Lactic Acid Bacteria (LAB) such as Lactobacillus acidophilus, Lactobacillus delbrueckii spp. bulgaricus and Bifidobacteria are important components of the normal intestinal microflora in humans and animals. These bacteria act as immunomodulators participating in generation and maintaining of at least three important activities of mucoid immune system: a) anti-microbial and pro-inflammatory action mediated by Th1-cytokines such as IL-12, TNF-alpha and IFN-; b) anti-inflammatory activities and oral tolerance, induced and maintained by Th2 cytokines, mainly IL-10 and TGF-ß; c) stimulation of adaptive immune responses, including local and systemic IgG and IgA synthesis being due mainly to IL-4 and IL-5 (1). Because in humans it would be difficult to create a system of studying direct interactions between the cells of mucoid immune system and the comensal bacteria in their natural environment, the use of peripheral blood mononuclear cells (PBMC) as the responder cells for LAB stimulation in vitro is a commonly acceptable alternative. It should be noted that interactions between commensal bacteria and blood leukocytes may occur due to a limited bacterial translocation through the epithelial barrier (2, 3). It may also be a reason for an increase of some non-specific (phagocytosis, Natural Killer cell activities) and specific (antibody synthesis) immune activities that have been observed in humans and animals following ingestion of different LAB strains (4). The cytokines synthesis patterns in the cells of immune system induced by LAB in vitro and in vivo have been shown to be strain-dependent (5, 6). However little is known how the composition of LAB strains may regulate the direction and strength of lymphocyte’s activation reflected either by pro-inflammatory, antimicrobial defence mechanisms or anti-inflammatory reactivities.

The aim of this study was to find out wheather three LAB strains (L. acidophilus, L. bulgaricus and B. bifidum), acting alone, in pairs or as a mixture would differ from each other in their abilities to induce lymphocytes’ activities in vitro measured by lymphocyte proliferation and cytokines synthesis profile.


MATERIAL AND METHODS
Bacteria

The bacteria were prepared according to the procedure described previously (7). We used three bacterial strains: L. delbrueckii ss. bulgaricus LbY-27, B. bifidum BB –12 R and L. acidophilus La-5R. The live bacteria strains were used as a stimulating agent in all experiments. The bacterial strains were received from Chr. Hansen, Dairy Ingredients Division, R&D Genetics & Microbiology, Horsholm, Denmark (7).

Isolation of cells

PBMC were obtained from heparinized blood of healthy volunteers (blood donors; median age 33 years, range 18-55 years), by Isopaque-Ficoll (Lymphoprep; Nycomed Pharma AS, Oslo, Norway) gradient centrifugation, as previously described (5, 6). Mononuclear cells were resuspended in culture medium made up of RPMI 1640 (Gibco, Paisley, UK) supplemented with 5% heat-inactivated fetal calf serum (Gibco). Freshly isolated PBMC were diluted in culture medium to a final concentration of 1×106 cells/ml.

Proliferation assay

The assay was made according to the procedure described previously (7, 8). Briefly, freshly isolated PBMC were diluted in culture medium to a final concentration of 1×106/ml and transfered in a volume of 180 µl to flat-bottomed 96–well microtitre trays (Costar, Cambridge, UK). Subsequently, 20 µl of culture medium alone (negative control), 20 µl of Concanavaline A (ConA) to a final concentration of 50 µg/ml (positive control) or 20 µl LAB in concentration that induced optimal proliferative response (L. bulgaricus+L. acidophilus - 4×106 bacteria/ml, mixture of bacteria and B. bifidum – 2×106 bacteria/ml, L. bulgaricus+B. bifidum, L. acidophilus+B. bifidum – 1×106 bacteria/ml or L. bulgaricus and L. acidophilus - 0.5×106 of bacteria/ml) were added. The cells were cultured in triplicates for 7 days. Lymphocyte proliferation was assessed by pulsing the cells with 1 µCi 3H-thymidine (Amersham, Little Chalfont, UK) for the last 16 h of the incubation period. The cultures were then harvested onto glass filter strips using an automated multisample harvester (Skatron, Lier, Norway) and analysed for 3H-thymidine incorporation by a liquid scintillation counting. Data are given as count per min (cpm) per 106 cells.

Cytokines assay

The assay was performed as previously described (7, 8). Briefly, cytokine concentration in cell culture supernatants (IFN-, TNF-alpha, IL-12 p40 and IL-10) was determined after 72 hours of bacterial stimulation of PBMC (1×106/ml) using ELISA (Opt-EIA system, Becton-Dickinson, San Diego, CA, USA), according to the manufacturer’s procedure. Dose-response experiments performed for each cytokine indicated that maximal secretion was obtained with 4×106 bacteria/ml (for L. bulgaricus+L. acidophilus), 2×106 (for mixture of bacteria and B. bifidum), 1×106 (for L. bulgaricus+B. bifidum, L. acidophilus+B. bifidum) and 0.5×106 (for L. bulgaricus and L. acidophilus), that also induced optimal proliferative response.

Statistics

The STATISTICA (version 6.0) computer software (StatSoft, USA) was used for the statistical analysis. Values were checked to fulfil the criteria of homogeneity according to Levene tests. For normal distribution, variables were analysed by Kolmogorov-Smirnov test with Lillefor’s correction. For those with non-normal distribution Mann-Whitney U-test was used. Statistical significance was considered at p<0.05.


RESULTS
Proliferative response PBMC

In order to find out if the composition of LAB bacteria strains would have an effect on their ability to induce lymphocyte proliferation, PBMC were stimulated with three different LAB strains used together (mixture of bacteria), in combinations with each other (pairs) or alone, and with a T cell-mitogen Con A, used as a positive control. All bacterial combinations were equally low inducers of lymphocytes’ proliferation (Fig. 1).

Pattern of cytokine expression in LAB-induced PBMC

The next set of experiments was performed to check if the LAB strains used alone or in combinations would differ in their capacity for induction of cytokine pattern in PBMC. Freshly isolated PBMC were activated for 72 hrs with mixture of three bacterial strains, single LAB strains and their pairs in conditions as described in Fig. 1. The amount of cytokines (IFN-, IL-12, IL-10 and TNF-alpha) secreted into the cell culture supernatants was determined by ELISA.

Fig. 1. Proliferative response in PBMC induced by mixture of bacteria, the pairs of LAB, single LAB strains and Con A (positive control). Values represent medians obtained from 10 (for mixture of three bacteria strains and L. acidophilus) and 12 (for remaining composition of LAB) independent experiments. Mann-Whitney U-test, statistically significant differences: a - vs. mixture of bacteria, the single and pairs of LAB strains.

The single LAB strains and their combinations effectively induced different cytokines synthesis pattern. First, L. acidophilus was much stronger inducer of IFN- and IL-12 than mixture of bacteria, L. bulgaricus or B. bifidum. Second, combination of L. acidophilus with L. bulgaricus or B. bifidum decreased IFN- synthesis to the level that was equal to that induced by mixture of bacteria itself, it also caused a significant reduction of IL-12 production. Third, all combinations and single strains of LAB were equally good inducers of both IL-10 and TNF-alpha. The results are presented in Figs 2-5.

Fig. 2. Induction of IFN- in PBMC stimulated by mixture of bacteria, the pairs or single LAB strains. Values represent medians obtained from 8 independent experiments. Mann-Whitney U-test, statistically significant differences: a - vs. L. acidophilus, b - vs. L. bulgaricus + B. bifidum.

Fig. 3. Induction of IL-12 in PBMC by mixture of bacteria, pairs of LAB strains and single LAB strains. Values represent medians obtained from 5 (for single bacteria strains and L. bulgaricus + L. acidophilus) and 6 (for remaining composition of Lactic Acid Bacteria) independent experiments. Mann-Whitney U-test, statistically significant differences: a - vs. mixture of all bacterial strains, b - vs. L. acidophilus, c - vs. L. bulgaricus, d - vs. L. bulgaricus + L. acidophilus.

Fig. 4. Effect of mixture of bacteria, the pairs and single LAB strains on TNF-alpha secretion in PBMC. Values represent medians obtained from 8 (for mixture of all bacteria strains and remaining composition of Lactic Acid Bacteria) and 5 (for single bacteria strains) independent experiments. Mann-Whitney U-test, statistically significant differences: a - vs. L. acidophilus, b - vs. L. bulgaricus.

Fig. 5. Effect of mixture of bacteria, the pairs and single LAB strains on IL-10 secretion in PBMC. Values represent medians obtained from 6 (for all composition of LAB) and 5 (for single bacteria strains) independent experiments. Mann-Whitney U-test, statistically significant differences: a - vs. mixture of bacteria, b - vs. L. bulgaricus.

DISCUSSION
The study was undertaken to find out if the composition of LAB bacterial strains used for PBMC stimulation would differ in their effect on: a) the level of lymphocytes’ proliferation and, b) the cytokine synthesis pattern. We found that the level of LAB strains-induced lymphocyte proliferative response in PBMC was equally low for both the single strains and their combinations (as compared with Con A-induced controls). The CD4+ T cells were the main responders (not shown) (7). Concerning this observation, some conflicting results have been reported in other studies. First, it has been shown that lymphocyte proliferation was increased in mice fed with different LAB (9) and the T-cell proliferative response in vitro to C. albicans, L. casei GG or B. animalis were stronger in LAB fed mice than in ones infected with C. albicans alone (10). Second, in humans consuming fermented milk products supplemented with either L. acidophilus La1 or B. bifidum Bb12, peripheral blood lymphocyte populations have remained not altered both in term of mitogen-induced proliferation and mode of surface receptors expression (11). Third, in vitro studies on human PBMC stimulated with L. johnsoni La1 and L. sakei LTH have shown moderate proliferative action of these bacteria in PBMC (12). Altogether these data and our observations indicate that a moderate proliferative response of lymphocytes to LAB strains possibly reflect a normal responsiveness of lymphocytes to microflora antigens, that renders them functionally active but not overstimulated (without harmful, proinflammatory activities) (Fig. 1).

In contrast to proliferation, the LAB-induced cytokines synthesis profile in PBMC was strictly dependent on the composition of the LAB strains. First, IFN- synthesis induced by mixture of bacteria and L. bulgaricus was equally low but L. acidophilus induced about 3-fold higher production of IFN-. Second, L. bulgaricus and B. bifidum inhibited L. acidophilus-induced IFN- synthesis. Third, the combination of L. bulgaricus + B. bifidum resulted in a very low synthesis of IFN- which was about 4-fold lower than that induced by mixture of bacteria and L. bulgaricus, and decreased by as much as 10-fold when compared with L. acidophilus-induced IFN- secretion (Fig. 2). Down regulation of L. acidophilus-induced high IFN- synthesis by the other 2 LAB strains used here may indicate the complex regulation of this cytokine synthesis and confirm anti-inflammatory action exerted by a combination of different LAB strains.

Relatively low mixture of bacteria-induced IFN- synthesis may reflect the complex mechanisms of control of this cytokine by means of different combinations of LAB that are normally present in the intestine. Rather low activation of IFN- expression by mixture of LAB strains possibly represents a normal cellular reactivity induced in vivo by a great variety of LAB strains normally present in digestive tract (13). Their presence is important for maintaining a Th2 profile of immune response which normally prevails in mucosal immune system (14).

This assumption can be further confirmed by the mode of IL-12 expression in PBMC induced either by mixture of bacteria, the pairs or single LAB strains. First, mixture of bacteria strains as well as L. bulgaricus were poor IL-12 inducers (identically as of IFN-) but again, L. acidophilus strongly induced IL-12 (8–fold more than mixture and 6-fold more than L. bulgaricus). Second, B. bifidum was moderate inducer of IL-12 having about 35% of L. acidophilus efficacy. Third, L. bulgaricus and B. bifidum inhibited L. acidophilus -mediated IL-12 synthesis (Fig. 3).

The data related to the pattern of IFN- and IL-12 expression in PBMC indicated that particular LAB strains differ in their capacity for IFN- and IL-12 induction. It has also been observed by others with respect to other LAB strains (L. johnsoni and L. sakei) which strongly induced IL-12 and IFN- (15). These cytokines are known to participate in enhancement of non-specific immunity and their release may be a reason of increased macrophage phagocytosis and NK cell activity both in mice injected intraperitoneally with Lactobacilli (16), and in humans, following oral administration of L. acidophilus or Bifidobacterium strain Bb12 (17, 18). Since both IL-12 and IFN- belong to a group of so called proinflammatory cytokines (able to induce harmful inflammatory reactions), simultaneously participating in immune reactions, its important to distinguish between inflammation and immunity. Inflammation, which is the local accumulation of leukocytes, fluid and plasma proteins, is quite distinct from immunity, defined as the ability to resist infection. So upregulation of inflammatory mediators (also proinflammatory cytokines) is not necessarily a desirable attribute for probiotics. Confirmatory to such notion is our finding that all three of LAB strains studied here showed the ability for strong induction of IL-10 as well as TNF-alpha, either used alone or in combinations (Figs 4-5). The main role of Lactobacilli-induced IL-10 would be to limit Th1-mediated proinflammatory response (reflected here by IFN- and IL-12 production). In contrast, TNF-alpha should participate rather in an enhancement of several cellular defence activities such as phagocytosis and cytotoxic reactions (20, 21).

On the basis of presented data we conclude that composition of LAB strains is a critical factor which makes possible the enhancement of: 1) cellular activities needed both for down regulation of potential proinflammatory reactions (due to IL-10) and, 2) defence mechanisms necessary for elimination of bacterial and viral pathogens (due to TNF-alpha expression).


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