potentiation of hydrogen peroxide, nitric oxide, and cytokine production in raw 264.7 macrophage...

International Journal of Food Microbiology 46 (1999) 231–241

Potentiation of hydrogen peroxide, nitric oxide, and cytokineproduction in RAW 264.7 macrophage cells exposed to human and

commercial isolates of Bifidobacterium

a ,c a ,b c d aSo Young Park , Geun Eog Ji , Young Tae Ko , Hoo Kil Jung , Zeynep Ustunol ,a ,*James J. Pestka

aDepartment of Food Science and Human Nutrition, 234 G.M. Trout Building, Michigan State University, East Lansing,

MI 48824-1224, USAbDepartment of Food Science and Nutrition, Seoul National University, Seoul 151-742, Korea

cDepartment of Food Science and Nutrition, Duksung University, Seoul, KoreadCentral Research Institute, Maeil Dairy Co., Ltd., Pyung Taek, Kyungki-Do, Korea

Received 15 June 1998; received in revised form 17 November 1998; accepted 24 November 1998

Abstract

Bifidobacteria have been previously shown to stimulate immune function and this may be mediated by macrophages. TheRAW 264.7 cell line was used here as a macrophage model to assess the effects of human and commercial Bifidobacteriumisolates on the production nitric oxide (NO), hydrogen peroxide (H O ) and the cytokines IL-6 and tumor necrosis factor2 2

(TNF)-a. Thirty three Bifidobacterium strains differentially stimulated the production of H O NO, TNF-a, and IL-6 in a2 2

dose-dependent manner in 24-h cultures. In the presence of lipopolysaccharide (LPS) the effects of bifidobacteria on NO andH O were masked and were less pronounced at the later stage of incubation. Co-stimulation of macrophages with both LPS2 2

and Bifidobacterium increased the production of IL-6 synergistically. In contrast, LPS reduced the ability of thebifidobacteria-induced macrophages to produce TNF-a. Our results demonstrated that both human and commercialBifidobacterium strains can stimulate H O , NO, TNF-a, and IL-6 production, and this effect was strain-dependent. The in2 2

vitro approaches employed here should be useful in further characterization of the effects of bifidobacteria on gastrointestinaland systemic immunity. 1999 Elsevier Science B.V. All rights reserved.

Keywords: Bifidobacterium; Macrophage; H O ; Nitric oxide; Cytokine2 2

1. Introduction intestinal tract of humans and animals. In breast-fedinfants, bifidobacteria comprise more than 90% of

Bifidobacterium spp. are nonpathogenic, Gram- the gut bacterial population (Bezkorovainy andpositive and anaerobic bacteria which inhabit the Miller-Catchpole, 1989; Mitsuoka, 1982), however,

their numbers gradually decrease over the life time* of the host. Bifidobacterium spp. are used in com-Corresponding author. Tel.: 1 1-517-353-1709; fax: 1 1-517-

353-8963; e-mail: [email protected]. mercial fermented dairy products and have been

0168-1605/99/$ – see front matter 1999 Elsevier Science B.V. All rights reserved.PI I : S0168-1605( 98 )00197-4

232 S.Y. Park et al. / International Journal of Food Microbiology 46 (1999) 231 –241

suggested to exert health promoting effects on the regulation of these mediators is critical for normalhost by maintaining intestinal microflora balances, physiological immune status.improving lactose tolerance, reducing serum choles- Characterization of the effects of Bifidobacteriumterol levels, increasing synthesis of vitamins, and on the production of macrophage mediators mayaiding anti-carcinogenic activity (Gopal et al., 1996; contribute to a better understanding of how thisHomma, 1988; Hughes and Hoover, 1991; Kurmann genus affects immune function at the cellular level.and Raric, 1991; Sanders, 1993). Other beneficial Our research groups are particularly interested in theeffects of the intake of Bifidobacterium are reported differential effects of Bifidobacterium isolates fromto include the reinforcement of immune function humans. In this study, we used the RAW 264.7(Yasui and Ohwaki, 1991; Yamazaki et al., 1991). murine macrophage model to evaluate the effects of

Bifidobacteria apparently enhance several immune human Bifidobacterium isolates on NO and H O ,2 2

functions, including macrophage and lymphocyte IL-6 and TNF-a production. The results showed thatactivation (Hatcher and Lambrecht, 1993; Sekine et Bifidobacterium can differentially upregulate theal., 1994c), antibody production (Lee et al., 1993; production of H O NO, TNF-a and IL-6 in dose-2 2

Link-Amster et al., 1994; Yasui et al., 1992; Yasui dependent fashion. In the presence of LPS,and Ohwaki, 1991), and the proliferative responses Bifidobacterium can selectively superinduce IL-6 butin spleen and Peyer’s patches (Hosono et al., 1997; can concurrently suppress the production of TNF-a.Kado-Oka et al., 1991; Lee et al., 1993; Takahashi etal., 1993; Yasui and Ohwaki, 1991). Bifidobacteriaingestion has been proposed to enhance resistance toinfection by pathogenic organisms (Duffy et al., 2. Materials and methods1994; Sasaki et al., 1994; Yasui et al., 1995) andpotentially prevent cancer (Fernandes and Shahani, 2.1. Bifidobacterium cultures1990; Rafter, 1995; Sekine et al., 1995, 1994a). Cellcomponents of Bifidobacterium which function as Twenty-four strains were recently isolated fromimmunomodifiers of the host reportedly include the faeces of healthy human subjects and identifiedpeptidoglycan, intracellular and extracellular poly- as Bifidobacterium spp. according to Bergey’s Manu-saccharide products, cell free extracts, and cell wall al of Systematic Bacteriology (Scardovi, 1986). B.preparations (Gomez et al., 1988; Hatcher and adolescentis 15703, B. longum 15707, and B. infantisLambrecht, 1993; Hosono et al., 1997; Namioka, 15697 were obtained from American Type Culture1985; Sekine et al., 1994a, 1995). However, at the Collection (Rockville, MD). Bifidobacterium Bf-1present time, there is not yet a clear understanding of and Bf-6 were kindly supplied by Michelle Malonethe molecular and cellular basis for immunostimula- of Sanofi Bio-Industries (Waukesha, WI). The identi-tion by bifidobacteria. fication and experimental use of BGN2, BGN3,

Macrophages play a major role in a host defense BGN4 and INT57 were reported previously (Choi etagainst infection and tumor formation. Of particular al., 1996). All strains were cultured and subculturedinterest, the production of nitric oxide (NO) and anaerobically in MRS broth (Difco, Detroit, MI)hydrogen peroxide (H O ) by macrophages mediates containing 5% (wt /vol) lactose (MRSL) at 378C2 2

killing or growth inhibition of tumor cells, bacteria, until late log phase. Cells were collected by centrifu-fungi and parasites (Lorsbach et al., 1993; Snyder gation at 1000 3 g for 15 mm at 48C and washedand Bredt, 1992; Schmidt and Walter, 1994). Macro- twice with 0.01 M phosphate buffered saline (pHphages may also regulate immunity via enhanced 7.2) followed by final washing with distilled water.production of several mediators such as tumor Cultures were dried (Speed-Vac Instruments, Inc.,necrosis factors (TNF)-a and interleukin (IL)-6 NY) and resuspended in Hanks’ buffered salt solu-(Marin et al., 1997). While these mediators play key tion (Sigma Chemical Co., St. Louis, MO) to thehomeostatic functional roles, they are potentially desired bacterial concentration on a dry weight basis.capable of injuring host tissues (Fukuo et al., 1995; For introduction into tissue cultures, bifidobacteriaLaskin and Pendino, 1995; Sarih et al., 1993). Thus, were heat-killed at 958C for 30 min. Heat-killed

S.Y. Park et al. / International Journal of Food Microbiology 46 (1999) 231 –241 233

cultures were aliquoted and stored at 2 808C until measured after 1-h incubation at 378C by fluoro-used. metric detection of DCF oxidation using a Cytofluor

II Model (Bioresearch, Bedford, MA) set at 484 nm2.2. Chemicals and reagents extinction /530 nm emission. A DCF calibration

curve was generated to quantitate the amount of DCFIFN-g, TNF-a, IL-6, purified antibodies to TNF-a oxidized.

or IL-6 (rat anti-mouse), and biotinylated rat anti-mouse TNF-a or IL-6 was obtained from PharMing- 2.5. NO determinationen (San Diego, CA). Salmonella typhimurium LPSwas from Sigma Chemical Company (St. Louis, Nitrite accumulation was used as an indication ofMO). 2,7-Dichlorofluorescein diacetate (DCF-DA) NO production. This procedure for NO determi-and dichlorofluorescein (DCF) were purchased from nation was based on the Griess reaction (Green et al.,Molecular Probes (Eugene, OR). Dulbecco’s modi- 1982). One hundred (100) ml of culture supernatantfied Eagle medium (DMEM) and fetal bovine serum or sodium nitrite standard (0 to 200 mM) was mixed(FBS) were obtained from Gibco Laboratories with an equal volume of Griess reagent (0.1% (w/v)(Chagrin Falls, IL). Tetramethylbenzidine (TMB) naphthyl ethylenediamine and 1% (w/v) sul-was from Fluka Chemical (Ronkonkoma, NY). fanilamide in 5% (v/v) phosphoric acid) in microti-

ter plate wells. After 5 min at room temperature the2.3. Cell culture optical density at 550 nm (OD ) was measured by550

a VMax microplate reader (Molecular Devices,The mouse macrophage cell line RAW 264.7 Menlo Park, CA). Samples of the culture media

(American Type Tissue Collection) was grown in incubated without macrophage were assayed forDMEM supplemented with 10% (v/v) FBS, 1 mM background levels of nitrite and these values weresodium pyruvate, 1% (v/v) NCTC-135, streptomycin subtracted from the values measured in the culture(100 mg/ml) and penicillin (100 U/ml). All cultures supernatant.were incubated at 378C in a humidified atmospherewith 5% CO . Cell number and viability were 2.6. TNF-a and IL-6 quantitation2

assessed by trypan blue dye exclusion (Strober,1991) on a Neubauer hemacytometer (American TNF-a and IL-6 were quantified by ELISA usingOptical, Buffalo, NY). Cells were grown to con- a modification of the procedure of Dong et al.fluence in sterile tissue culture dishes and gently (1994). Briefly, microtiter strip wells (Immunolon IVdetached by repeated pipetting. For experiments, Removawell; Dynatech Laboratories, Chantilly, VG)cells were cultured in triplicate at a density of 5 3 were coated overnight at 48C with 50 ml of 1 mg/ml

510 cells per ml in 96-well flat-bottomed tissue purified antibodies to TNF-a or IL-6 antibodies (ratculture plates (Costar, Cambridge MA). Cultures anti-mouse) in 0.1 M sodium bicarbonate buffer (pHcontaining Bifidobacterium with and without LPS 8.2). Wells were incubated with 300 ml of 3% (v/v)were incubated for various time intervals and ana- bovine serum albumin (BSA) in 0.01 M PBS (pHlyzed for H O , NO, TNF-a and IL-6. 7.2) containing 0.2% (v/v) Tween 20 (PBST) at2 2

378C for 30 min to block nonspecific protein bind-2.4. H O determination ing. Standard recombinant murine TNF-a, IL-6 and2 2

samples, diluted in 10% (v/v) FBS RPMI-1640,The method of Tiku et al. (1990) was used for were added in 50-ml aliquots to appropriate wells

H O determination. After culturing in 96-well and incubated at 378C for 1 h. After washing four2 2

plates, cell culture supernatants were removed and times with PBST biotinylated rat anti-mouse TNF-aattached cells were washed twice with 200 ml of 0.01 or IL-6 antibodies were diluted in BSA-PBST to 1M phosphate (pH 7.2) buffered saline (pH 7.2, PBS) mg/ml and 1.5 mg/ml, respectively, and 50 ml wereand then resuspended in 200 ml PBS containing 4 added and incubated at room temperature for 1 h.mM DCF-DA. Intracellular production of H O was Plates were washed six times and incubated with 502 2


ml of streptavidin–horseradish peroxidase conjugate(1.5 mg/ml in BSA–PBST) at room temperature for1 h. After washing eight times, bound peroxidaseconjugate was detected by adding 100 ml per wellsolution of substrate consisting of 0.1 mg/ml TMB,and 100 ml of 1% H O in 25 ml of 0.1 M citric-2 2

phosphate buffer (pH 5.5). An equal volume of 6 NH SO was added to stop the reaction. The plates2 4

were read at 450 nm on a Vmax Kinetic MicroplateReader (Molecular Devices, Menlo Park, CA). TNF-a and IL-6 were quantitated using Vmax Software(Molecular Devices).

2.7. Statistical analysis

Data were analyzed by Student–Newman–Keuls(SNK) test following one way analysis of variance(ANOVA) using the Sigmastat Statistical AnalysisSystem (Jandel Scientific, San Rafael, CA). A prob-

Fig. 1. Potentiation of H O production by bifidobacteria. RAW2 25ability of P , 0.05 in the two-tailed test was used in 264.7 cells (5 3 10 per ml) were cultured with and without LPS

the criterion for statistical significance. (20 ng/ml) for 24 h in the presence of various bacterial con-centrations. Data are mean6SD of triplicate cultures. Valuesmarked with asterisk differ significantly from control values (P ,

0.05). ND 5 not detectable.3. Results

3.1. Effect of Bifidobacterium on H O and NO2 2

production

Hydrogen peroxide and NO are important macro-phage mediators because they act as reactive oxygenand nitrogen intermediates during oxygen-dependentphagocytosis (Kim et al., 1996). The effects ofBifidobacterium exposure on H O and NO pro-2 2

duction in RAW 264.7 cells were evaluated usingfluorometric H O analysis and Griess assay, respec-2 2

tively. As examples of the typical patterns observedfor all strains, the effects of Bifidobacterium SJ32and Bifidobacterium Int-57 on H O and NO pro-2 2

duction, are displayed in Figs. 1 and 2, respectively.Various Bifidobacterium strains induced the pro-

duction of H O (Table 1). The maximal level was2 2

generally observed at 30–100 mg bacteria per mlwhereas at 300 mg bacteria per ml the production ofH O tended to decline slightly except in a few2 2

strains. Marked differences between strains wereobserved. LPS at a concentration of 20 ng/ml alonealso induced production significantly. However when Fig. 2. Potentiation of NO production by bifidobacteria. Ex-co-stimulated with Bifidobacterium and LPS, the perimental conditions are as described in Fig. 1 legend.


Table 1aEffect of Bifidobacterium on H O production by unstimulated (LPS 2 ) and stimulated (LPS 1 ) RAW 264.7 cells2 2

bStrain H O relative change (fold control)2 2

LPS 2 (mg/ml bacteria) LPS 1 (mg/ml bacteria)

11 33 100 300 11 33 100 300c c c c cBf1 1.4 1.7 1.8 1.6 1.3 1.5 1.7 1.4c c c c cBf6 1.3 1.7 1.6 1.5 1.2 1.4 1.6 1.3

c c c cBGN2 1.2 1.3 1.5 1.5 1.1 1.3 1.4 1.0c c c cBGN3 1.3 1.7 1.8 1.6 1.2 1.4 1.5 1.4

c c c c c cBGN4 1.6 1.7 1.8 1.7 1.3 1.7 1.7 1.8cCN2 0.9 1.2 1.5 1.4 0.9 1.0 1.0 0.9

cHJ3O 1.2 1.5 1.3 1.4 1.3 1.2 1.7 1.2cInt57 1.3 1.5 1.5 1.4 1.1 1.3 1.3 1.2c cJH2 1.3 1.3 1.5 2.2 1.2 1.4 1.2 1.1

JS8 1.0 1.0 1.3 1.5 0.8 0.9 1.0 0.8c cJS9 1.5 1.7 1.6 1.3 1.1 1.2 1.6 1.3

cMS1 1.0 1.1 1.2 1.5 0.8 0.9 1.0 1.3c c c cSI31 1.4 1.6 1.3 1.2 1.4 1.5 1.7 1.6c c c c cSJ32 1.5 1.7 2.2 2.0 1.2 1.2 1.4 1.3

c cB. adolescentis ATCC 15703 1.2 1.3 1.5 1.6 1.2 1.3 1.4 1.6c c c c cB. infantis ATCC 15697 1.4 1.7 1.5 1.4 1.4 1.8 2.0 1.6

B. longum ATCC 15707 0.9 1.0 1.0 1.2 0.8 1.0 0.9 1.0a 5RAW 264.7 cells (5 3 1O cells per ml) were cultured with 20 ng/ml of lipopolysaccharide and various concentration of bifidobacteria for24 h.b H O relative change was calculated by dividing experimental data by control values (n 5 3).2 2c P , 0.05.

level of H O was not significantly higher than centrations in the absence or presence of LPS and2 2

Bifidobacterium alone. cytokine secretion in culture supernatants was moni-The effects of Bifidobacterium exposure on NO tored by ELISA. The patterns observed for

production in RAW cells was evaluated in 24-h Bifidobacterium BGN-3 and Bifidobacterium infantiscultures (Table 2). Bifidobacterium exposure in the ATCC 15697 on TNF-a and IL-6 stimulation, arerange of 11 to 300 mg bacteria per ml markedly displayed in Figs. 3 and 4, respectively, and areincreased the production of NO. Strains which representative of all the strains.produced a high level of H O also exhibited In non-LPS treated cells, exposure to bifidobac-2 2

increased NO production. Generally, NO production teria affected supernatant TNF-a (Table 3) in aincreased with increasing concentrations of dose-dependent manner. The degree of TNF-aBifidobacterium in the range of 11 to 300 mg stimulation significantly differed between strains.bacteria per ml as exemplified in Fig. 2. LPS alone BGN4, JH2, E2-18, SJ32, and BF1 showed relativelyalso induced NO production significantly (Fig. 2). strong stimulation and strains SH5, CN2, MS1, JS8Further stimulation of NO production in LPS (20 and UN4 showed relatively weaker stimulating ac-ng/ml) stimulated RAW cells were observed. tivity. When co-stimulated with LPS (20 ng/ml) and

low concentrations of Bifidobacterium (10 mg/ml),TNF-a levels were higher than Bifidobacterium

3.2. Cytokine production by unstimulated and LPS- alone as exemplified in Fig. 3. However, at highstimulated RAW 264.7 cells concentrations of Bifidobacterium (250 mg/ml), the

level was lower than Bifidobacterium alone.To assess the effects of bifidobacteria on TNF-a Bifidobacterium, at concentration of 10–250 mg/

and IL-6 production by macrophages, RAW 264.7 ml, also induced the production of IL-6 dose depen-cells were incubated with a range of bacteria con- dently (Table 4). When costimulated with LPS (20


Table 2aEffect of Bifidobacterium on NO production by unstimulated (LPS 2 ) and stimulated (LPS 1 ) RAW 264.7 cells

bStrain NO relative change (fold control)


11 33 100 300 11 33 100 300c c c c c c c cBf1 2.7 3.2 4.1 3.8 1.8 2.0 1.9 2.0c c c c c cBf6 1.6 2.9 3.8 37 1.1 1.3 1.9 2.1

c c c c cBGN2 1.4 2.2 3.2 34 1.2 1.4 1.7 1.9c c c c c cBGN3 2.3 2.7 3.1 3.0 1.4 1.7 1.6 1.6c c c c c c c cBGN4 2.8 33 4.1 44 1.5 1.7 1.8 2.0

cCN2 1.3 1.1 1.4 3.6 1.3 1.4 1.4 1.4c c c c c cHJ30 1.6 2.0 2.4 3.0 1.5 1.7 1.8 1.8

c c c c cInt57 1.4 2.4 34 43 1.4 1.6 1.7 2.2c c c c c c cJH2 2.8 4.6 44 45 1.4 1.6 1.6 1.8

c cJS8 1.1 1.3 1.4 3.0 0.8 0.9 0.9 1.4c c c c c c cJS9 2.0 3.2 34 33 1.6 1.8 1.9 2.0

c cMS1 1.0 0.8 1.6 3.2 1.4 1.2 1.4 1.4c c c c c c cSI31 1.8 2.5 2.5 2.6 1.3 1.7 1.8 1.9c c c c c cSJ32 2.5 3.2 3.8 4.2 1.2 1.3 1.6 1.5

c c c c c cB. adolescentis ATCC 15703 1.4 1.3 2.4 3.8 1.5 1.8 1.7 1.6c c c c c c cB. infantis ATCC 15697 1.9 2.7 2.8 2.9 1.3 1.7 1.8 1.9

c cB.longum ATCC 157O7 1.2 1.4 2.3 3.9 1.2 1.0 1.2 1.0a 5RAW 264.7 cells (5 3 10 cells per ml) were cultured with 20 ng/ml of lipopolysaccharide and various concentration of bifidobacteria for24 h.b N0 relative change was calculated by dividing experimental data by control values (n 5 3).c P , 0.05.

as exemplified in Fig. 4. Strains showing highng/ml) the production of IL-6 increased to a muchinduction of TNF-a also generally showed highgreater level than either LPS or Bifidobacteriuminduction of IL-6.alone at all the Bifidobacterium concentrations tested

Fig. 3. Potentiation of IL-6 production by bifidobacteria. Ex- Fig. 4. Potentiation of TNF-a production by bifidobacteria.perimental conditions are as described in Fig. 1 legend. Experimental conditions are as described in Fig. 1 legend.


Table 3aEffect of Bifidobacterium on TNF-a production by LPS non-stimulated (LPS 2 ) and stimulated (LPS 1 ) RAW 264.7 cells

bStrain TNF-a relative change (fold control)


10 50 250 10 50 250c c c cBf1 151.2 224.1 295.7 1.2 3.0 1.2c c cBf6 1.3 37.2 121.0 1.8 1.2 1.6c c cBGN2 1.5 10.5 38.1 2.1 1.5 0.8

c c c c c cBGN3 394 114.8 216.7 4.5 5.2 3.3c c c c cBGN4 554 43.8 54.2 12.3 12.9 5.8

c c c c c cBMH3 3.1 934 326.1 3.3 4.9 3.4c c c c cCH4 53 92.7 192.3 4.0 3.2 2.2

c c c c cCN1 1.6 19.9 65.3 2.2 2.5 1.9c c cCN2 1.4 37 56.5 1.3 2.2 0.5c c c c cE15 116.9 644 111.4 3.2 2.6 1.8c c c c cE17 156.4 102.0 109.0 3.6 3.3 1.8c c c c c cE2–18 162.3 144 167.6 5.4 4.2 37c c c c c cEH17 1.4 32.3 174.0 2.8 3.4 1.6

c c c c c cHJ30 54 70.1 158.2 2.8 2.5 3.2c c c c cInt57 1.2 70.0 169.0 4.9 3.2 2.4

c c c c c cJH2 181.3 253.7 3775 5.9 3.9 4.0c c c c c cJO3 2.7 128.3 316.0 3.2 2.6 3.1c c c c c cJS8 1.8 4.2 208.7 2.3 3.7 3.9c c c c c cJS9 9.0 166.8 248.3 2.3 4.4 6.3c c c c cKI 2.4 56.3 190.2 3.1 2.0 2.3c c cKI2 2.6 399 129.4 1.5 2.2 1.7c c cKJ 3.8 65.3 132.4 1.8 2.1 1.6

c c c c cMS1 0.8 3.0 553 39 34 2.1c c cMS5 3.0 110.4 173.2 1.6 1.8 0.6c c cSH1 30.2 115.8 90.0 1.5 1.9 0.4c c c c cSH2 2.6 3.7 38.6 2.3 2.1 1.2

c c cSH5 1.3 2.4 58.2 1.3 2.7 1.5c c cSI31 25.4 459 149.2 1.5 1.3 2.8c c c c c c5J32 124.3 178.1 254.8 2.8 45 4.0c c c cUN4 1.4 15.5 185.3 2.8 1.7 0.4c c c cB. adolescentis ATCC 15703 77.6 152.4 195.8 1.5 0.9 2.5c c c cB. infantis ATCC 15697 14.5 96.0 70.3 1.9 2.3 0.7c c c c c cB. longum ATCC 15707 102.5 111.5 161.2 2.2 3.2 2.5

a 5RAW 264.7 cells (5 3 10 cells per ml) were cultured with 20 ng/ml of lipopolysaccharide and various concentrations of bifidobacteria for24 h.b TNF-a relative change was calculated by dividing experimental data by control values (n 5 3).c P , 0.05.

4. Discussion stimulate macrophages and T cells (Hatcher andLambrecht, 1993; Sekine et al., 1994a,b). There is

Bifidobacteria and other lactic acid bacteria have extensive evidence that cytokines play pivotal rolesbeen previously shown to stimulate immune function in host defense, inflammatory responses, and au-(Lee et al., 1993; Kado-Oka et al., 1991; Gomez et toimmune disease (Abbas et al., 1994; Akira et al.,al., 1988). Furthermore, bifidobacteria and other 1993). Sekine et al. (1995) have proposed criticallactic acid bacteria can improve antitumor activity of roles for cytokines in the antitumor promotingthe host (Fernandes and Shahani, 1990; Rafter, 1995; properties of B. infantis. However, the mechanismsSekine et al., 1995, 1994b). It has been suggested whereby bifidobacteria may modulate the immunethat these activities may arise from their ability to response remains unclear.


Table 4aEffect of Bifidobacterium on IL-6 production by LPS non-stimulated (LPS 2 ) and stimulated (LPS 1 ) RAW 264.7 cells

bStrain IL-6 relative change (fold control)


10 50 250 10 50 250c c c c cBf6 1.2 13.2 65.5 3.6 14.7 36.2

c c c c cBGN2 1.1 43 48.2 2.7 8.7 26.8c c c c cBGN3 1.6 6.3 83.0 3.6 17.8 55.2

c c c c c cBGN4 434 70.3 959 18.9 50.0 33.0c c c cBMH3 10.2 6.5 89.3 6.0 20.7 41.7

c c c c c cCH4 1.6 22.3 51.6 5.1 20.1 31.5c c c c cCN1 2.0 7.6 43.0 2.9 12.7 24.5c c c c cCN2 1.1 2.1 22.4 2.4 10.6 20.5

c c c c c cE15 21.0 18.3 143.4 14.0 26.9 35.8c c c c c cE17 19.1 345 794 11.4 21.4 31.8

c c c c cE2–18 23.0 48.8 114.5 18.5 31.1 44.7c c c c cEH17 1.1 9.2 69.3 3.2 13.2 37.8c c c c cHJ3O 1.6 13.9 53.7 47 10.1 26.0c c c c cInt57 1.1 6.3 48.6 11.5 16.1 29.7

c c c c c cJH2 31.5 57.5 74.4 32.4 43.3 64.3c c c cJO3 1.5 5.3 75.6 5.5 11.3 4.4c c cJS8 0.9 1.3 22.8 2.7 6.1 9.9

c c c c cJS9 5.0 29.5 105.1 6.7 19.9 49.4c c c c c cKI 2.4 23.9 65.4 9.8 25.6 34.3

c c c c cKI2 1.6 16.8 51.5 2.6 15.6 22.5c c c c c cKJ 2.5 26.8 61.8 3.2 16.3 16.0

c c c cMS1 1.0 1.2 4.1 2.6 16.1 9.5c c cMS5 1.1 6.1 33.3 3.8 17.2 25.9

c c c c c cSH1 14.4 36.9 58.8 10.3 27.1 33.6c c c c cSH2 1.4 9.2 69.5 33 13.7 31.6

c c cSH5 1.0 1.1 7.3 2.0 5.5 15.8c c c c c cSI31 3.8 35.2 93.0 4.8 18.6 26.0c c c c c cSJ32 60.8 70.9 136.8 24.1 38.9 45.1

c c c cUN4 1.0 2.1 20.2 3.1 47 14.6c c c c c cB. adolescentis ATCC 15703 34.5 47.3 108.4 17.7 31.6 51.6c c c c c cB. infantis ATCC 15697 7.2 48.4 78.7 8.0 29.1 38.7c c c c c cB. longum ATCC 15707 26.9 47.4 69.3 9.4 29.9 30.6

a 5RAW 264.7 cells (5 3 10 cells per ml) were cultured with 20 ng/ml of lipopolysaccharide and various concentration of bifidobacteria for24 h.b IL-6 relative change was calculated by dividing experimental data by control values (n 5 3).c P , 0.05.

Macrophages are important regulatory and effector degree of macrophage activation. In the presentcells that play a central role in cell-mediated im- study, exposure of RAW 264.7 cell line to humanmunity because they present antigen and mediate bifidobacteria isolates resulted in marked increases ofinflammatory, tumoricidal and microbicidal activity H O , NO, TNF-a, and IL-6 production. These2 2

(Abbas et al., 1994). These functions can be altered results are consistent with previous reports thatby a variety of stimulatory or suppressive signals bifidobacteria enhance production of TNF-a and IL-(Yu et al., 1990) and are influenced by many 6 by cells of the monocyte lineage (Miettinen et al.,environmental factors. Numerous macrophage func- 1996; Marin et al., 1997; Solis Pereyra and Lemon-tions are mediated through the release of different nier, 1993; Sekine et al., 1994b). The enhancementcytokines (Cavaillon, 1994). Therefore, cytokine of NO and H O production by Bifidobacterium was2 2

production is likely to be a good indicator of the newly noted in this study.


The effect of bifidobacteria on TNF-a production the future mechanistic characterization of the effectsby RAW 264.7 cells was very dramatic, particularly of human bifidobacteria on gastrointestinal immunityin the absence of LPS stimulation. TNF-a mediates a and the possibility for enhancing the health benefitswide variety of biological activities (Vassalli, 1992). using bifidobacteria products. Further research isThe induction of NO and H O by Bifidobacterium needed to determine whether the in vitro effects2 2

may be a consequence of TNF-a stimulation as this observed here will also occur in animals ingestingcytokine is known to be an inducer for NO and H O these bacteria.2 2

(Hoffman and Weinberg, 1987; Lowenstein et al.,1993). The decreased production of H O and TNF-2 2

a in LPS co-treated cultures at high concentrations Acknowledgementsof Bifidobacterium suggests that TNF-a and H O2 2

might be susceptible to saturation or feedback mech- This work was supported by research grantsanisms. supported by the National Dairy Council, the USDA-

The increased production of macrophage TNF-a, NRICGP, the Michigan State University Crop andH O and NO by bifidobacteria observed in this2 2 Food Bioprocessing Center, the Ministry of Sciencestudy suggests that these may be key factors for the and Technology of Korea (G7 project, 1995) and byincreased phagocytosis and inhibition of tumor cell Research and Development Center of the Koreanproliferation observed by various investigators Department of Agriculture (1996). We would also(Duffy et al., 1994; Fernandes and Shahani, 1990; thank Dr. James R. Clarke, Michigan State Universi-Rafter, 1995; Sasaki et al., 1994; Sekine et al., 1995, ty, East Lansing, Michigan for critical reading of this1994a; Yasui et al., 1995). Moderate production of manuscript.cytokines, H O and NO induced by bifidobacteria2 2

used in foods could have a beneficial effect inmaintaining an immunological balance and increas- Referencesing resistance to infections without the induction ofsecondary effects that occur with cytokine treatment.

Abbas, A.K., Lichtman, A.H., Pobe, J.S., 1994. Cytokines:However, it should be noted that high concentration Cellular and Molecular Immunology, second ed. W.B. Saun-of TNF-a, H O and NO causes cachexia, tissue ders Co., Philadelphia, PA, p. 240.2 2

Akira, S., Taga, T., Kishimoto, T., 1993. Interleukin-6 in biologyinjury, disseminated intravascular coagulation andand medicine. Adv. Immunol. 54, 1–78.shock (Abbas et al., 1994). Therefore, the control of

Bezkorovainy, A., Miller-Catchpole, R., 1989. Biochemistry andthose macrophage mediators in vivo is very im-Physiology of Bifidobacteria, CRC Press, Boca Raton, FL.

portant. One way of controlling the levels of these Cavaillon, J.M., 1994. Cytokines and macrophages. Biomed.mediators might involve the selection of the appro- Pharmacother. 48, 445–453.

Choi, Y.J., Kim, C.J., Park, S.Y., Ko, Y.T., Jeong, H.K., Ji, G.E.,priate strains which elicit the desired level based on1996. Growth and b-glucosidase activity of Bifidobacterium. J.experimental studies.Microbiol. Biotechnol. 6, 255–259.Although bifidobacteria are now routinely incorpo-

Dong, W., Azcona-Olivera, J.I., Brooks, K.H., Linz, J.E., Pestka,rated into dairy foods or pharmaceuticals, statements J.J., 1994. Elevated gene expression and production of inter-of specific benefits from these products need to be leukins 2, 4, 5, and 6 during exposure to vomitoxin (deox-

ynivalenol) and cycloheximide in the EL-4 thymoma. Toxicol.further backed by mechanistic and clinical studies.Appl. Pharmacol. 127, 282–290.This study provides some insight into the mecha-

Duffy, L.C., Zielezny, M.I., Riepenhoff-Talty, M., Dxyja, D.,nisms by which these organisms may provide healthSayahtaheri-Altaie, S., Griffiths, E., Ruffin, D., Barrett, H.,

benefits. The results reported here suggest that both Rossman, J., Ogra, P.L., 1994. Effectiveness of Bifidobac-human and commercial isolates of bifiobacteria terium bifidum in mediating the clinical course of murine

rotavirus diarrhea. Pediatr. Res. 35, 690–695.increase the secretion of several macrophage medi-Fernandes, C.F., Shahani, K.M., 1990. Anticarcinogenic andators and thus could potentially modulate the host

immunological properties of dietary lactobacilli. J. Food Prot.immune response. The results also indicate that this53, 704–710.

stimulatory capacity may be related to dose, strain Fukuo, K., Inoue, T., Morimoto, S., Nakahashi, T., Yasuda, O.,and morphology of the Bifidobacterium strain. The in Kitano, S., Sasada, R., Ogihara, T., 1995. Nitric oxidevitro approaches employed here should be useful in mediates cytotoxicity and basic fibroblast growth factor release


in cultured vascular smooth muscle cells. A possible mecha- Molecular basis for the synergy between interferon-gammanism of neo vascularization in atherosclerotic plaques. J. Clin. and lipopolysaccharide. J. Biol. Chem. 268, 1908–1913.Invest. 95, 669–676. Lowenstein, C.J., Alley, E.W., Raval, P., Snowman, A.M., Snyder,

S.H., Russel, S.W., Murphy, W.J., 1993. Macrophage nitricGomez, E., Melgar, M.M., Silva, G.P., Portoles, A., Gil, I., 1988.oxide synthase gene: two upstream regions mediate inductionExocellular products from Bifidobacterium adolescentis asby interferon-g and lipopolysaccharide. Proc. Natl. Acad. Sci.immunomodifiers in the lymphoproliferative responses ofU.S.A. 90, 9730–9734.mouse splenocytes. FEMS Microbiol. Lett. 56, 47–52.

Marin, M.L., Lee, J.H., Murtha, J., Ustunol, Z., Pestka, J.J., 1997.Gopal, A., Shah, N.P., Roginski, H., 1996. Bile tolerance,Differential cytokine production in clonal macrophage andtaurocholate deconjugation and cholesterol removal by Lac-T-cell lines cultured with bifidobacteria. J. Dairy Sci. 80,tobacillus acidophilus and Bifidobacterium. Milchwissenschaft2713–2720.51, 619–623.

Miettinen, M., Vuopio-Varkila, J., Varkila, K., 1996. Production ofGreen, L.G., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok,human necrosis factor alpha, interleukin-6 and interleukin-10J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, andis induced by lactic acid bacteria. Infect. Immun. 64, 5403–[N] nitrate in biological fluids. Anal. Biochem. 126, 131–138.5405.Hatcher, G.E., Lambrecht, R.S., 1993. Augmentation of macro-

Mitsuoka, T., 1982. Recent trends in research on intestinal flora.phage phagocytic activity by cell-free extracts of selectedBifidobact. Microfl. 1, 3–24.lactic acid-producing bacteria. J. Dairy Sci. 76, 2485–2492.

Namioka, S., 1985. Immunoresponsiveness of newborn piglets andHoffman, M., Weinberg, J.B., 1987. Tumor necrosis factor-apeptidoglycan derived from Bifidobacterium. Bifodobact. Mi-induces increased hydrogen peroxide production and Fccrofl. 4, 3–14.receptor expression, but not increased Ia antigen expression by

peritoneal macrophages. J. Leuk. Biol. 42, 704–707. Rafter, J.J., 1995. The role of lactic acid bacteria in colon cancerprevention. Scand. J. Gastroenterol. 30, 497–502.Homma, N., 1988. Bifidobacteria as a resistance factor in human

beings. Bifidobact. Microfl. 7, 35–43. Sarih, M., Souvannavong, V., Adam, A., 1993. Nitric oxidesynthase induces macrophage death by apoptosis. Biochem.Hosono, A., Lee, J., Ametani, A., Natsume, M., Hirayama, M.,Biophys. Res. Commun. 191, 503–508.Adachi, T., Kaminogawa, S., 1997. Characterization of a

water-soluble polysaccharide fraction with immunopotentiat- Sanders, M.E., 1993. Effect of consumption of lactic cultures oning activity from Bifidobacterim adolescentis M 101-4. Biosci. human health. Adv. Food Nutr. Res. 37, 67–130.Biotech. Biochem. 61, 312–316. Sasaki, T., Fukami, S., Namioka, S., 1994. Enhanced resistance of

mice to Escherichia coli infection induced by administrationHughes, D.B., Hoover, D.G., 1991. Bifidobacteria: Their potentialof peptidoglycan derived from Bifidobacterium thermophilum.for use in American dairy products. Food Technol. 45, 74–83.J. Vet. Med. Sci. 53, 433–437.Kado-Oka, Y., Fujiwara, S., Hirota, T., 1991. Effects of bifidobac-

Schmidt, H.H.H.W., Walter, U., 1994. NO at work. Cell 78,teria cells on mitogenic response of splenocytes and several919–925.functions of phagocytes. Milchwissenshaft 46, 626–630.

Scardovi, V., 1986. Genus Bifidobacterium. In: Sneath, P.H.A.,Kim, Y.-M., Billiar, T.R., Lancaster, J.R. Jr., 1996. ReactiveMair, N.S., Sharpe, M.E., Holt, J.G., (Eds.), Bergey’s Manualoxygen and nitrogen metabolites and related enzymes. In:of Systematic Bacteriology, vol. 2, Williams and Wilkins,Hezenberg, L.A., Weir, D.M., Herzenberg, L.A., Blackwell, C.Baltimore, p. 1418–1434.(Eds.), Weir’s Handbook of Experimental Immunology, vol. 4.

The Integrated Immune System, p. 171.1–171.10. Sekine, K., Ohta, J., Onishi, M., Tatsuki, T., Shimokawa, Y.,Toida, T., Kawashima, T., Hashimoto, Y., 1995. Analysis ofKurmann, J.A., Raric, J.L., 1991. The health potential of productsantitumor properties of effector cells stimulated with a cellcontaining bifidobacteria. In: Robinson, R.K. (Ed.), Therapeu-wall preparation (WPG) of Bifidobacterium infantis. Biol.tic Properties of Fermented Milks. Elsevier Appl. Sci., Lon-Pharm. Bull. 18, 148–153.don, England, p. 117.

Sekine, K., Watanabe-Sekine, E., Ohta, J., Toida, T., Tatsuki, T.,Laskin, D.L., Pendino, J., 1995. Macrophages and inflammatoryKawashima, T., Hashimoto, Y., 1994. Induction and activationmediators in tissue injury. Annu. Rev. Pharmacol. Toxicol. 35,of tumoricidal cells in vivo and in vitro by the bacterial cell655–677.wall of Bifidobacterium infantis. Bifidobact. Microfl. 13, 65–Lee, J., Ametani, A., Enomoto, A., Sato, Y., Motoshima, H., Ike,77.F., Kaminogawa, S., 1993. Screening for the immunopotentiat-

Sekine, K., Watanabe-Sekine, E., Toida, T., Kasashima, T.,ing activity of food microorganisms and enhancement of theKataoka, T., Hashimoto, Y., 1994. Adjuvant activity of the cellimmune response by Bifidobacterium adolescentis M101-4.wall of Bifidobacterium infantis for in vivo immune responsesBiosci. Biotech. Biochem. 57, 2127–2132.in mice. Immunopharmacol. Immunotoxicol. 16, 589–609.Link-Amster, H., Rochat, F., Saudan, K.Y., Mignot, O., Aesch-

Sekine, K., Kasashima, T., Hashimoto, Y., 1994. Comparison oflimann, J.M., 1994. Modulation of a specific humoral immunethe TNF-a levels induced by human-derived Bifidobacteriumresponse and changes in intestinal flora mediated throughlongum and rat-derived Bifidobacterium animalis in mousefermented milk intake. FEMS Immunol. Med. Microbiol. 10,peritoneal cells. Bifidobact. Microfl. 13, 79–89.55–64.

Snyder, S.H., Bredt, D.S., 1992. Biological roles of nitric oxide.Lorsbach, R.B., Murphy, W.J., Lowenstein, C.J., Snyder, S.H.,Sci. Amer. 266, 68–77.Russell, S.W., 1993. Expression of the nitric oxide synthase

gene in mouse macrophages activated for tumor cell killing. Solis Pereyra, B., Lemonnier, D., 1993. Induction of human


cytokines by bacteria used in dairy foods. Nutr. Res. 13, Yasui, H., Kiyoshima, J., Ushijima, H., 1995. Passive protection1127–1140. against rotavirus-induced diarrhea of mouse pups born to and

Strober, W., 1991. Trypan blue exclusion test for cell viability. In: nursed by dams fed Bifidobacterium breve YIT 4064. J. Inf.Coligan, J.E., Kruisbeek, A.M., Marguiles, D.H., Shevach, Dis. 172, 403–409.E.M., Strober W. (Eds.), Current Protocols in Immunology, Yasui, H., Ohwaki, M., 1991. Enhancement of immune responseGreene Pub. and Wiley-Interscience, New York, pp. A.3.3–4. in Peyer’s patch cells cultured with Bifidobacterium breve. J.

Takahashi, T., Oka, T., Iwana, H., Kuwata, T., Yamamoto, Y., Dairy Sci. 74, 1187–1195.1993. Immune response of mice to orally administered lactic Yasui, H., Nagaoka, N., Mike, A., Hayakawa, K., Ohwaki, M.,acid bacteria. Biosci. Biotech. Biochem. 57, 1557–1560. 1992. Detection of Bifidobacterium strains that induce large

Tiku, M.L., Liesch, J.B., Robertson, F.M., 1990. Production of quantities of IgA. Microbial Ecol. Health Dis. 5, 155–162.hydrogen peroxide by rabbit articular chondrocytes: Enhance- Yu, S.F., Koerner, T.J., Adams, D.O., 1990. Gene regulation inment by cytokines. J. Immunol. 145, 690–696. macrophage activation: Differential regulation of genes encod-

Vassalli, P., 1992. The pathophysiology of tumor necrosis factors. ing for tumor necrosis factor, interleukin-1, JE, and KC byAnnu. Rev. Immunol. 10, 411–452. interferon-gamma and lipopolysaccharide. J. Leuk. Biol. 48,

Yamazaki, S., Tsuyuki, S., Akashiba, H., Kamimura, H., Kimura, 412–419.M., Kawashima, T., Ueda, K., 1991. Immune response ofBifidobacterium-monoassociated mice. Bifidobact. Microfl. 10,19–31.

potentiation of hydrogen peroxide, nitric oxide, and cytokine production in raw 264.7 macrophage...

Documents