Biol. Pharm. Bull. 33(2) 289—293 (2010)

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Establishment of an in Vitro Peyer’s Patch Cell Culture System Correlative
to in Vivo Study Using Intestine and Screening of Lactic Acid Bacteria
Enhancing Intestinal Immunity
Hekui JIN,a Fumiko HIGASHIKAWA,b Masafumi NODA,b Xingrong ZHAO,a Yasuyuki MATOBA,a
Takanori KUMAGAI,a and Masanori SUGIYAMA*,aa

Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical Sciences, Hiroshima
University; and b Department of Molecular Nutrition Sciences, Graduate School of Biomedical Sciences, Hiroshima
University; 1–2–3 Kasumi, Minami-ku, Hiroshima 734–8551, Japan.
Received September 7, 2009; accepted November 9, 2009; published online November 11, 2009
Some lactic acid bacteria (LAB) are known as representative of probiotics. To screen LAB effective toenhance intestinal immunity, in the present study, we developed an accurate and convenient in vitro evaluation
system using Peyer’s patch cells (PP-cells) isolated from the mice intestine. We observed that the amount of
immunoglobulin A (IgA) produced by PP-cells co-cultured with LAB was well correlative to that in PP-cells,
0.888,intestine and feces isolated from live mice after oral administration of LAB [correlation coefficient (r)0.883, and 0.920, respectively]. In addition, using this in vitro system, we suggest that the IgA level of PP-cells coculturing with plant-derived LAB might be more enhanced than with animal-derived LAB.
Key words lactic acid bacterium; intestinal immunity; immunoglobulin A; probiotic

Some lactic acid bacteria (LAB) function as probiotics.
The beneficial functions brought by LAB on gastrointestinal
conditions, such as constipation, diarrhea, inflammatory
bowel disease, Helicobacter pylori infection, lactose intolerance, and colon cancer, have been reported.1,2) LAB havealso been found to modulate systemic and/or intestinal immune responses, including allergies,3—10) although their application in medical treatment is still far from possible. It is
important to accumulate information about numerous LAB
strains to screen the most appropriate and effective LAB foreach purpose.
According to the circumstances of their isolated sources,
LAB can be roughly classified into two groups: one is fromanimal sources, such as raw milk, cheese, or intestine, andthe other is from plant sources, such as grasses, vegetables,or fruits. Animal-derived LAB have been traditionally used
for fermented foods, such as yogurt and cheese. On the other
hand, plant-derived LAB are used for Japanese traditional
fermented dishes and miso. Lactococcus (Lc.) lactis, Lactobacillus (Lb.) bulgaricus, and Streptococcus salivarius subsp.
thermophilus are typical animal-derived LAB. Lb. plantarum
and Lb. brevis are representatives of plant-derived LAB. But,
it is also true that Lc. lactis was isolated from many kinds offlowers (unpublished data). We insist that plant-derived LAB,
in general, must be more resistant to rigorous environments
than animal-derived LAB. Indeed, the plant-derived LAB
strains newly isolated by our group, Lb. plantarum SN13T,
SN35N, and Lb. brevis 925A, were found to be more viable
in artificial gastric fluid and bile than animal-derived LAB,
like Lc. lactis, Lb. bulgaricus, and Lb. acidophilus (unpublished data). Our previous clinical study has demonstrated
that the yogurt made by Lb. plantarum SN13T improves constipation, serum lipid levels, and liver function more efficiently than the yogurt made by animal-derived LAB.11) It is
known that even the same species of LAB show different
probiotic properties. Considering the advantage of plantderived LAB resistant to digestive fluids in gastrointestinal
tract, it will be significant to screen various plant-derived∗ To whom correspondence should be addressed.

LAB strains which show excellent probiotic activities.
Gastrointestinal mucosa is the primary site of antigen
invasions. The gut-associated lymphoid tissue (GALT) is
composed of Peyer’s patches, peripheral lymphoid tissues,and the appendix. The GALT plays an important role in
the immune system of the gastrointestinal tract.12,13) Immunoglobulin A (IgA) is the most abundant in the body of all
immunoglobulin isotypes as well as the predominant immunoglobulin isotype in the mucosal surface of the gastrointestinal tract.12,14) Peyer’s patch, where IgA is produced byplasma cells, is considered as the most important defense site
in intestinal immunity to prevent microbial invasion.15—18)
The effect of probiotics on intestinal immunity has been investigated using laboratory animals; IgA assessments wereconducted using isolated intestine, feces, or a culture of exvivo Peyer’s patch cells (PP-cells) after oral administration of
LAB.5,19,20) This method is, however, not suitable for thescreening of hundreds of LAB strains at once and consumes
an excessive number of laboratory animals. Therefore, a system in which PP-cells are co-cultured with LAB may be aconvenient and useful tool for estimating the influence of
LAB on intestinal immunity.
To evaluate how accurate an in vitro PP-cells culture system is by comparing with the in vivo experiments using animals, in this study, we investigated the correlation between invitro PP-cells culture system and in vivo oral administrationin terms of the change of IgA production under the LAB influences.
MATERIALS AND METHODS
Animals Female BALB/cA (SPF) mice, 6 to 7 weeks ofage at commencement, were purchased from Japan CLEA
(Shizuoka, Japan) and housed in plastic cages under 12 h
light/dark cycles. Animal experiments were conducted in accordance with the “Guidelines for the Care and Use of Laboratory Animals” established by Hiroshima University. Mice
were sacrificed by ether anesthetization.

e-mail: sugi@hiroshima-u.ac.jp© 2010 Pharmaceutical Society of Japan

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Table 1.

Vol. 33, No. 2
LAB Strains Used in This Study
LAB species

Pediococcus pentosaceus LP28
Enterococcus avium G-15
Enterococcus mundtii 15-1A
Lactobacillus plantarum SN13Ta)
Lactobacillus brevis 925A
Lactococcus lactis 527
Lactobacillus bulgaricus B-5b
Streptococcus salivarius
subsp. thermophilus 510

Isolated source

Abbreviation

Plant (longan, fruit)
Plant (carrot’s leaves)
Plant (mibuna, vegetable)
Plant (Nahm)
Plant (kimchi)
Animal (yogurt)
Animal (yogurt)
Animal (yogurt)

LP28
G-15
15-1A
SN13T
925A
527
B-5b
510

Streptococcus lactis 527, Lactobacillus bulgaricus B-5b, and Streptococcus salivarius subsp. thermophilus 510 have been used as a starter for yogurt production by adairy company (Nomura Dairy Co., Japan). These strains have been purchased from thefoundation of the Japan Dairy Technical Association. Streptococcus lactis is now taxonomically classified into Lactococcus lactis. In our laboratory, Lactococcus lactis 527,
Lactobacillus bulgaricus B-5b, and Streptococcus salivarius subsp. thermophilus 510
have been published as Lactococcus lactis A6, Lactobacillus bulgaricus C6, and Streptococcus thermophilus 510, respectively.11) a) SN13T was isolated from Nahm, the fermented sausage made in Thailand. To make Nahm, the pig minced meat is wrapped andfermented with a leaf of the tropical vegetation, like banana.

Bacterial Cell Preparation The LAB strains used in thecurrent study are summarized in Table 1. Five plant-derived
LAB strains, Pediococcus (P.) pentosaceus LP28 (LP28), Enterococcus (E.) avium G-15 (G-15), E. mundtii 15-1A (151A), Lb. plantarum SN13T (SN13T), and Lb. brevis 925A
(925A), were newly isolated in our laboratory. Three typicalanimal-derived LAB strains, Lc. lactis 527 (527), Lb. bulgaricus B-5b (B-5b), and Streptococcus salivarius subsp. thermophilus 510 (510), were kindly provided from Nomura
Dairy Products Co., Ltd., Japan. All LAB strains were cultured in De Man-Rogosa-Sharpe (MRS) broth (Merck KGaA,
Darmstadt, Germany) and lyophilized for preservation. Heatkilled LAB were prepared by autoclaving at 120 °C for
20 min followed by washing three times with phosphatebuffered saline (PBS).
PP-Cell Preparation Preparation of primary cultures of
PP-cells was performed as described previously21) with thefollowing slight modifications. BALB/cA mice were sacrificed
by ether, and their small intestines were placed in a petri dishfilled with PBS containing penicillin (100 U/ml; Invitrogen,
Carlsbad, CA, U.S.A.) and streptomycin (100 m g/ml; Invitrogen) on ice. Visible Peyer’s patches were carefully isolated
from the small intestinal wall and placed in an ice-cold complete medium, an RPMI-1640 medium (Invitrogen) containing 5% fetal bovine serum (FBS, Invitrogen), 50 m M 2-mercaptoethanol, 100 U/ml penicillin, and 100 m g/ml streptomycin. To obtain a single PP-cell suspension, the Peyer’s
patches were digested with type 1 collagenase (70 U/ml; Wako
Pure Chemical Industries, Ltd., Tokyo, Japan) dissolved in
the same complete medium and incubated for 60 min at
37 °C. After filtration through a 100 m m nylon mesh (Cell
Strainers, BD Biosciences, Bedford, MA, U.S.A.), the PPcells were washed three times with a complete RPMI-1640
medium. Cell viability was assessed by trypan blue exclusion.
Co-culture of PP-Cells with LAB A total of 5.0106PP-cells in 100 m l of the complete medium supplemented
with 5 m g/ml of concanavalin A (Sigma, St. Louis, MO,
U.S.A.) and 50 m g/ml of lipopolysaccharide (LPS, Sigma)
were added to each well of a 96-well tissue culture plate (BD
Biosciences), and then each LAB strain suspended in 100 m lof the same medium was added at final concentrations of
2.5104, 2.5105, and 2.5106 colony forming unit (cfu)/
ml. PP-cells and LAB were co-cultured for 16 h or 24 h at
37 °C in a humidified atmosphere with 5% CO2, and the IgAlevels of each supernatant were then assessed using an enzyme-linked immunosorbent assay (ELISA) kit for IgA according to the manufacturer’s instructions (Bethyl Laboratories, Inc., Montgomery, TX, U.S.A.). To determine the effectof secretions from LAB, each LAB strain was incubated at
2.5106 cfu/ml in a complete medium at 37 °C for 24 h, andthe supernatants were then prepared by 0.22 m m-filter sterilization. The LAB supernatants were added to the PP-cells at
1 : 1 ratio, and IgA production was measured after 16 h of incubation at 37 °C. For the experiment of heat-killed LAB,
PP-cells were co-cultured with them instead of with living
LAB.
In Vivo Analyses of the Intestinal IgA Productivity
Using Mice Mice were orally administered each LAB
strain for 7 d with their drinking water, which contained LAB
at a concentration of 1.0108 cfu/ml. Control mice received
only autoclaved distilled water instead of the LAB suspension. The drinking water containing LAB was changed twice
a day to keep it fresh. Fresh feces were collected by isolating
mice in an individual cage for defecation on day 3, day 5,
and day 7. The feces were stirred in PBS containing 0.1 mg/
ml trypsin inhibitor and 50 mM ethylenediaminetetraacetate(EDTA) at 4 °C overnight and centrifuged at 9300 g for 10
min. The resulting supernatant was used to measure thequantity of IgA contained in fecal extracts. After feces werecollected on day 7, mice were sacrificed. Peyer’s patches andwhole intestines were obtained from the mice. After both organs were dispersed into PBS and centrifuged, each supernatant fluid was used to assess the IgA level.
RESULTS
PP-cells and LAB were alive in an RPMI-1640 medium
containing 5 m g/ml concanavalin A and 50 m g/ml LPS atleast for 24 h at 37 °C. Under this condition, the live LAB did
not affect the viability of PP-cells (data not shown). The IgAlevel produced by PP-cells was significantly increased when
co-cultured with the plant-derived LP28, G-15, 15-1A, or
SN13T strain in a dose-dependent manner (Fig. 1). In contrast, all animal-derived LAB, such as 527, B-5b, and 510,
and a plant-derived strain, 925A, did not enhance or even decreased the IgA production by PP-cells.
After oral administration of each LAB strain to mice every
day for 7 d, we obtained the feces, Peyer’s patches, and thewhole intestine excluding Peyer’s patches, and then usedthem for the determination of IgA productivity. Ingestion of
LAB via drinking water was 4.00.5108 cfu/day/mouse.
Secretion and excretion of IgA into feces were remarkably
elevated by the consumption of LP28, G-15, 15-1A, andSN13T, when compared with the others tested in the present
study. In fact, these four LAB strains enhanced the IgA production of PP-cells in in vitro. The IgA levels were continuously increased until day 7 (Fig. 2A). Similarly, these four
LAB strains significantly enhanced the IgA level in PP-cells
and the whole intestine after oral administration, whereas theother strains, 925A, 527, B-5b, and 510, did not (Figs. 2B,
C). Thus, the amounts of IgA produced by PP-cells co-cul-February 2010

291

Fig. 1. Effects of Various LAB Strains on IgA Production by Co-culture
with PP-Cells
Each LAB strain or medium alone was mixed with PP-cells (2.5107 cells/ml) in acomplete RPMI-1640 medium containing 5% FBS, 50 m M 2-mercaptoethanol, 100 U
penicillin/ml, 100 m g streptomycin/ml, 5 m g concanavalin A/ml, and 50 m g LPS/ml.
LAB cells were added at 2.5104, 2.5105, and 2.5106 cfu/ml. They were incubatedfor 16 h at 37 °C and analyzed to determine the IgA level contained in supernatants.
Each bar represents the meanS.D. (n4). ∗ p0.05, ∗∗ p0.01 (Dunnett’s multiple
comparison test).

tured with LAB are correlative to those produced in feces, invivo PP-cells, and in vivo whole intestine, after oral consumption of LAB. The correlation coefficients between invitro and in vivo studies using feces, Peyer’s patch, and intestine were 0.920, 0.888, and 0.883, respectively (Fig. 3), suggesting that this simple and convenient in vitro system is accurate and useful for screening any LAB effect on the intestinal immune response.
To analyze a phenomenon in which the level of IgA produced by PP-cells is enhanced by co-culturing with the plantderived LAB, we made an attempt to determine whether
some LAB secrete the stimulating substances. When each
LAB-culture filtrate, which was prepared through a membrane filter (pore size: 0.22 m m), was cultured with PP-cells,
the filtrates from the G-15 and SN13T cultures enhanced theIgA production by Peyer’s patch (Fig. 4A). This indicates that
only the G-15 and SN13T strains in eight LAB strains used
may release some substances effective to intestinal immunity.
Although a plant-derived 925A strain and animal-derived
LAB (527, B-5b, and 510) were ineffective, this observation
is agreeable with the result obtained by the in vitro direct coculture of LAB and PP-cells. We also assessed whether anincrement in IgA production was observed when each of various LAB sterilized for 20 min at 120 °C was added to the
PP-cell culture. Figure 4B shows that the increment of IgAproduction was observed only in the case of heat-killed G-15
and SN13T cells. On the other hand, PP-cells stimulated IgA
production only when co-cultured with the LP28 and 15-1A
strains in the living state. Thus, the mechanism to enhance
the level of IgA produced by PP-cells varies by species ofplant-derived LAB. In addition, in the present study, it wasobserved that the enhancements of IgA production by co-culture with plant-derived LAB were completely abolishedwhen CD4 T cells were depleted from the total PP-cells(data not shown).
DISCUSSION
Some LAB strains, which are effective on intestinal immu-

Fig. 2. Amounts of IgA Produced in Feces at Days 3, 5 and 7 (A), PPCells at Day 7 (B), and Intestine at Day 7 (C) after Oral Administration ofEach LAB Strain or Vehicle Alone as a Control
Each bar represents the meanS.D. of five mice. ∗ p0.05, ∗∗ p0.01 (Dunnett’s
multiple comparison test).

nity, are recognized as representative probiotics. PP-cells are
the most important defense mechanism providing intestinal
immunity. In the present study, we established a system which
rapidly and accurately detects the probiotic effect of LAB on
the intestinal immune response by using PP-cells co-cultured
with LAB. This method will be a substitutive method for thetime-consuming in vivo studies which require the sacrifice ofa number of experimental animals. The amount of IgA produced by PP-cells co-cultured with LAB was well correlatedwith that by PP-cells, intestine, and feces isolated from live
mice after oral administration of LAB.
In this study, we evaluated the effect of LAB on intestinal
immunity using five plant-derived LAB and three animal-

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Fig. 4. Effects of Various LAB Strains under Different Conditions on IgA
Production by the PP-Cell Culture(A) PP-cells were incubated with the LAB culture filtrate after eliminating the cellmass through a 0.22 m m pore size filter. (B) PP-cells were incubated with heat-killed
LAB. They were incubated in a complete RPMI-1640 medium containing 5% FBS,
50 m M 2-mercaptoethanol, 100 U penicillin/ml, 100 m g streptomycin/ml, 5 m g concanavalin A/ml, and 50 m g LPS/ml for 16 h, and the IgA levels of the supernatants were
measured. Each bar represents the meanS.D. (n4). ∗∗ p0.01 (Dunnett’s multiple
comparison test).

Fig. 3. Correlations between in Vitro and in Vivo Results Regarding theChange of IgA Production(A) In vitro PP-cell culture versus feces; (B) in vitro PP-cell culture versus in vivo
PP-cells isolated from mice after oral administration of LAB; (C) in vitro PP-cell culture versus intestine. The correlation coefficients were 0.920 (A), 0.888 (B), and 0.883(C). Each point represents the meanS.D.

derived LAB strains. As a result, we observed that plant-derived LAB are more effective on intestinal immuno-stimulation than animal-derived LAB strains. To study how a certain
LAB strain can stimulate IgA production by PP-cells, we
compared the direct and indirect effect of LAB on PP-cells.
Some substances as a cell wall component of LAB may function as immune stimulants in many cases, as shown in reports
according to which heat-killed LAB are able to stimulate theimmune response.3,9,10) It has also been reported that the immunomodulatory activity of Bifidobacterium bifidum was notdue to components secreted from bacteria.5) Gram-positivebacteria, such as LAB, contain lipoteichoic acids as a compo-

nent of the cell walls, which function as major immunostimulatory factors and agonists of the toll-like receptor 2.22—24)
In this study, we showed that the the G-15 and SN13T culture
filtrates enhanced the IgA level produced by PP-cells. Thissuggests that some substances enhancing IgA production by
PP-cells may be secreted from the G-15 and SN13T cells.
Furthermore, the heat-killed LAB, G-15 and SN13T, also
exhibited the IgA-enhancing effect, suggesting that the result
is consistent with those reported previously.3,9,10) Interestingly, the supernatant fluids from LP28 and 15-1A cultures
and the heat-killed LP28 and 15-1A cell mass were ineffective for enhancing IgA production. The heat-unstable substances such as proteins, which are contained in these LAB,
might play a role in enhancing the IgA level produced by PPcells. Judging from these observations, these plant-derived
LAB may have multiple mechanisms to stimulate intestinal
immunity. Antigen-specific IgA has been reported to be produced from the plasma cells located in PP-cells in both T
cell-dependent and -independent manners.25) The incrementof IgA-producing cells brought by LAB is not always correlative with that of CD4 T cells.6,7) Under the presence of
SN13T or G-15, the expression levels of interleukin-6 (IL-6),
IL-10, and/or tumor growth factor-b (TGF-b ) in PP-cells
were enhanced until 2—3 times. In addition, when CD4 T
cells were depleted from PP-cells, the IgA increments by cocultured with the plant-derive LAB was completely disap-

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peared (data not shown), suggesting that the enhancements ofintestinal immunity may be T cell-dependent.
In conclusion, this in vitro co-culturing system of PP-cellswith LAB is convenient and accurate to evaluate probiotic influence on intestinal immunity, suggesting that this system
may be a useful tool to screen the IgA-enhancing LAB at one
time. Using this in vitro system, we found that Lb. plantarum
SN13T and E. avium G-15 are useful as probiotics to enhance intestinal immunity.
Acknowledgements Experiments were carried out inpart at the Research Center for Molecular Medicine and theAnalysis Center of Life Science at Hiroshima University.
This work was supported by Hiroshima Biocluster (Cooperative Link of Unique Science and Technology for Economy
Revitalization), Japan (M. S.).
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