris KVS20 (10). These findings suggested that CSP may stimulate the function of MIlS in vivo through the activation of lymphoid tissue, such as spleen and lymph nodes. In the present study, we focused on immunological effects of CSP on spleen cells in which a mitogenic activity with CSP was precisely evaluated.
Abbreviation key: Bf =fraction enriched with B cells, Con A = concanavalin A, CSP = slime products, CWC = cell-wall components,
Received March 30, 1992. Accepted July 20. 1992. ILaboratory of Animal Products Chemistry. 2Laboratory of Animal Morphology. 1992 J Dairy Sci 75:2946-2951
MATERIALS AND METHODS MIce
Specific pathogen-free C57BU6 and BALB/c (+/+. nul+, and nulnu) mice were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan). All mice were male and were used at 6 to 7 wk of age. 2946
2947
B-CELL MITOGENIC ACTIVITY
Preparation of Whole Cell, Cell·Wall, and CSP of L lect/s ssp. cremorls KVS20
Lactococeus laetis ssp. eremoris KVS20, isolated from Scandinavian ropy sour milk, viili (12), was used. The lyophylized preparation of whole cells (WCP) and CSP from the cultures of L. laetis ssp. eremoris KVS20 was obtained by the method previously reported (10, 11). Cell-wall components (CWC) of L. laetis ssp. cremoris KVS20 were prepared according to the method described by Park et al. (20). Spleen Cell Cultures
Spleen cells were prepared aseptically from spleen by gentle mincing and tapping on a 200-mesh stainless steel screen in RPMI-I640 medium (Nissui Seiyaku Co., Ltd., Tokyo, Japan). The cells were washed twice with cold RPMI medium, and erythrocytes were lysed with 50 mM Tris·HCI buffer (pH 7.6) containing NH4CI (27). The cells were suspended at a concentration of 2 x 106 cells/ml in RPMI1640 medium supplemented with 100 IU/ml of penicillin, 100 J.Lg/ml of streptomycin, 2.1 mM L-glutamine, and 2% fetal calf serum. The T- and B-cell fractions were separated by filtration through a nylon wool column (25). Spleen cell suspension was penetrated in nylon fiber (Wako Pure Chemicals Inc., Ltd., Osaka, Japan) packed with a plastic syringe (Terumo, Tokyo, Japan) and incubated for 1 h at 37"C under an atmosphere of 95% air and 5% C02. Effluent cells were collected by addition of warm RPMI medium to the column and used as the fraction enriched with T cells (Te). After the column was washed, the nylon wool was squeezed with stainless steel forceps several times, and the remaining cells were collected with warm RPMI medium and used as the fraction enriched with B cells (Be)' Mitogen
Lipopolysaccharide (LPS) from Escherichia coli 01l1:B4 and concanavalin A (COD A) were obtained from Sigma Chemical Co. (St. Louis, MO) and were appropriately diluted before use. Mitogen Responses
Mitogenic responses were determined by tritiated thymidine ([3H]TdR) incorporation
into spleen cells in triplicate (17). An aliquot of spleen cell suspension (.1 ml) was put into a well of a round-bottom microculture plate (AtS Nunc, Roskilde, Denmark). An equal volume of RPMI-I640 containing various concentrations of WCP, CWC, and CSP was added to each well. All cultures were incubated for 24 to 120 h at 37"C under an atmosphere of 95% air and 5% C02. The cultures were pulsed for 6 h with 9.25 kBq of [3H]TdR (Amersham Japan, Tokyo, Japan) before the end of experimental periods and harvested on the glass filter (Labo Mash, Labo Science Co., Ltd., Tokyo, Japan). The amount of [3H]TdR incorporated into spleen cells was determined in a scintillation counter (Beckman Instruments Inc., Palo Alto, CA). The stimulation index (81) was determined as follows: SI = (counts per minute in treated - counts per minute in background)/(counts per minute in control counts per minute in background). LPS Content In CSP
To examine the contamination of LPS in CSP, the content in CSP was assayed by using an endotoxin assay kit (Endospecy; Seikagaku Co., Tokyo, Japan) (19). Statistics
The significance between the means of SI and a control were analyzed by Dunnett's option (3) of the GLM procedure in SAS (23). The optimal concentration of CSP was determined by REG procedure in SAS using an equation f(x) = -.OOO512x2 + .118807x .686806, where f(x) =SI, and x =dose (micrograms per milliliter). RESULTS
Mitogenic Activity of L mctls ssp. cremorls KVS20 Preparations
When WCP, CWC, and CSP were added to the cultures of spleen cells of C57BU6 mice at
a concentration of 10, 50, and 100 J.Lg/ml, WCP (50 J.Lg/ml) and CSP (50 and 100 J.Lglml) significantly enhanced mitogenic activity (Table 1). The SI of CSP was higher than that of WCP, but it was not as high as LPS (20 J.Lg/ ml) and Con A (2 J.Lglml). To eliminate the Journal of Dairy Science Vol. 75. No. 11. 1992
2948
KITAZAWA ET AL.
TABLE I. Mitogenic activity of preparations of Lactococcus lactis ssp. cremoris KVS20. Stimulation index2
Dose
Sample'
X
(JJ.glmI) 10 50 100
1.87 1.93· 1.65
.20 .10 .27
CWC
10 50 100
1.05 1.11 1.06
.01 .01 .06
CSP
10 50 100
1.05 4.92·· 5.32··
.13
20 2
22.60*· 31.78··
.93 2.85
=
(JJ.gIml) CSP
'WCP Whole cell lyophylized preparation, ewc cell-wall components. CSP slime products. LPS lipopolysaccharide. Con A = concanavalin A.
=
12.5 25 50 75 100 200 1000
LPS Con A
.17 .32
= =
Stimulation index2
Dose
Sample'
SD
WCP
LPS Con A
TABLE 2. Dose response on mitogenic activity of slime products.
20 2
X 1.04 1.84 3.40·· 5.55·· 6.29··· 2.55·· 1.14 31.38·· 31.78"·
SD .12 .13 .16 .17 .07 .03 .05 4.26
2.77
'CSP = Slime products, LPS = lipopolysaccharide. Con A = concanavalin A. 2Stimulation index represent the arithmetic mean of triplicate samples.
"p < .01 (against control). •••p < .001 (against control).
2Stimulation index represents the arithmetic mean of triplicate samples. • p < .05 (against control).
up < .01 (against control).
possibility that mitogenic activity of esp was due to contamination by endotoxin (LPS), LPS content in esp was analyzed. and the minimal concentration needed to induce mitogenic activity was determined. The content was less than 1 ng/IOO Ilg of esp. In addition, 10 ng! rol of LPS did not significantly (P > .05) stimulate [3H]TdR incorporation into spleen cells in this mitogenic assay system. The results indicated that esp induced mitogenic activity to murine spleen cells. Properties of CSP~nduced Mitogenic Responses
To estimate the precise properties of the mitogenic responses of esp. the optimal concentration and the response pattern for the mitogenic activity were determined in spleen cell cultures of C57BU6 mice. The CSP significantly (P < .01) induced the mitogenic activity of spleen cells at a range of 50 to 200 Ilg/ml. The highest activity (SI of 6.29) was obtained with a concentration of 100 Ilglml (Table 2). The optimal concentration of CSP for mitogenic activity was estimated as 116 Ilg/ml by the regression analysis. Addition of 100 Ilg/ml of CSP induced significant (P < Journal of Dairy Science Vol. 75. No. II, 1992
7 6
5 H
l/l
4 3
2 1 0 0
24 48 72
120
S t ixnul. a.t ion. ( h ) Figure 1. Response pattern of slime products (CSP)induced mitogenic activity. The results are expressed as the mean and standard deviation of the stimulation index (51).•p < .05 and "P < .01 (against control).
2949
B-CELL MITOGENIC ACTIVITY TABLE 3. Comparative mitogenic activity of slime products to fractionated spleen cells. Fractioned spleen cells 2
Stimulation index! CSp3
X
-
SD
5.74** 9.43* 1.42
Con A
LPS
(100 JLglml) -
-
.34 1.35 .42
(20 JLglml) -
X
SD
14.50** 28.53** 1.74
.29
-
(2 Ilglml) SD 22.30** .02 2.19* .32 144.45*** 1.16
X
.77
.01
!Stimulation index represents the arithmetic mean of triplicate samples. 2NC 3(;SP
= Unfractioned
= slime
spleen cells, B£
products, LPS
= fraction
enriched with B cells, T£
= lipopolysaccharide, Con
= concanavalin
A
= fraction
enriched with T cells.
A.
*p < .05 (against control). "p < .01 (against control). ***p < .001 (against control).
.05) mitogenic activity at 24 h, and peak ac· tivity occurred at 48 h (Figure 1). Thereafter, the activity gradually decreased until the end of the experimental period. The results obtained indicate that CSP is an effective mitogen for murine spleen cells. Identification of Lymphocytes ResponsIve to CSP
To determine the contribution of T and B cells to CSP-induced mitogenic responses, Te and Be were cultured with the optimal concen-
(IJ. 9/11.1)
nu/+
---
I[ Control
* ij **H' ** H
75.23
50 75 100
***r--D \ ***r---o ) 75
tration of CSP. The mitogenicity of Te and Be was fully induced by Con A and LPS, respectively. The mitogenic activity of CSP depended on Be, but not on Te (Table 3), indicating that CSP is a possible mitogen to B cells. To confinn further that CSP is a B-cell mitogen, spleen cells of BALB/c athymic nu/nu mice and the littermate nu/+ mice were cultured with CSP (50 to 100 I!g/ml). The mitogenic activity of LPS occurred in both the cultures of nu/+ and athymic nu/nu mice, but Con A did not induce mitogenic activity in the cultures of athymic nu/nu mice that were defi-
ConA 2
22.14
81***
LPS 20
3( ~
30 15
10
S I
5
o
o
5
10
15 20
25
S I =
Figure 2. The B- and T-cell dependency of slime products (CSP)-induced mitogenic activity. Con A Concanavalin A. LPS = lipopolysaccharide. SI stimulation index. *p < .05. **p < .01, and ***p < .001 (against control).
=
Journal of Dairy Science Vol. 75, No. II, 1992
2950
KITAZAWA ET AL.
cient in T cells. The CSP caused mitogenic activity in both cultures from nu/+ and athymic nu/nu mice (Figure 2), and the activity was compatible with that obtained from C57BU6 mice. The results verified that CSP is a potent B-cell mitogen. DISCUSSION
Whole cell preparations of some dairy lactic acid bacteria that do not produce CSP have been reported to induce mitogenic activity to spleen cells (9, 24). In the present study, we demonstrated significant mitogenic activity of CSP, which is a cell surface component of slime-forming L lactis ssp. cremoris KVS20, dairy lactic acid bacteria, to murine lymphocytes of spleen. However, WCP and CWC of L. lactis ssp. cremoris KVS20 without CSP had little or no mitogenic activity to spleen cells. This suggests that the difference in mitogenic activity of lactic acid bacteria depends on the difference in cell components. The CSP induced mitogenic activity to BE but not to TE. Furthermore, CSP activity to spleen cells from athymic nu/nu mice was as high as that to BE' The small content of LPS in CSP eliminated the possibility that the mitogenic activity of CSP to spleen cells was caused by the contamination with LPS. These findings confirm that CSP is an effective Bcell mitogen. A previous study (10) indicated that intraperitoneal injection of CSP induced an antigen presenting M~ and a cytotoxic M~ and that CSP had no effect on the induction of cytotoxic M~ in vitro. Lymphokines, which are released from stimulated lymphocytes, render cytotoxic M~ (6, 7, 16, 21, 22). Therefore, CSP conceivably has the ability to stimulate lymphocytes in which the released lymphokines activate M~. In this study, CSP stimulated B cells as a mitogen. The findings may support the idea that CSP induces cytotoxic MI!I through the activation of lymphocytes. Various polysaccharides separated from Kampo medicine (14, 26), Dictyophora indusiata (8), and Nocardia (15) have been widely accepted as potent mitogens. A novel polysaccharide from Nocardia asteroides was reported to be a mitogen specific for B cells (15). Analysis showed that a major component Journal of Dairy Science Vol. 75, No. 11, 1992
of CSP is a phosphopolysaccharide (data not shown) that is similar to the CSP of L. lactis ssp. cremoris SBT0495 (18). Therefore, the substances of mitogenic activity of CSP may be the polysaccharide. We presented herein evidence that CSP is a potent mitogen to murine B cells, although the activity is not as high as LPS. Because ropy sour milk (viiH) containing CSP has long been consumed as a food, it is important to investigate whether CSP stimulates immune responses when ingested. Some dairy lactic acid bacteria augment mitogenic response (2) and antibody production (28) of intestinallymphoid tissue cells (peyer's patches). These cells come into direct contact with dietary antigens (1). Therefore, to establish whether dairy products containing CSP are beneficial food additives, it is necessary to examine the immunological effects on Peyer's patches. ACKNOWLEDGMENTS
The authors thank Nobuko Kumano, Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai, Japan, for her useful advice. The authors also thank Kuniji Yamaki, Laboratory of Animal Genetics, Faculty of Agriculture, Tohoku University, Sendai, Japan, for his helpful discussions in statistical analysis. This work was partially supported by a grant-in-aid for encouragement of young scientists (number 01760224) from the Ministry of Education, Science, and Culture of Japan and the Morinaga HOshikai to H. Kitazawa. REFERENCES 1 Bienestock, J., and A. D. BeCus. 1980. Mucosal immunology. Immunology 41:249. 2 De Simone, C., R. Vesely, R. Negri, B. B. Salvadori. S. zanzoglu, A. Cilli, and L. Lucci. 1987. Enhancement of immune response of murine Peyer's patches by a diet supplemented with yogurt. Immunopharmacol. Immunotoxicol. 9:87. 3 Dunnett, C. W. 1955. A multiple comparisons procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50:1096. 4 Fernandes. C. F.• and K. M. Shahani. 1990. Anticarcinogenic and immunological properties of dietary lactobacilli. J. Food Prot. 53:704. 5 Fernandes, C. F., K. M. Shahani. and M. A. Amer. 1987. Therapeutic role of dietary lactobacilli and lactobacilli fermented dairy products. Fed. Eur. Microbiol. Soc. Microbiol. Rev. 46:343.
B-CELL MITOGENIC ACI'lVITY
6 Fidler, I. I., and A. Raz. 1981. The induction of tumoricidal capacities in mouse and rat macrophages by Iympholdnes. Lympholdnes 3:345. 7 Groot, J. W. De, R. A. De Weger, R. J. Vandebriel, and W. Den Otter. 1989. Differences in the induction of macrophage cytotoxicity by the specific T lymphocyte factor, specific macrophage arming factor (SMAFl, and the lymphokine, macrophage activating factor (MAF). Immunobiology 179:131. 8 Hara, C., Y. Kumazawa, K. Inagaki, M. Kaneko, T. Kiho, and S. Ukai. 1991. Mitogenic and colonystimulating factor-inducing activities of polysaccharide fractions from the fruit bodies of Dicryophora indusiata fisch. Chern. Pharm. Bull. 39:1615. 9 Kadooka, Y., S. Fujiwara, and T. Hirota. 1991. Effects of Bijidobacteria cells on mitogenic response of splenocytes and several functions of phagocytes. Milchwissenschaft 46:626. 10 Kitazawa, H., T. Itoh, and T. Yamaguchi. 1991. Induction of macrophage cytotoxicity by slime products produced by encapsulated lActoeoccus 1JJctis ssp. cremoris. Anim. Sci. Techno!. 62:861. 11 Kitazawa, H., M. Nomura, T. Itoh, and T. Yamaguchi. 1991. Functional alteration of macrophages by a slime-forming lActococcus lactis ssp. cremoris. I. Dairy Sci. 74:2082. 12 Kitazawa, H., T. Toba, T. Itoh, N. Kumano, and S. Adachi. 1990. Antitumor activity of ropy sour milks in murine solid tumor. Anim. Sci. Techno!. 61:1033. 13 Kitazawa, H., T. Toba, T. Itoh, N. Kumano, S. Adachi, and T. Yamaguchi. 1991. Antitumoral activity of slime-forming, encapsulated 1Actococcus lactis ssp. cremoris isolated from Scandinavian ropy sour milk ''villi''. Anim. Sci. Techno!. 62:277. 14 Kiyohara, H., N. Takemoto, Y. Komatsu, H. Kawamura, E. Hosoya, and H. Yamada. 1991. Characterization of mitogenic pectic polysaccharides from Kampo (Japanese herbal) medicine "JuzenTaibo-To". Planta Med. 57:254. 15 Kurisaki, T.• K. Munemura, K. Kobayashi, M. Yamamoto, M. Hayashi, A. Nagai, and M. Yamasaki. 1991. E-15, A novel polysaccharide mitogen from Nocardia specific for B cells. Agric. BioI. Chem. 55: 2987. 16 Meltzer, M. S. 1981. Macrophage activation fortumor cytotoxicity: characterization of priming and trigger signals during Iymphokine activation. I. Immuno!. 127:179.
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17 Miura, M., T. Saito, H. Kiatazawa, T. Itoh, and T. Inamoto. 1991. Specific adsorption of immunoglobulin G in bovine colostrum on sephadex and its mitogenic activity on mouse splenocytes. Agric. Bio!. Chern. 55:3103. 18 Nakajima, H.• S. Toyada, T. Toba, T. Itoh, T. Mukai. H. Kitazawa, and S. Adachi. 1990. A novel phospbopolysaccharide from slime-forming 1Actococcus lactis subspecies cremoris SBT 0495. I. Dairy Sci. 73: 1472. 19 Obayashi, T., 1985. A new chromogenic endotoxinspecific assay using recombined limulus coagulation enzymes and its clinical applications. Clin. Chim. Acta 149:55. 20 Park, C. I., H. Sugawara, and T. Itoh. 1991. Fractionation and characterization of structural components of lactobacilli cell walls. Milchwissenschaft 46:87. 21 Pels, E., R. A. De Weger, and W. Den Otter. 1984. Lymphocyte induced macrophage cytotoxicity inducing lymphocyte. Immunobiology 166:84. 22 Ruco, L. P., and M. S. Meltzer. 1978. Macrophage activation for tumor cytotoxicity: increased lymphokine responsiveness of peritoneal macrophages during acute inflammation. J. Immuno!. 120:1054. 23 SAS4P/STAT User's Guide: Release 6.03 Edition. 1988. REG procedure, p. 773. SAS Inst., Inc., Cary, NC. 24 Shimizu, T., I. Mifuchi, and T. Yokokura. 1981. Mitogenic effect of lactobacilli on murine lymphocytes. Chern. Pharm. Bull. 29:3731. 25 Trizio, D., and G. Cudkowicz. 1974. Separation of T and B lymphocytes by nylon wool columns: evaluation of efficacy by functional assays in vivo. J. Immunol. 113:1093. 26 Yamada, H., H. Kiyohara, J.-e. Cyong, N. Takemoto, Y. Komatsu. H. Kawamura, M. Aburada, and E. Hosoya. 1990. Fractionation and characterization of mitogenic and anti-eomplementary active fractions from Kampo (Japanese herbal) medicine "IounTaibo-To". Planta Med. 56:386. 27 Yamaguchi, T., Y. Kuroda, M. Saito, T. Ebina, F. Hoshino, and N. Ishida. 1984. Immune interferon production by TH69, a lyophilized preparation of Streptococcus faecaiis, in murine spleen cell cultures. Microbio!. Immuno!. 28:601. 28 Yasui, H., A. Mike, and M. Ohwalci. 1989. Immunogenicity of Bifidobacterium breve and change in antibody production in Peyer's patches after oral administration. I. Dairy Sci. 72:30.
Iournal of Dairy Science Vol. 75, No. 11, 1992
[3H]TdR = tritiated thymidine, LPS = lipopolysaccharide. Mff = macrophage, SI = stimulation index, T f = fraction enriched with T cells, WCP = whole celllyophylized preparation.
The mitogenic activities of whole cell lyophylized preparations, cell-wall components, and slime products obtained from Lactococcus lactis ssp. cremoris KVS20 were examined on murine spleen cells. Whole cell lyophylized preparations and slime products significantly (P < .05) stimulated mitogenic responses of the cells. The highest activity was induced by slime products in which the optimal concentration was 116 Jlglml. The significant (P < .05) increase of mitogenic activity induced by slime products occurred at 24 h, and the peak response was obtained 48 h after the stimulation. The activity was much higher in the fraction enriched with B cells than in the fraction enriched with T cells. In addition, slime products induced mitogenic activity to spleen cells of athymic nulnu mice. The chemical analysis of lipopolysaccharide and the minimal concentration for mitogenic response eliminated the possibility that the activity of slime products may be due to the contamination of lipopolysaccharide. The data demonstrate that slime products are a potent B-cell-dependent mitogen. (Key words: Lactococcus lacns ssp. cremoris, slime products, mitogenic activity, B-cell-dependent mitogen)
INTRODUCTION
The immunobiological effects with dairy lactic acid bacteria and their products have been an area of great interest (4, 5). In previous studies (12, 13), we reported that Scandinavian ropy sour milk, viili, and one of the starter cultures--the slime-forming, encapsulated Lactococcus lactis ssp. cremoris (designated L. lachs ssp. cremoris KVS20}-exhibited antitumoral activity against sarcoma180 in vivo. In addition, an intraperitoneal injection of L. lactis ssp. crenwris KVS20 and the slime products (CSP) induced cytotoxic macrophage (Mff) in a short-term study (10. 11). However, CSP failed to render thioglycollate-induced MIlS cytotoxic by the stimulation in vitro unlike L. lactis ssp. cremoris KVS20 (10). These findings suggested that CSP may stimulate the function of MIlS in vivo through the activation of lymphoid tissue, such as spleen and lymph nodes. In the present study, we focused on immunological effects of CSP on spleen cells in which a mitogenic activity with CSP was precisely evaluated.
Abbreviation key: Bf =fraction enriched with B cells, Con A = concanavalin A, CSP = slime products, CWC = cell-wall components,
Received March 30, 1992. Accepted July 20. 1992. ILaboratory of Animal Products Chemistry. 2Laboratory of Animal Morphology. 1992 J Dairy Sci 75:2946-2951
MATERIALS AND METHODS MIce
Specific pathogen-free C57BU6 and BALB/c (+/+. nul+, and nulnu) mice were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan). All mice were male and were used at 6 to 7 wk of age. 2946
2947
B-CELL MITOGENIC ACTIVITY
Preparation of Whole Cell, Cell·Wall, and CSP of L lect/s ssp. cremorls KVS20
Lactococeus laetis ssp. eremoris KVS20, isolated from Scandinavian ropy sour milk, viili (12), was used. The lyophylized preparation of whole cells (WCP) and CSP from the cultures of L. laetis ssp. eremoris KVS20 was obtained by the method previously reported (10, 11). Cell-wall components (CWC) of L. laetis ssp. cremoris KVS20 were prepared according to the method described by Park et al. (20). Spleen Cell Cultures
Spleen cells were prepared aseptically from spleen by gentle mincing and tapping on a 200-mesh stainless steel screen in RPMI-I640 medium (Nissui Seiyaku Co., Ltd., Tokyo, Japan). The cells were washed twice with cold RPMI medium, and erythrocytes were lysed with 50 mM Tris·HCI buffer (pH 7.6) containing NH4CI (27). The cells were suspended at a concentration of 2 x 106 cells/ml in RPMI1640 medium supplemented with 100 IU/ml of penicillin, 100 J.Lg/ml of streptomycin, 2.1 mM L-glutamine, and 2% fetal calf serum. The T- and B-cell fractions were separated by filtration through a nylon wool column (25). Spleen cell suspension was penetrated in nylon fiber (Wako Pure Chemicals Inc., Ltd., Osaka, Japan) packed with a plastic syringe (Terumo, Tokyo, Japan) and incubated for 1 h at 37"C under an atmosphere of 95% air and 5% C02. Effluent cells were collected by addition of warm RPMI medium to the column and used as the fraction enriched with T cells (Te). After the column was washed, the nylon wool was squeezed with stainless steel forceps several times, and the remaining cells were collected with warm RPMI medium and used as the fraction enriched with B cells (Be)' Mitogen
Lipopolysaccharide (LPS) from Escherichia coli 01l1:B4 and concanavalin A (COD A) were obtained from Sigma Chemical Co. (St. Louis, MO) and were appropriately diluted before use. Mitogen Responses
Mitogenic responses were determined by tritiated thymidine ([3H]TdR) incorporation
into spleen cells in triplicate (17). An aliquot of spleen cell suspension (.1 ml) was put into a well of a round-bottom microculture plate (AtS Nunc, Roskilde, Denmark). An equal volume of RPMI-I640 containing various concentrations of WCP, CWC, and CSP was added to each well. All cultures were incubated for 24 to 120 h at 37"C under an atmosphere of 95% air and 5% C02. The cultures were pulsed for 6 h with 9.25 kBq of [3H]TdR (Amersham Japan, Tokyo, Japan) before the end of experimental periods and harvested on the glass filter (Labo Mash, Labo Science Co., Ltd., Tokyo, Japan). The amount of [3H]TdR incorporated into spleen cells was determined in a scintillation counter (Beckman Instruments Inc., Palo Alto, CA). The stimulation index (81) was determined as follows: SI = (counts per minute in treated - counts per minute in background)/(counts per minute in control counts per minute in background). LPS Content In CSP
To examine the contamination of LPS in CSP, the content in CSP was assayed by using an endotoxin assay kit (Endospecy; Seikagaku Co., Tokyo, Japan) (19). Statistics
The significance between the means of SI and a control were analyzed by Dunnett's option (3) of the GLM procedure in SAS (23). The optimal concentration of CSP was determined by REG procedure in SAS using an equation f(x) = -.OOO512x2 + .118807x .686806, where f(x) =SI, and x =dose (micrograms per milliliter). RESULTS
Mitogenic Activity of L mctls ssp. cremorls KVS20 Preparations
When WCP, CWC, and CSP were added to the cultures of spleen cells of C57BU6 mice at
a concentration of 10, 50, and 100 J.Lg/ml, WCP (50 J.Lg/ml) and CSP (50 and 100 J.Lglml) significantly enhanced mitogenic activity (Table 1). The SI of CSP was higher than that of WCP, but it was not as high as LPS (20 J.Lg/ ml) and Con A (2 J.Lglml). To eliminate the Journal of Dairy Science Vol. 75. No. 11. 1992
2948
KITAZAWA ET AL.
TABLE I. Mitogenic activity of preparations of Lactococcus lactis ssp. cremoris KVS20. Stimulation index2
Dose
Sample'
X
(JJ.glmI) 10 50 100
1.87 1.93· 1.65
.20 .10 .27
CWC
10 50 100
1.05 1.11 1.06
.01 .01 .06
CSP
10 50 100
1.05 4.92·· 5.32··
.13
20 2
22.60*· 31.78··
.93 2.85
=
(JJ.gIml) CSP
'WCP Whole cell lyophylized preparation, ewc cell-wall components. CSP slime products. LPS lipopolysaccharide. Con A = concanavalin A.
=
12.5 25 50 75 100 200 1000
LPS Con A
.17 .32
= =
Stimulation index2
Dose
Sample'
SD
WCP
LPS Con A
TABLE 2. Dose response on mitogenic activity of slime products.
20 2
X 1.04 1.84 3.40·· 5.55·· 6.29··· 2.55·· 1.14 31.38·· 31.78"·
SD .12 .13 .16 .17 .07 .03 .05 4.26
2.77
'CSP = Slime products, LPS = lipopolysaccharide. Con A = concanavalin A. 2Stimulation index represent the arithmetic mean of triplicate samples.
"p < .01 (against control). •••p < .001 (against control).
2Stimulation index represents the arithmetic mean of triplicate samples. • p < .05 (against control).
up < .01 (against control).
possibility that mitogenic activity of esp was due to contamination by endotoxin (LPS), LPS content in esp was analyzed. and the minimal concentration needed to induce mitogenic activity was determined. The content was less than 1 ng/IOO Ilg of esp. In addition, 10 ng! rol of LPS did not significantly (P > .05) stimulate [3H]TdR incorporation into spleen cells in this mitogenic assay system. The results indicated that esp induced mitogenic activity to murine spleen cells. Properties of CSP~nduced Mitogenic Responses
To estimate the precise properties of the mitogenic responses of esp. the optimal concentration and the response pattern for the mitogenic activity were determined in spleen cell cultures of C57BU6 mice. The CSP significantly (P < .01) induced the mitogenic activity of spleen cells at a range of 50 to 200 Ilg/ml. The highest activity (SI of 6.29) was obtained with a concentration of 100 Ilglml (Table 2). The optimal concentration of CSP for mitogenic activity was estimated as 116 Ilg/ml by the regression analysis. Addition of 100 Ilg/ml of CSP induced significant (P < Journal of Dairy Science Vol. 75. No. II, 1992
7 6
5 H
l/l
4 3
2 1 0 0
24 48 72
120
S t ixnul. a.t ion. ( h ) Figure 1. Response pattern of slime products (CSP)induced mitogenic activity. The results are expressed as the mean and standard deviation of the stimulation index (51).•p < .05 and "P < .01 (against control).
2949
B-CELL MITOGENIC ACTIVITY TABLE 3. Comparative mitogenic activity of slime products to fractionated spleen cells. Fractioned spleen cells 2
Stimulation index! CSp3
X
-
SD
5.74** 9.43* 1.42
Con A
LPS
(100 JLglml) -
-
.34 1.35 .42
(20 JLglml) -
X
SD
14.50** 28.53** 1.74
.29
-
(2 Ilglml) SD 22.30** .02 2.19* .32 144.45*** 1.16
X
.77
.01
!Stimulation index represents the arithmetic mean of triplicate samples. 2NC 3(;SP
= Unfractioned
= slime
spleen cells, B£
products, LPS
= fraction
enriched with B cells, T£
= lipopolysaccharide, Con
= concanavalin
A
= fraction
enriched with T cells.
A.
*p < .05 (against control). "p < .01 (against control). ***p < .001 (against control).
.05) mitogenic activity at 24 h, and peak ac· tivity occurred at 48 h (Figure 1). Thereafter, the activity gradually decreased until the end of the experimental period. The results obtained indicate that CSP is an effective mitogen for murine spleen cells. Identification of Lymphocytes ResponsIve to CSP
To determine the contribution of T and B cells to CSP-induced mitogenic responses, Te and Be were cultured with the optimal concen-
(IJ. 9/11.1)
nu/+
---
I[ Control
* ij **H' ** H
75.23
50 75 100
***r--D \ ***r---o ) 75
tration of CSP. The mitogenicity of Te and Be was fully induced by Con A and LPS, respectively. The mitogenic activity of CSP depended on Be, but not on Te (Table 3), indicating that CSP is a possible mitogen to B cells. To confinn further that CSP is a B-cell mitogen, spleen cells of BALB/c athymic nu/nu mice and the littermate nu/+ mice were cultured with CSP (50 to 100 I!g/ml). The mitogenic activity of LPS occurred in both the cultures of nu/+ and athymic nu/nu mice, but Con A did not induce mitogenic activity in the cultures of athymic nu/nu mice that were defi-
ConA 2
22.14
81***
LPS 20
3( ~
30 15
10
S I
5
o
o
5
10
15 20
25
S I =
Figure 2. The B- and T-cell dependency of slime products (CSP)-induced mitogenic activity. Con A Concanavalin A. LPS = lipopolysaccharide. SI stimulation index. *p < .05. **p < .01, and ***p < .001 (against control).
=
Journal of Dairy Science Vol. 75, No. II, 1992
2950
KITAZAWA ET AL.
cient in T cells. The CSP caused mitogenic activity in both cultures from nu/+ and athymic nu/nu mice (Figure 2), and the activity was compatible with that obtained from C57BU6 mice. The results verified that CSP is a potent B-cell mitogen. DISCUSSION
Whole cell preparations of some dairy lactic acid bacteria that do not produce CSP have been reported to induce mitogenic activity to spleen cells (9, 24). In the present study, we demonstrated significant mitogenic activity of CSP, which is a cell surface component of slime-forming L lactis ssp. cremoris KVS20, dairy lactic acid bacteria, to murine lymphocytes of spleen. However, WCP and CWC of L. lactis ssp. cremoris KVS20 without CSP had little or no mitogenic activity to spleen cells. This suggests that the difference in mitogenic activity of lactic acid bacteria depends on the difference in cell components. The CSP induced mitogenic activity to BE but not to TE. Furthermore, CSP activity to spleen cells from athymic nu/nu mice was as high as that to BE' The small content of LPS in CSP eliminated the possibility that the mitogenic activity of CSP to spleen cells was caused by the contamination with LPS. These findings confirm that CSP is an effective Bcell mitogen. A previous study (10) indicated that intraperitoneal injection of CSP induced an antigen presenting M~ and a cytotoxic M~ and that CSP had no effect on the induction of cytotoxic M~ in vitro. Lymphokines, which are released from stimulated lymphocytes, render cytotoxic M~ (6, 7, 16, 21, 22). Therefore, CSP conceivably has the ability to stimulate lymphocytes in which the released lymphokines activate M~. In this study, CSP stimulated B cells as a mitogen. The findings may support the idea that CSP induces cytotoxic MI!I through the activation of lymphocytes. Various polysaccharides separated from Kampo medicine (14, 26), Dictyophora indusiata (8), and Nocardia (15) have been widely accepted as potent mitogens. A novel polysaccharide from Nocardia asteroides was reported to be a mitogen specific for B cells (15). Analysis showed that a major component Journal of Dairy Science Vol. 75, No. 11, 1992
of CSP is a phosphopolysaccharide (data not shown) that is similar to the CSP of L. lactis ssp. cremoris SBT0495 (18). Therefore, the substances of mitogenic activity of CSP may be the polysaccharide. We presented herein evidence that CSP is a potent mitogen to murine B cells, although the activity is not as high as LPS. Because ropy sour milk (viiH) containing CSP has long been consumed as a food, it is important to investigate whether CSP stimulates immune responses when ingested. Some dairy lactic acid bacteria augment mitogenic response (2) and antibody production (28) of intestinallymphoid tissue cells (peyer's patches). These cells come into direct contact with dietary antigens (1). Therefore, to establish whether dairy products containing CSP are beneficial food additives, it is necessary to examine the immunological effects on Peyer's patches. ACKNOWLEDGMENTS
The authors thank Nobuko Kumano, Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai, Japan, for her useful advice. The authors also thank Kuniji Yamaki, Laboratory of Animal Genetics, Faculty of Agriculture, Tohoku University, Sendai, Japan, for his helpful discussions in statistical analysis. This work was partially supported by a grant-in-aid for encouragement of young scientists (number 01760224) from the Ministry of Education, Science, and Culture of Japan and the Morinaga HOshikai to H. Kitazawa. REFERENCES 1 Bienestock, J., and A. D. BeCus. 1980. Mucosal immunology. Immunology 41:249. 2 De Simone, C., R. Vesely, R. Negri, B. B. Salvadori. S. zanzoglu, A. Cilli, and L. Lucci. 1987. Enhancement of immune response of murine Peyer's patches by a diet supplemented with yogurt. Immunopharmacol. Immunotoxicol. 9:87. 3 Dunnett, C. W. 1955. A multiple comparisons procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50:1096. 4 Fernandes. C. F.• and K. M. Shahani. 1990. Anticarcinogenic and immunological properties of dietary lactobacilli. J. Food Prot. 53:704. 5 Fernandes, C. F., K. M. Shahani. and M. A. Amer. 1987. Therapeutic role of dietary lactobacilli and lactobacilli fermented dairy products. Fed. Eur. Microbiol. Soc. Microbiol. Rev. 46:343.
B-CELL MITOGENIC ACI'lVITY
6 Fidler, I. I., and A. Raz. 1981. The induction of tumoricidal capacities in mouse and rat macrophages by Iympholdnes. Lympholdnes 3:345. 7 Groot, J. W. De, R. A. De Weger, R. J. Vandebriel, and W. Den Otter. 1989. Differences in the induction of macrophage cytotoxicity by the specific T lymphocyte factor, specific macrophage arming factor (SMAFl, and the lymphokine, macrophage activating factor (MAF). Immunobiology 179:131. 8 Hara, C., Y. Kumazawa, K. Inagaki, M. Kaneko, T. Kiho, and S. Ukai. 1991. Mitogenic and colonystimulating factor-inducing activities of polysaccharide fractions from the fruit bodies of Dicryophora indusiata fisch. Chern. Pharm. Bull. 39:1615. 9 Kadooka, Y., S. Fujiwara, and T. Hirota. 1991. Effects of Bijidobacteria cells on mitogenic response of splenocytes and several functions of phagocytes. Milchwissenschaft 46:626. 10 Kitazawa, H., T. Itoh, and T. Yamaguchi. 1991. Induction of macrophage cytotoxicity by slime products produced by encapsulated lActoeoccus 1JJctis ssp. cremoris. Anim. Sci. Techno!. 62:861. 11 Kitazawa, H., M. Nomura, T. Itoh, and T. Yamaguchi. 1991. Functional alteration of macrophages by a slime-forming lActococcus lactis ssp. cremoris. I. Dairy Sci. 74:2082. 12 Kitazawa, H., T. Toba, T. Itoh, N. Kumano, and S. Adachi. 1990. Antitumor activity of ropy sour milks in murine solid tumor. Anim. Sci. Techno!. 61:1033. 13 Kitazawa, H., T. Toba, T. Itoh, N. Kumano, S. Adachi, and T. Yamaguchi. 1991. Antitumoral activity of slime-forming, encapsulated 1Actococcus lactis ssp. cremoris isolated from Scandinavian ropy sour milk ''villi''. Anim. Sci. Techno!. 62:277. 14 Kiyohara, H., N. Takemoto, Y. Komatsu, H. Kawamura, E. Hosoya, and H. Yamada. 1991. Characterization of mitogenic pectic polysaccharides from Kampo (Japanese herbal) medicine "JuzenTaibo-To". Planta Med. 57:254. 15 Kurisaki, T.• K. Munemura, K. Kobayashi, M. Yamamoto, M. Hayashi, A. Nagai, and M. Yamasaki. 1991. E-15, A novel polysaccharide mitogen from Nocardia specific for B cells. Agric. BioI. Chem. 55: 2987. 16 Meltzer, M. S. 1981. Macrophage activation fortumor cytotoxicity: characterization of priming and trigger signals during Iymphokine activation. I. Immuno!. 127:179.
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17 Miura, M., T. Saito, H. Kiatazawa, T. Itoh, and T. Inamoto. 1991. Specific adsorption of immunoglobulin G in bovine colostrum on sephadex and its mitogenic activity on mouse splenocytes. Agric. Bio!. Chern. 55:3103. 18 Nakajima, H.• S. Toyada, T. Toba, T. Itoh, T. Mukai. H. Kitazawa, and S. Adachi. 1990. A novel phospbopolysaccharide from slime-forming 1Actococcus lactis subspecies cremoris SBT 0495. I. Dairy Sci. 73: 1472. 19 Obayashi, T., 1985. A new chromogenic endotoxinspecific assay using recombined limulus coagulation enzymes and its clinical applications. Clin. Chim. Acta 149:55. 20 Park, C. I., H. Sugawara, and T. Itoh. 1991. Fractionation and characterization of structural components of lactobacilli cell walls. Milchwissenschaft 46:87. 21 Pels, E., R. A. De Weger, and W. Den Otter. 1984. Lymphocyte induced macrophage cytotoxicity inducing lymphocyte. Immunobiology 166:84. 22 Ruco, L. P., and M. S. Meltzer. 1978. Macrophage activation for tumor cytotoxicity: increased lymphokine responsiveness of peritoneal macrophages during acute inflammation. J. Immuno!. 120:1054. 23 SAS4P/STAT User's Guide: Release 6.03 Edition. 1988. REG procedure, p. 773. SAS Inst., Inc., Cary, NC. 24 Shimizu, T., I. Mifuchi, and T. Yokokura. 1981. Mitogenic effect of lactobacilli on murine lymphocytes. Chern. Pharm. Bull. 29:3731. 25 Trizio, D., and G. Cudkowicz. 1974. Separation of T and B lymphocytes by nylon wool columns: evaluation of efficacy by functional assays in vivo. J. Immunol. 113:1093. 26 Yamada, H., H. Kiyohara, J.-e. Cyong, N. Takemoto, Y. Komatsu. H. Kawamura, M. Aburada, and E. Hosoya. 1990. Fractionation and characterization of mitogenic and anti-eomplementary active fractions from Kampo (Japanese herbal) medicine "IounTaibo-To". Planta Med. 56:386. 27 Yamaguchi, T., Y. Kuroda, M. Saito, T. Ebina, F. Hoshino, and N. Ishida. 1984. Immune interferon production by TH69, a lyophilized preparation of Streptococcus faecaiis, in murine spleen cell cultures. Microbio!. Immuno!. 28:601. 28 Yasui, H., A. Mike, and M. Ohwalci. 1989. Immunogenicity of Bifidobacterium breve and change in antibody production in Peyer's patches after oral administration. I. Dairy Sci. 72:30.
Iournal of Dairy Science Vol. 75, No. 11, 1992
