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Clin Exp Immunol. May 1999; 116(2): 276–282.
PMCID: PMC1905267

Lactobacilli from human gastrointestinal mucosa are strong stimulators of IL-12 production

Abstract

Interaction of macrophages with bacteria is a stimulus for production of cytokines such as IL-10 and IL-12. IL-12 stimulates T cell and natural killer (NK) cell cytotoxicity and interferon-gamma (IFN-γ) production. IL-10 opposes the T cell-stimulating action of IL-12, decreases the release of proinflammatory cytokines from macrophages, and stimulates B cells. We have studied the capacity of human intestinal isolates from the three Lactobacillus species dominating on the human gastrointestinal mucosa, L. plantarum, L. rhamnosus and L. paracasei ssp. paracasei, to induce production of IL-10 and IL-12 from human blood mononuclear cells, or monocytes. Whole killed lactobacilli were potent stimulators of IL-12 over a wide range of bacterial concentrations. Lactobacillus paracasei gave the highest levels of IL-12 (1.5 ng/ml in response to 5 × 106 bacteria/ml), roughly 10 times more than obtained by stimulation with L. rhamnosus or L. plantarum. Escherichia coli induced on average < 50 pg/ml of IL-12 regardless of the bacterial concentration used. The secretion of free p40 subunit IL-12 followed the same pattern as the secretion of p70 (bioactive IL-12) with regard to the efficiency of different bacteria as stimulators. Escherichia coli was the most efficient trigger of IL-10 production, inducing 0.5 ng/ml IL-10 after stimulation with 5 × 106 bacteria/ml. Lactobacillus rhamnosus induced the highest levels of IL-10 among the lactobacilli (0.5 ng/ml) compared with 0.1 ng/ml evoked by L. plantarum or L. paracasei, but 10 times more bacteria were required for optimal stimulation than with E. coli. When neutralizing anti-IL-10 antibodies were added to the cultures, the IL-12-inducing capacity of L. rhamnosus was increased markedly, while that of E. coli remained low. The results show that mucosa-associated lactobacilli can be potent stimulators of IL-12, and thus potentially of cell-mediated immunity, if they pass over the gut epithelial barrier and interact with cells of the gut immune system.

Keywords: Lactobacillus, Escherichia coli, cytokines, IL-10, IL-12

INTRODUCTION

The Lactobacillus genus is very heterogeneous, comprising around 70 recognized species and subspecies [1]. Lactobacilli are members of the normal indigenous flora of the oral cavity, small and large intestine, and female genital tract [2,3]. The Lactobacillus species which dominate on the human gastrointestinal mucosa are: L. plantarum, L. rhamnosus and L. paracasei ssp. paracasei, which have been isolated from 52%, 26% and 17% of healthy individuals, respectively [3]. The same species seem to dominate in the oral cavity and in the intestine [3].

Lactobacilli can translocate, i.e. pass viable across the intestinal barrier [46]. Still, they rarely cause serious infection, except for rare cases in individuals with a pre-existing structural heart disease or severe immunodeficiency [7,8]. The capacity to translocate enables lactobacilli to influence cells of the immune system. Lactobacilli have also been used as probiotics, i.e. live bacterial food supplement aimed at, for example, improving certain immune effector functions. Both tumouricidal effects [9] and enhanced phagocytosis in man [10] and mice [11] have been attributed to the ingestion of lactobacilli. Lactobacilli can also function as adjuvants and vectors for vaccine delivery, promoting both cell-mediated immunity and antibody responses [1215]. In vitro, several species of lactobacilli have been shown to trigger the release of IL-6 and tumour necrosis factor-alpha (TNF-α) from human blood mononuclear cells, in amounts comparable to those induced by Escherichia coli [16].

Two important immunoregulatory cytokines produced by cells of the innate defence system in response to bacteria are IL-12 and IL-10. IL-12 stimulates interferon-gamma (IFN-γ) production from T cells and natural killer (NK) cells and increases their cytotoxicity [17]. Functional IL-12 is a heterodimer of 70 kD (p70) formed by the covalent assembly of two chains of approximate molecular weights of 40 kD (p40) and 35 kD (p35), respectively. The production of p70 and p40 are regulated independently [18]. An excess of p40 is produced, the function of which is unclear.

The functions of IL-12 are in many aspects opposed by the cytokine IL-10, which has potent anti-inflammatory properties [1921], abrogates the secretion of IFN-γ from T helper cells [22] and stimulates B cell growth and differentiation [23]. The secretion of IL-12 in response to certain stimuli is decreased in the presence of recombinant IL-10 [24].

In the present study we investigate the IL-12 and IL-10 response by human peripheral blood mononuclear cells (PBMC) to L. plantarum, L. rhamnosus and L. paracasei isolated from oral or rectal mucosa of healthy volunteers. The responses are compared with those induced by E. coli.

MATERIALS AND METHODS

Bacteria

The lactobacilli were chosen from a selection of Lactobacillus strains isolated from the oral cavity or from rectal biopsies of healthy volunteers [3]. Bacterial samples were taken with a cotton-tipped swab from the back of the tongue, and biopsies were taken from the rectal mucosa during rectoscopy. Lactobacilli were isolated on the selective medium Rogosa agar and frozen after no more than two passages after isolation from the mucosa. Two isolates each of L. plantarum, L. paracasei and L. rhamnosus were included, one of which derived from the oral and one from the rectal mucosa (Table 1). The two L. plantarum strains expressed a mannose-specific adhesin [25], mediating adherence to the colonic cell line HT-29 (Table 1). For comparison, two E. coli strains were used. Escherichia coli O6:K13:H1 (no. 20561 CCUG; Culture Collection of the University of Göteborg) was isolated from a case of cystitis and expresses type 1 fimbriae mediating mannose-specific adherence (Table 1). Escherichia coli MS101, a derivative of the rough laboratory strain K12 transformed with the gene encoding for the K5 capsule [26], was kindly provided by I. Roberts (University of Manchester, UK).

Table 1
Characterization and source of bacteria used to stimulate human peripheral blood mononuclear cells

For the experiments, lactobacilli were cultured on Rogosa for two days and E. coli on tryptic soy agar overnight. The bacteria were harvested in PBS, washed and suspended at a concentration of 109/ml, corresponding to an optical density (OD) of 0.96 at 597 nm (Vitatron; Bergström Instruments, Göteborg, Sweden). The bacteria were killed by exposure to UV light for 15 min, which was confirmed by a negative viable count.

Preparation and culture of mononuclear cells

PBMC were obtained from healthy blood donors by density gradient centrifugation (Lymphoprep; Nyegaard, Oslo, Norway) for 20 min at 1000 g. After two washes with ice-cold pyrogen-free RPMI 1640 medium (Gibco, Edinburgh, UK), the cells were suspended in RPMI supplemented with 5% inactivated human serum from donors of the AB blood group (Sigma, St Louis, MO), 50 mm gentamycin (Sigma) and 2 mml-glutamine (Gibco). Cultures with a final volume of 200 μl/well were set up in flat-bottomed 96-well microtitre plates (Nunc, Roskilde, Denmark) with 2 × 106 mononuclear cells and 5 × 104–5 × 107 bacteria/ml, corresponding to 0.025–25 bacteria per mononuclear cell. As a T cell mitogen, we used phytohaemagglutinin (PHA; Sigma) at a concentration of 10 μg/ml. The cell cultures were incubated for 7 days at 37°C in a humidified atmosphere supplemented with 5% CO2. Cytokine concentrations in the supernatants were quantified on days 1, 3, 5 and 7.

Monocytes were obtained by plating 2 × 106 mononuclear cells suspended in RPMI with 5% fresh homologous serum in flat-bottomed 96-well plates for tissue culture (Nunc). After incubation at 37°C for 30 min, non-adherent cells were removed by gentle flushing of the wells with warm medium. The adherent monocyte monolayers were then stimulated with bacteria as described above. Flow cytometry analysis of the amount of cells positive for CD14 indicated that approx. 70% of the monocytes were retained from the mononuclear cell mixture by this procedure.

IL-12 determination

IL-12 concentrations were determined using a sandwich ELISA specific for p70. Microtitre plates (Nunc) were coated with anti-IL-12 antibodies overnight at 4°C with monoclonal mouse IgG1 anti-human IL-12 (clone B-P24; Diaclone, Besancon, France, unknown concentration) diluted 1:200 in PBS. The plates were washed and blocked with 5% bovine serum albumin (BSA; Sigma) in PBS for 2 h at room temperature. The plates were emptied and left to dry at room temperature overnight. Supernatants were diluted 1:5 or 1:25 in PBS with 1% BSA and incubated 2 h together with biotinylated monoclonal mouse IgG1 anti-human IL-12 diluted 1:100 in PBS with 1% BSA (clone B-T21; Diaclone, unknown concentration). After washing, the plates were incubated with streptavidin–horseradish peroxidase (HRP) (Diaclone) diluted 1:6700 for 20 min. The plates were washed, and incubated for 20 min with tetramethylbenzidine (TMB) substrate (Diaclone). The reaction was stopped with 1 m H2SO4 and the colour reaction was measured at 450 nm. The limit of detection of the assay was 10 pg/ml.

For the measurement of both free IL-12 p40 subunit and the heterodimer p70, an ELISA kit (cat. no. KHC0122; Biosource, Camarillo, CA) was used according to the manufacturer's instructions. Supernatants were diluted 1:5 or 1:25. The limit of detection of the assay was 10 pg/ml.

IL-10 determination

Supernatant concentrations of IL-10 were measured using a sandwich ELISA. Polystyrene microtitre plates (Maxisorp; Nunc) were coated overnight at 4°C with 2 μg/ml of purified rat IgG1 anti-human IL-10 (Pharmingen, San Diego, CA) diluted in bicarbonate buffer. After washing the plates with PBS–Tween, the supernatants were diluted 1:2 in a dilution buffer and incubated at room temperature for 3 h. As a standard, recombinant human IL-10 (R&D Systems, Abingdon, UK) with a purity of 99% was used. After washing, 2 μg/ml of biotinylated rat IgG2a anti-human IL-10 (Pharmingen) was added and the plates were incubated for 2 h at 37°C. The plates were washed and alkaline phosphatase-conjugated extravidin (Sigma), diluted 1:2000, was added. The plates were incubated for 1 h at room temperature, washed and incubated with the substrate p-nitrophenyl phosphate in diethanolamine buffer pH 9.8. The colour reaction was measured at 405 nm in a spectrophotometer (Titertec Multiscan; Flow Labs, MacLean, VA). The limit of detection of the assay was 10 pg/ml.

Blocking of IL-10

For inhibition experiments, rat IgG1 anti-IL-10 antibodies (Pharmingen) or rat IgG1 control antibodies (Pharmingen) were added to the cell cultures at a final concentration of 1, 5 or 10 μg/ml before the addition of bacteria (5 × 106 bacteria/ml). After 1 day of culture, supernatants were tested for IL-10 and IL-12 by ELISA.

Statistical analysis

The statistical significance of the difference in cytokine production in response to bacteria was evaluated by the Wilcoxon signed rank test, comparing the responses to both isolates of one bacterial species with the response to both isolates of another species.

RESULTS

Production of IL-12 by PBMC and monocytes stimulated with lactobacilli or E. coli

Different concentrations of whole UV-killed lactobacilli or E. coli were used to stimulate PBMC from nine blood donors. Maximal IL-12 responses occurred on day 1 (data not shown). All six strains of lactobacilli were potent stimulators of IL-12 production, compared with E. coli(Fig. 1). Among the lactobacilli, L. paracasei ssp. paracasei induced the highest IL-12 response (> 1000 pg/ml), while the L. plantarum and L. rhamnosus strains gave lower levels (200 pg/ml), a difference which was statistically significant (L. paracasei versus L. plantarum or L. rhamnosus, P < 0.001) The lactobacilli were active at low bacterial concentrations. Thus, optimal responses were obtained with 5 × 106 or 5 × 105 lactobacilli/ml (2.5 or 0.25 bacteria/cell, respectively), and substantial IL-12 production often occurred with as few bacteria as 5 × 104/ml, which represents 0.025 bacteria/mononuclear cell (Fig. 1). At the highest bacterial concentration, 5 × 107/ml, IL-12 responses were low. Escherichia coli induced < 50 pg/ml of IL-12 regardless of bacterial concentration (Fig. 1).

Fig. 1
Concentration of IL-12 p70 in supernatants of mononuclear cell (MNC) cultures stimulated for 24 h with whole killed lactobacilli or Escherichia coli, or with phytohaemagglutinin (PHA; 10 μg/ml). Non-stimulated cultures are shown as control. Bacterial ...

Monocytes purified from the PBMC mixture by adherence yielded approximately four times the concentration of IL-12 in the supernatants compared with unfractionated PBMC (data not shown). The relative efficiency of the different bacteria, as well as the different bacterial doses, were identical using PBMC or monocytes.

Production of total IL-12 (p40 + p70) versus bioactive IL-12 (p70)

In addition to the functional p70 heterodimer, free p40 chain of IL-12 is produced in response to certain stimuli. We were therefore interested in whether the pattern of secretion of functional IL-12 p70, versus p40, differed between lactobacilli and E. coli. One high inducer of IL-12 p70 (L. paracasei 34D) and one of the low inducers of p70 (L. rhamnosus 50A) as well as E. coli O6:K13:H1 were used in different concentrations to stimulate PBMC from one donor. The supernatants were tested with an ELISA that measures both p40 and p70. The relative responses to the various bacteria and bacterial concentrations were the same, whether only p70, or both p40 and p70, were measured. Thus, L. paracasei induced the highest levels followed by L. rhamnosus, whereas E. coli gave rise to the lowest levels (Table 2).

Table 2
Secretion of bioactive (p70) versus total (p40 + p70) IL-12 in response to lactobacilli and E. coli

Production of IL-10 by PBMC and monocytes in response to lactobacilli or E. coli

IL-10 responses were maximal on day 1 after stimulation with bacteria or PHA (data not shown). Escherichia coli was a potent inducer of IL-10, triggering high levels (500 pg/ml) at a concentration of 5 × 106 bacteria/ml (2.5 bacteria/mononuclear cell). Equally high levels of IL-10 were obtained with the two L. rhamnosus strains, but optimal responses required 10 times higher bacterial concentrations, 5 × 107/ml (Fig. 2). Lactobacillus plantarum or L. paracasei induced approx. 100 pg/ml of IL-10, a bacterial concentration of 5 × 106/ml or 5 × 107/ml being equally efficient (Fig. 2). The response to 5 × 107 bacteria/ml of L. rhamnosus was significantly higher than that to the same amounts of L. plantarum or L. paracasei (P < 0.001 for both).

Fig. 2
Concentration of IL-10 in supernatants from 24 h mononuclear cell (MNC) cultures from nine blood donors stimulated with whole killed lactobacilli or Escherichia coli, or with phytohaemagglutinin (PHA; 10 μg/ml). Bacterial concentrations ranged ...

Monocytes purified by adherence produced roughly half as much IL-10 as did whole PBMC. The dose dependence as well as the relative efficiency of the seven bacterial strains tested was, however, identical for monocytes and PBMC (data not shown).

The effect on IL-12 production of inhibition of IL-10

IL-10 has been shown to suppress the production of IL-12 [24]. We therefore tested whether blocking of IL-10 would permit an increased production of IL-12 from blood mononuclear cells when stimulated with the different bacterial species. Indeed, the amounts of IL-12 induced by L. rhamnosus 7D increased two-fold after inhibition of IL-10 (P = 0.102). Conversely, the production of IL-12 after stimulation with E. coli O6:K13:H1 was minimal, even in the presence of blocking antibodies against IL-10 (Fig. 3).

Fig. 3
IL-12 concentrations in supernatants from blood mononuclear cells stimulated for 24 h with Lactobacillus rhamnosus 7D or Escherichia coli O6:K13:H1 in the presence of 5 μg/ml of neutralizing antibodies to IL-10 or control IgG1 antibodies. The ...

DISCUSSION

The results of the present study show that lactobacilli of the type colonizing the human gastrointestinal mucosa are potent inducers of IL-12. All three species dominant on the human mucosa, L. plantarum, L. rhamnosus and L. paracasei ssp. paracasei [3], triggered high levels of IL-12 from human blood mononuclear cells. Regardless of whether they were isolated from the oral or rectal mucosa, the two strains from each species behaved similarly. Among the lactobacilli, L. paracasei was the most efficient inducer of IL-12, but all the tested lactobacilli were much more potent inducers of IL-12 than E. coli.

Monocytes seemed to be the major type of cell producing IL-12 in the blood. Monocytes retrieved from the blood mononuclear cells by adherence gave approximately four-fold higher IL-12 levels in the supernatant than unfractionated mononuclear cells, despite the fact that we extracted only roughly 70% of the CD14+ monocytes from the mononuclear cells by the adherence procedure. Thus, IL-12 could be consumed by cells in the non-adherent population, e.g. T cells, NK cells or B cells. Alternatively, certain cell types in the blood might down-regulate the production of IL-12 by monocytes. A third possibility is that the adherence procedure primes for IL-12 production.

Regarding IL-10, stimulation with L. rhamnosus gave rise to much higher levels than L. paracasei and L. plantarum. Lactobacillus rhamnosus gave similar levels of IL-10 to E. coli, but 10 times more bacteria than with E. coli were required for maximal responses. Thus, with the exception of L. plantarum, which induced relatively low levels of both IL-12 and IL-10, the other bacteria showed an inverse relation between IL-12 and IL-10 inducing capacity. Accordingly, IL-10 has been shown to suppress IL-12 formation in vitro [24]. Indeed, when a blocking anti-IL-10 MoAb was added directly to the cultures, the IL-12 levels in cultures stimulated with lactobacilli rose two-fold. In contrast, blocking of IL-10 caused no increase of IL-12 production in E. coli-stimulated cultures. The effectiveness of the antibody was shown in concomitant experiments, where it was shown to strongly increase IFN-γ production in cultures stimulated with E. coli (unpublished observations).

The above results suggest that lactobacilli contain certain components that trigger the production of IL-12 which are absent, or only present in low amounts, in E. coli. Another possible reason for the inability of E. coli to induce IL-12 in the presence of blocking anti-IL-10 antibody is that E. coli induce other down-regulating factors, i.e. prostaglandins [27]. Candidates for stimulation of IL-12 include lipotheichoic acid (LTA), which is found only in Gram-positive bacteria [1], and peptidoglycan, which is present in much larger amounts in Gram-positive than Gram-negative bacteria. Whether Gram-positive bacteria are generally better in stimulating IL-12 than Gram-negative bacteria remains to be tested. Support for this notion derives from the observation that Gram-positive staphylococci are much better stimulators of IL-12 than E. coli lipopolysaccharide (LPS) or mycobacteria [28] and that the Gram-positive pneumococci give rise to 30 times more IL-12 than the Gram-negative Haemophilus influenzae [29,30].

IL-12 enhances the cytotoxicity mediated by NK cells against tumours [17], which may be of relevance to the capacity of certain lactobacilli strains to promote anti-tumour functions in mice [9,31]. IL-12 also stimulates macrophages, through the action of IFN-γ [17], which may be one reason why ingestion of lactobacilli leads to a transient up-regulation of phagocytic function of blood mononuclear cells [10], and why administration of lactobacilli to rats with galactosamine-induced liver damage promotes clearance of bacteria from the blood [32]. We have also noted signs of T cell activation in the intestinal mucosa in vivo in response to colonization with lactobacilli. Thus, germ-free rats colonized with L. plantarum and E. coli showed increased numbers of cells expressing the IL-2 receptor (CD25) in the intestinal mucosa, compared with rats colonized with E. coli alone [5]. IL-12 decreases IL-4 production [33]. Accordingly, certain lactobacilli suppress IgE production in vivo and in vitro [3436].

Lactobacilli form part of the mucosal flora of the gastrointestinal tract from the mouth to the rectum. In the large intestine, lactobacilli are found in equal numbers to E. coli and in higher numbers than enterococci [3]. In the small intestine they probably constitute an even larger part of the bacterial population, since enterobacteria and anaerobes are scarce at this site. Since certain lactobacilli can adhere to human intestinal epithelial cells [3,25] and are able to translocate over the gut barrier [4,5], they occupy a position favourable for interaction with cells in the gut-associated immune system. One of the reasons why they scarcely cause disease might be their potent ability to induce the production of IL-12 and thereby activate macrophages to a more effective clearance of the bacteria, decreasing their infectivity. It is likely that an induction of IL-10 (as seen with E. coli especially) would be a more effective strategy by bacteria to evade host defences, since IL-10 down-regulates inflammation [1921].

Lactobacilli, of the same types as those found on the intestinal mucosa, are also found in high numbers in fermented foods, including wine, olives and cheese [37]. Such fermented foods constituted, until recently, a large part of the human diet. Upon ingestion of fermented foods, lactobacilli may be taken up via the Peyer's patches and trigger cytokine production. Cytokines induced by lactobacilli colonizing the gastrointestinal tract, or present in the blood, may thus be important regulators of the gut-associated immune system.

Note added in proof

Recently Miettinen et al. demonstrated induction of IL-12 p70 production by PBMC after stimulation with live lactobacilli. Infect Immun 1998; 66:6058–62.

Acknowledgments

The skilful technical assistance of Ingela Kinell and Eva Ågren is greatly appreciated. The study was supported by grants from the Swedish Council for Forestry and Agricultural Research and the Swedish Medical Research Council (no. K98-06X-12612-01A). We thank Siv Ahrné and Göran Molin, Department of Food Technology, for supplying the lactobacilli strains and Ian Roberts for providing the E. coli MS101 strain.

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