Streptococcus pyogenes and Lactobacillus rhamnosus differentially ...

Published on February 13, 2004 as DOI:10.1189/jlb.1003461

Streptococcus pyogenes and Lactobacillus rhamnosus differentially induce maturation and production of Th1-type cytokines and chemokines in human monocyte-derived dendritic cells Ville Veckman,1 Minja Miettinen, Jaana Pirhonen, Jukka Sire´n, Sampsa Matikainen, and Ilkka Julkunen Department of Microbiology, National Public Health Institute, Helsinki, Finland

Abstract: Dendritic cells (DCs) are the most efficient antigen-presenting cells and thus, have a major role in regulating host-immune responses. In the present study, we have analyzed the ability of Gram-positive, pathogenic Streptococcus pyogenes and nonpathogenic Lactobacillus rhamnosus to induce the maturation of human monocyte-derived DCs. Stimulation of DCs with S. pyogenes resulted in strong expression of DC costimulatory molecules CD80, CD83, and CD86 accompanied with a T helper cell type 1 (Th1) cytokine and chemokine response. S. pyogenes also induced interleukin (IL)-2 and IL-12 production at mRNA and protein levels. In addition, IL-23 and IL-27 subunits p40, p19, p28, and EBI3 were induced at mRNA level. In contrast, L. rhamnosus-stimulated DCs showed only moderate expression of costimulatory molecules and produced low levels of cytokines and chemokines. Furthermore, no production of IL-2 or IL-12 family cytokines was detected. Bacteriainduced DC maturation and especially cytokine and chemokine production were reduced when bacteria were heat-inactivated. Our results show that human monocyte-derived DCs respond differently to different Gram-positive bacteria. Although pathogenic S. pyogenes induced a strong Th1-type response, stimulation with nonpathogenic L. rhamnosus resulted in development of semi-mature DCs characterized by moderate expression of costimulatory molecules and low cytokine production. J. Leukoc. Biol. 75: 000 – 000; 2004.

INTRODUCTION

tosis, and induction of cytokine and chemokine production [1]. As a result of the maturation process, DCs exit peripheral tissues and migrate to secondary lymphoid organs, where antigen presentation to T cells takes place [2]. The type of maturation stimulus as well as cytokines and chemokines produced by the DCs dictates whether adaptive immune response is directed toward T helper cell type 1 (Th1), Th2, or tolerance. Interleukin (IL)-12 is an important Th1 skewing cytokine, which is composed of p35 and p40 subunits. Recently, two new IL-12 cytokine family members have been described. IL-23 is a heterodimer of p19 and p40 subunits, and it induces the proliferation of memory T cells [3]. IL-27 consists of p28 and EBI3 subunits, and it induces the proliferation of naı¨ve T cells [4]. Currently, little data exist about the ability of bacteria to induce the expression of IL-23 or IL-27. Another key cytokine of the adaptive immune response is IL-2, which induces the proliferation of T and natural killer (NK) cells. Previously, it has been thought that IL-2 is only produced by lymphocytes. However, recent reports indicate that DCs are also able to produce IL-2 in response to microbial stimulation [5, 6]. Streptococcus pyogenes or group A streptococcus is a human pathogen causing, e.g., erysipelas, myositis, and skin infections [7]. S. pyogenes produces several exoenzymes and exotoxins, which promote the pathogenesis of the bacteria [8]. Some of the streptococcal exotoxins are potent superantigens that induce spontaneous T cell activation, which can lead to streptococcal toxic shock syndrome [7]. We and others have reported that S. pyogenes induces Th1-type immune response in monocytes, macrophages, and streptococci-infected tissues [9 –11]. Lactobacillus rhamnosus GG is a nonpathogenic lactic acid bacterium, which is widely used in various probiotic products. There is in vitro and in vivo evidence that probiotic bacteria, especially L. rhamnosus, have immunostimulatory effects. In vivo effects include reduced susceptibility to atopy or allergy after administration of probiotic products

Dendritic cells (DCs) are professional antigen-presenting cells, which reside in an immature stage in peripheral tissues. Contact with an antigen, i.e., a bacterium or an allergen, induces a maturation process, which is accompanied by functional and phenotypical changes. These include enhanced expression of cell-surface costimulatory molecules, reduction of DC endocy-

1 Correspondence: Department of Microbiology, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland. E-mail: [email protected] Received October 8, 2003; revised December 23, 2003; accepted January 8, 2004; doi: 10.1189/jlb.1003461

Key Words: gram-positive bacteria 䡠 IL-2 䡠 IL-23 䡠 IL-27

Journal of Leukocyte Biology Volume 75, May 2004 1

Copyright 2004 by The Society for Leukocyte Biology.

[12, 13]. In addition, it has been shown that probiotic bacteria reduce inflammatory responses in milk-hypersensitive subjects [14]. The reduced susceptibility to atopy and allergy could result from the ability of probiotic bacteria to modulate immune response from Th2 to Th1 [13]. Despite these findings, the molecular mechanisms behind the immunostimulatory effects of probiotic bacteria have been poorly characterized. As DCs have a major role in regulating innate and adaptive immune responses, the proposed immunostimulatory effects of probiotic bacteria could result from an interplay between DCs and the bacteria. Currently, there are only few published observations about the interactions between lactic acid bacteria and DCs [15, 16]. In the present work, we have compared the ability of two Gram-positive bacteria, pathogenic S. pyogenes and nonpathogenic L. rhamnosus, to induce DC maturation, which was characterized by analyzing the expression of DC costimulatory molecules, cytokine, and chemokine production and DC endocytotic activity in response to bacterial stimulation. Our results indicate that S. pyogenes is a more potent stimulator of DC maturation than L. rhamnosus. Although S. pyogenes induced a Th1-type response, stimulation of DCs with L. rhamnosus resulted in a weak cytokine and chemokine response and only moderate expression of costimulatory molecules.

MATERIALS AND METHODS Bacterial strains S. pyogenes serotype T1M1 (IH32030), isolated from a child with bacteremia, was obtained from the collection of National Public Health Institute (Helsinki, Finland), and L. rhamnosus GG (American Type Culture Collection, Manassas, VA, 53103) was from Valio R&D (Helsinki, Finland). Bacteria were stored in skimmed milk at –70°C and passaged three times as described previously [17] before they were used in stimulation experiments. S. pyogenes was grown in TY medium supplemented with 0.2% glucose [18] and L. rhamnosus in MRS medium (Difco, Detroit, MI). For stimulation experiments, bacteria were grown to logarithmic growth phase, and the number of bacterial cells was determined by counting in a Petroff-Hauser counting chamber.

DC purification Monocytes were purified from freshly collected, leukocyte-rich buffy coats obtained from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki). Human peripheral blood mononuclear cells were isolated by a density gradient centrifugation over Ficoll-Paque gradient (AmershamPharmacia Biotech, Uppsala, Sweden) as described previously [19]. Mononuclear cells were collected, and monocytes were further purified by centrifugation over Percoll gradient (Amersham-Pharmacia Biotech). Percoll gradients of 34%, 47.5%, and 60% (v/v) were made by mixing Percoll with RPMI-1640 medium (Sigma Chemical Co., St. Louis, MO) supplemented with 0.6 ␮g/ml penicillin, 60 ␮g /ml streptomycin, 2 mM L-glutamine, 20 mM HEPES, and 10% fetal calf serum (FCS; Integro BV, Dieren, The Netherlands). Mononuclear cells were suspended to 34% Percoll solution, and the three Percoll layers were mixed. Cells were centrifuged at 1700 g for 35 min, and the top layer containing monocytes was collected. Next, cells were washed twice with serum-free RPMI-1640 medium with supplements as above, and the remaining T or B cells were depleted by using anti-CD3 and anti-CD19 magnetic beads (Dynal, Oslo, Norway). After beading, cells were washed once with RPMI-1640 medium and counted. Monocytes were allowed to adhere to plastic six-well plates (Falcon, Becton Dickinson, Franklin Lakes, NJ) for 1 h at 37°C in RPMI-1640 medium without FCS (2.5⫻106 cells/well). After incubation, nonadherent cells were removed, and the wells were washed with phosphatebuffered saline (PBS). Monocytes were allowed to differentiate to immature

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DCs for 6 days in RPMI 1640 with the same supplements as mentioned plus 10% FCS, 10 ng/ml recombinant human (rh) granulocyte macrophage-colony stimulating factor (Leucomax, Schering-Plough, Innishannon, Ireland), and 20 ng/ml rhIL-4 (R&D Biosystems, Abingdon, UK). Fresh media (1 ml/well) was added every 2 days. Cultured cells were CD1asemi, CD14–, CD80low, CD83–, and CD86semi, and they showed a typical DC morphology (data not shown).

Stimulation experiments To minimize inter-individual variation, all experiments were performed with cells obtained from three to four donors. Stimulation experiments were conducted in RPMI-1640 medium containing 10% FCS. The optimal stimulation dose of S. pyogenes and L. rhamnosus was determined to be 5:1 bacteria:DC ratio (see Fig. 1). In certain experiments, bacteria were heat-inactivated by boiling for 15 min. After stimulation, cells and cell-culture supernatants were collected and pooled. Cells were used for isolation of total cellular RNA or for flow cytometric analysis [fluorescein-activated cell sorter (FACS)]. Supernatants were stored at –20°C and used for cytokine and chemokine quantification by enzyme-linked immunosorbent assay (ELISA).

RNA isolation and analysis For isolation of total cellular RNA, stimulated cells were collected, washed once with PBS, and lysed in guanidinium isothiocyanate [20] followed by centrifugation through a CsCl cushion [21]. RNA was quantified photometrically, and samples containing equal amounts (10 ␮g) of total cellular RNA were size-fractionated on 1% formaldehyde-agarose gels and transferred to Hybond-N nylon membranes (Amersham-Pharmacia Biotech). To control equal sample loading, ethidium-bromide staining was used. The probes for CD80, CD83, and CD86 were cloned from cDNA obtained by reverse transcriptasepolymerase chain reaction (RT-PCR) using total cellular RNA from S. pyogenes-stimulated DCs as template. The primers used for CD80 cloning were 5⬘-ACGCTTGGATCCGGGAACACCTGGCTGAAGTGACG-3⬘ and 5⬘CAGGGCGGATCCTTTCCCTTCTCAATCTCTCATTC-3⬘. The CD83 primers were 5⬘-CTCCATGGATCCATTATCCTTGCTATGATGATGGT-3⬘ and 5⬘-TAAAAAGGATCCATCAACTTGGTATCCGTTTTACT-3⬘. The CD86 primers were 5⬘-GACGTTGGATCCAGCTTGTCTGTTTCATTCCCTGA-3⬘ and 5⬘ACTTTTGGATCCACGCTGGGCTTCATCAGATCTTTCAGG-3⬘. EBI3 and p28 probes were cloned from cDNA obtained by RT-PCR using total cellular RNA from Sendai-infected macrophages as template. The primers used for EBI3 cloning were 5⬘-GCTTGTAACGGATCCAGTACTTCA-3⬘ and 5⬘-ATTGCTCCTGGATCCTGCCGCCTG-3⬘. p28 was cloned with 5⬘-AGAGGAGCTGGATCCAGGACACCT-3⬘ and 5⬘-GGTGTCTGGGGATCCCCAAGGCCC-3⬘ primers. The IL-2 probe was PCR-cloned from full-length IL-2 cDNA [22] with 5⬘-AGTCTTGCACGGATCCCAAACAGTG-3⬘ and 5⬘-AAATTCTACAATGGATCCTGTCTCA-3⬘ primers. The probes for CCL19 and CCL20 were a kind gift from Dr. Zlotnik. The probes for p19, p35, and p40 have been described previously [23, 24]. The probes for Northern blot analysis were labeled with [␣-32P]deoxy-adenosine 5⬘-triphosphate (3000 Ci/mmol; Amersham-Pharmacia Biotech) using a random-primed DNA-labeling kit. Hybridizations were performed in Ultrahyb buffer (Ambion, Austin, TX). After hybridization, membranes were washed three times with 1⫻ saline sodium citrate/0.1% sodium dodecyl sulfate at 42°C for 30 min and once at 65°C for 30 min. Membranes were exposed to Kodak X-Omat AR films (Eastman Kodak, Rochester, NY) at –70°C with intensifying screens.

Cytokine- and chemokine-specific ELISAs Cytokine and chemokine levels from cell-culture supernatants were analyzed by the sandwich-ELISA method as described previously [10]. Tumor necrosis factor ␣ (TNF-␣), chemokine ligand 5 (CCL5)/regulated on activation, normal T expressed and secreted, CXC ligand 9 (CXCL9)/monokine induced by interferon-␥ (IFN-␥), and CXCL10/IFN-inducible protein 10 levels were determined with antibody pairs and standards obtained from BD PharMingen (San Diego, CA). IL-2, CCL19/macropahge-inflammatory protein-3␤ (MIP-3␤), and CCL20/MIP-3␣ levels were determined with a Duoset kit (R&D Biosystems) and IL-12 p70 levels with an IL-12 Elipair kit (BioSite, Ta¨ by, Sweden).

Flow cytometry (FACS) For FACS analysis, the cells from three to four blood donors were pooled after stimulation experiments. Cells were washed once with cold PBS, and nonspe-

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Fig. 1. The effect of bacterial dose on DC maturation. DCs were stimulated with indicated bacteria:DC ratios for 24 h. After stimulation, the cells from four different donors were pooled, washed, and stained with antibodies against CD80, CD83, and CD86. The expression of costimulatory molecules was analyzed by flow cytometry. Values represent mean fluorescence intensities (MFIs). Results from one representative experiment are shown. Dotted lines indicate respective isotype controls.

cific binding of antibodies was prevented by incubating cells with 2% FCS in PBS for 15 min. The expression of costimulatory molecules was analyzed by staining DCs with fluorescein isothiocyanate (FITC)-conjugated anti-CD80, anti-CD83, and anti-CD86 antibodies (Caltag Laboratories, Burlingame, CA). Respective FITC-conjugated mouse isotype controls were used. Cells were stained on ice for 35 min, washed twice with PBS ⫹ 2% FCS, and fixed with 2% paraformaldehyde for 15 min. Next, the cells were washed and suspended into PBS ⫹ 2% FCS. Cells were analyzed with FACScan using Cellquest software (Becton Dickinson).

FITC– dextran uptake DC endocytotic activity was analyzed by a FITC– dextran uptake method [25]. After stimulation, DCs were incubated for 2 h with 1 mg/ml FITC– dextran FD-40S (Sigma Chemical Co.). Next, DCs were collected, washed three times with PBS, and fixed with 3% paraformaldehyde for 15 min. FITC– dextran uptake was analyzed by flow cytometry as described above.

ized the effect of bacterial dose on the expression of CD80, CD83, and CD86. DCs were stimulated with increasing amounts of L. rhamnosus or S. pyogenes for 24 h, and the expression of costimulatory molecules was analyzed by flow cytometry. Stimulation with S. pyogenes induced the expression of CD83 and CD86 better than stimulation with L. rhamnosus (Fig. 1). The highest MFIs were observed in between 5:1 and 25:1 bacteria:DC ratios for L. rhamnosus and S. pyogenes. When bacterial dose was increased to 25:1 or higher, the number of dead DCs increased (data not shown). Bacteriainduced expression of costimulatory molecules did not further increase, even if the stimulation time was extended (data not shown). As result, 5:1 bacteria:DC ratio was selected for further experiments.

Streptococci- and Lactobacilli-induced cytokine and chemokine production RESULTS Effect of bacterial dose on DC maturation First, we determined an optimal bacterial dose for stimulation experiments. As maturation of DCs is accompanied with enhanced expression of costimulatory molecules, we character-

DC maturation is accompanied by the production of TNF-␣, CCL19, and CCL20 [1, 2]. Furthermore, the two latter chemokines characterize DC maturation stage, as CCL20 and CCL19 are produced by DCs at early and at late maturation stages, respectively. We were also interested in the ability of Gram-positive bacteria to induce the produc-

Veckman et al. Streptococci- and Lactobacilli-induced DC maturation

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tion of IL-2, IL-12, CXCL9, and CXCL10, the key factors involved in proliferation, polarization, and chemotaxis of Th1 cells [26, 27]. To characterize the kinetics of bacteriainduced cytokine and chemokine production, DCs were stimulated with L. rhamnosus or S. pyogenes at a 5:1 bacteria:DC ratio. Cell-culture supernatants were collected at different time-points, and cytokine and chemokine levels were determined by ELISA. In S. pyogenes-stimulated DCs, TNF-␣, IL-2, CXCL10, and CCL20 production was detectable at 6 h, and IL-12, CCL5, CCL19, and CXCL9 production was measurable at 12 h after stimulation (Fig. 2). The production of all the cytokines and chemokines analyzed increased toward 24 h. In contrast to S. pyogenes, L. rhamnosus stimulation resulted in a detectable production of only TNF-␣ and CCL20 (Fig. 2). Moreover, L. rhamnosus-induced production of these cytokines and chemokines was very low compared with S. pyogenes-stimulated DCs.

Bacteria-induced mRNA expression of costimulatory molecules and CCL19 and CCL20 To further analyze Streptococci- and Lactobacilli-induced DC maturation, we studied the kinetics of bacteria-induced upregulation of CD80, CD83, CD86, CCL19, and CCL20 genes by Northern blotting. Stimulation of DCs with S. pyogenes resulted in enhanced mRNA expression of CD83 and CCL20 genes at 6 h after stimulation, and CD80, CD86, and CCL19 mRNA expression was enhanced after 12 h stimulation (Fig. 3). The mRNA expression of costimulatory molecules and of CCL20 remained elevated up to 48 h, whereas CCL19 mRNA expression peaked at 24 h and was down-regulated at 48 h. In L. rhamnosus-stimulated DCs, enhanced mRNA expression of CD83 was seen at 6 h after stimulation (Fig. 3). L. rhamnosus also induced CCL20 mRNA expression transiently at 6 –12 h after stimulation. No up-regulation of CD80, CD86,

Fig. 2. Bacteria-induced cytokine and chemokine production. DCs were stimulated with live L. rhamnosus or S. pyogenes at a 5:1 bacteria:DC ratio. Cell-culture supernatants were collected at indicated time-points, and cytokine and chemokine levels were detected by ELISA. Results are means of three individual experiments, each performed with cells from four different donors. Error bars represent standard deviations. Note the differences in scales.

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Effect of heat inactivation on bacteria-induced DC maturation

Fig. 3. Bacteria-induced mRNA expression of CD80, CD83, CD86, CCL19, and CCL20. DCs were stimulated with live L. rhamnosus or S. pyogenes at a 5:1 bacteria:DC ratio for times indicated in the figure. After stimulation, total cellular RNA was collected, and mRNA expression of CD80, CD83, CD86, CCL19, and CCL20 was analyzed by Northern blotting. Ethidium-bromide staining was used to control equal sample loading. Results from one of three experiments performed are shown.

or CCL19 genes was seen in L. rhamnosus-stimulated DCs compared with unstimulated DCs.

Our results showed a difference between live, nonpathogenic L. rhamnosus and pathogenic S. pyogenes in their ability to induce DC maturation. To analyze whether these differences were related to metabolic activity of the bacterium, i.e., to the production of extracellular compounds as has been shown for S. pyogenes [7, 8], we performed experiments with heat-inactivated bacteria and studied their effect on DC maturation. DCs were stimulated with live or heat-inactivated L. rhamnosus or S. pyogenes at a 5:1 bacteria:DC ratio for 24 h. After stimulation, the expression of costimulatory molecules, cytokine, and chemokine production and the degree of DC endocytosis were analyzed. L. rhamnosus-induced expressions of CD83 and CD86 were reduced by 62% and 33% by heat inactivation, respectively. The corresponding figures for S. pyogenes were 67% and 56% (Fig. 5A). L. rhamnosus- or S. pyogenes-induced increase in CD80 expression was low (Figs. 1 and 5A), but heat inactivation of bacteria completely prevented this enhanced CD80 expression (Fig. 5A). Heat inactivation almost completely abolished the ability of S. pyogenes to induce cytokine and chemokine production (Fig. 5B). The production levels of TNF-␣, IL-2, IL-12, CCL5, CXCL9, and CXCL10 were reduced in response to heat-inactivated S. pyogenes. The amounts produced in response to

Bacteria-induced mRNA expression of IL-2, IL-12, IL-23, and IL-27 Recent reports indicate that DCs are able to produce several important T cell-modulating cytokines. These include IL-2, IL-12, and the two novel IL-12 cytokine family members IL-23 and IL-27 [3–5]. Currently, there are no data concerning the ability of Gram-positive bacteria to induce the production of IL-2, IL-23, or IL-27 in human monocyte-derived DCs. Thus, we performed a Northern blot analysis. Stimulation of DCs with S. pyogenes resulted in enhanced IL-2 mRNA expression already after 3 h stimulation (Fig. 4A). S. pyogenes-induced IL-2 mRNA expression peaked at 6 and 12 h and was down-regulated at 24 h. No IL-2 mRNA expression was detected in unstimulated or L. rhamnosus-stimulated DCs. S. pyogenes-stimulated DCs showed transient p35 mRNA expression between 12 and 24 h (Fig. 4B). S. pyogenes was able to induce strong p40 mRNA expression already at 6 h. The p40 mRNA expression peaked at 12 h and was down-regulated but detectable at 48 h. S. pyogenes-induced p19 mRNA expression was detected at a low level after 6 h stimulation, and the p19 mRNA expression peak was at 48 h. S. pyogenes also induced p28 and EBI3 mRNA expression. The p28 mRNA expression peaked at 6 h and was down-regulated thereafter. The EBI3 mRNA expression was enhanced after 12 h S. pyogenes stimulation. L. rhamnosus stimulation resulted in detectable mRNA expression of only p40 and EBI3. The mRNA expression levels of these genes were very low compared with those of S. pyogenes-stimulated DCs.

Fig. 4. Bacteria-induced mRNA expression of (A) IL-2 and (B) members of the IL-12 cytokine family. DCs were stimulated with L. rhamnosus or S. pyogenes for indicated times, and total RNA was collected and analyzed by Northern blotting. IL-12 consists of p35 and p40 subunits, and IL-23 is a heterodimer of p19 and p40. IL-27 is composed of p28 and EBI3. Ethidiumbromide staining was used to control equal sample loading.


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Fig. 5. Effect of heat inactivation on bacteriainduced expression of DC costimulatory molecules. DCs were stimulated with live (UN), heat-inactivated (HI), L. rhamnosus (LR), or S. pyogenes (SP) for 24 h. (A) After stimulation, the expression of CD80, CD83, and CD86 was analyzed by flow cytometry. Values indicate MFIs. Results from one of three experiments performed are shown. (B) Effect of heat inactivation on bacteria-induced cytokine and chemokine production. Results are means of three individual experiments each performed with cells from four different donors. Error bars indicate standard deviations. Note the differences in scales. (C) Effect of heat inactivation on bacteria-induced DC endocytosis. DCs were stimulated for 24 h as described above, and FITC– dextran uptake was analyzed by flow cytometry. Results are presented as a percentage of fluorescence intensity (FITC– dextran uptake) compared with unstimulated, immature DCs (100%). Results are means of three individual experiments, and error bars represent standard deviations.

heat-inactivated S. pyogenes were similar to those induced by live L. rhamnosus (Fig. 5B). After antigen uptake, the endocytotic capacity of DCs gradually decreases [2]. Thus, the degree of endocytotic activity can be considered as a marker for DC maturation. To analyze the effect of L. rhamnosus or S. pyogenes stimulation on DC endocytosis, DCs were stimulated with live or heat-inactivated bacteria for 24 h. After stimulation, DCs were incubated with FITC– dextran for 2 h, washed, and analyzed by flow cytometry. Stimulation of DCs with live or heat-inactivated L. rhamnosus reduced DC endocytosis by 20% compared with unstimulated, immature DCs (Fig. 5C). When DCs were stimulated with live S. pyogenes, DC endocytosis was ⬃55% lower than in unstimulated DCs. Heat-inactivated S. pyogenes reduced DC endocytosis by 40%. The difference in DC endocytosis between live and heat-inactivated S. pyogenes was, however, not statistically significant. 6


DISCUSSION In the present study, we demonstrate that stimulation of monocyte-derived DCs with Gram-positive, pathogenic S. pyogenes or nonpathogenic L. rhamnosus results in distinct responses. S. pyogenes was able to induce efficient DC maturation, as characterized by enhanced expression of costimulatory molecules and reduction of DC endocytosis. In addition, stimulation of DCs with S. pyogenes resulted in the production of TNF-␣, IL-2, IL-12, CCL5, CCL19, CCL20, CXCL9, and CXCL10. S. pyogenes also induced the mRNA expression of the recently identified cytokines IL-23 and IL-27. In contrast to S. pyogenes, interaction of L. rhamnosus with DCs resulted in only a moderate up-regulation of costimulatory molecules and a very low production of TNF-␣ and CCL20. S. pyogenes stimulation induced a rapid and strong IL-2 production at mRNA and protein level. Transient IL-2 produchttp://www.jleukbio.org

tion has previously been described in murine DCs stimulated with Escherichia coli, yeast, or microbial components [5, 6]. DC-produced IL-2 has been shown to be important in enhancing T cell proliferation [5], and it has been speculated that DC-derived IL-2 could play a role in activating NK cells in the early phases of immune response [28]. Our results show that in addition to murine DCs, human monocyte-derived DCs are able to produce IL-2 in response to Gram-positive bacteria. However, significant differences between different bacterial species exist, as in contrast to S. pyogenes, no IL-2 was detected at mRNA or protein level in L. rhamnosus-stimulated DCs. The difference in the ability of S. pyogenes and L. rhamnosus to induce IL-2 production could result from a different ability to induce the activation of transcription factors. IL-2 gene expression has been studied thoroughly in T cells, where nuclear factor of activated T cells (NFAT) and activated protein-1 (AP-1) transcription factors have been shown to be crucial for IL-2 gene expression [29, 30]. As Cyclosporin A, a selective NFAT inhibitor, reduces IL-2 expression in DCs (ref. [6] and data not shown), it is likely that NFAT transcription factors, most likely in concert with AP-1, regulate IL-2 gene expression in DCs as well. However, further studies are required to characterize the regulation of IL-2 production in DCs. IL-12 has long been known to be an important cytokine in skewing T cell differentiation toward Th1 type. Recently, two other IL-12-related cytokines, designated IL-23 and IL-27, were described. The biological roles of IL-12, IL-23, and IL-27 are distinct but overlapping. IL-27 induces the proliferation of naı¨ve T cells, and IL-23 has been shown to activate memory T cells [3, 4]. It is currently known that human and murine DCs produce IL-23 and IL-27 in response to lipopolysaccharide challenge [3, 4]. However, no data on the ability of Grampositive bacteria to induce IL-23 or IL-27 exist. As in the case of IL-2 production, we found that only S. pyogenes was able to induce efficient mRNA expression of IL-12, IL-23, and IL-27 subunits in human monocyte-derived DCs. From the mRNA expression data, it could be interpreted that IL-12 and IL-27 are produced relatively early during S. pyogenes infection, and IL-23 is produced at later phases. Our results suggest that S. pyogenes-stimulated DCs may efficiently activate naı¨ve and memory T cells. S. pyogenes was also able to induce CXCL9 and CXCL10 production in DCs. These chemokines attract NK cells and Th1-polarized T cells, as both of these cell types express the chemokine receptor CXCR3 [27, 31, 32]. Thus, S. pyogenes-infected DCs are likely to recruit these cell types to the site of inflammation. As S. pyogenes-stimulated DCs also produce IL-2, IL-12, IL-23, and IL-27, it could be speculated that in vivo, these DCs create a cytokine milieu that polarizes adaptive immune response toward Th1 type. We have previously shown that stimulation of human mononuclear cells or macrophages with L. rhamnosus results in efficient cytokine and chemokine response. In these cells, L. rhamnosus induces the production of TNF-␣, IL-12, IL-18, IFN-␣/␤, and Th1-attracting chemokines CCL2 and CXCL10 [10, 11, 33]. As an indication of L. rhamnosus-induced DC maturation, we detected some enhancement of CD83 and CD86 expression and reduction of DC endocytosis. However, the L. rhamnosus-induced cytokine and chemokine response in DCs

was low. Only TNF-␣ and CCL20 were detected, and the production levels were significantly lower than in S. pyogenesstimulated DCs. These results are in line with recent observations by Christensen et al. and Braat et al. [15, 16]. In addition, we observed that L. rhamnosus was unable to induce the expression of IL-2, IL-12, IL-23, or IL-27. This most likely decreases the potency of L. rhamnosus-stimulated DCs to activate and polarize T cells. Heat inactivation almost completely abolished the S. pyogenes-induced cytokine and chemokine production. The difference between live and heat-inactivated bacteria in the ability to induce expression of DC costimulatory molecules was less dramatic. Moreover, the endocytotic activity of DCs was similarly reduced in response to live and heat-inactivated bacteria. Thus, live and heat-inactivated bacteria induced functional maturation of DCs but showed different ability to induce cytokine and chemokine gene expression. These results indicate that the metabolic activity of bacteria, at least in the case of S. pyogenes, may be required for efficient cytokine and chemokine production. One possibility is that heat inactivation prevents the production of streptococcal factors such as exoenzymes or exotoxins, which could play a role in enhancing DC activation. However, further experiments are required to clarify this observation. Another possibility is that the distinct responses between live and heat-inactivated bacteria resulted from heat-induced modification of bacterial cell structures. This could affect the ability of DCs to recognize bacteria by their pattern-recognition molecules and thus, the efficiency by which transcriptional pathways are activated in DCs. In the present study, we have shown that human monocytederived DCs distinguish between nonpathogenic and pathogenic Gram-positive bacteria and respond to them differently. Stimulation of DCs with L. rhamnosus leads to a moderate increase in the expression of cell-surface costimulatory molecules and to a weak cytokine and chemokine response. In contrast, S. pyogenes strongly induced DC maturation accompanied by high Th1-type cytokine and chemokine production. These different DC responses most likely result in the development of distinct adaptive immune responses as well.

ACKNOWLEDGMENTS This work was supported by the Medical Research Council of the Academy of Finland, the Finnish Cancer Foundation, the Sigrid Juselius Foundation, and the Maud Kuistila Foundation. We are grateful to Mari Aaltonen, Hanna Valtonen, and Teija Westerlund for expert technical assistance.

REFERENCES 1. Banchereau, J., Steinman, R. M. (1998) Dendritic cells and the control of immunity. Nature 392, 245–252. 2. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B., Palucka, K. (2000) Immunobiology of dendritic cells. Annu. Rev. Immunol. 18, 767– 811. 3. Oppmann, B., Lesley, R., Blom, B., Timans, J. C., Xu, Y., Hunte, B., Vega, F., Yu, N., Wang, J., Singh, K., Zonin, F., Vaisberg, E., Churakova, T., Liu, M., Gorman, D., Wagner, J., Zurawski, S., Liu, Y., Abrams, J. S., Moore, K. W., Rennick, D., de Waal-Malefyt, R., Hannum, C., Bazan,


7

4.

5.

6. 7. 8. 9. 10.

11.

12. 13. 14.

15. 16.

17. 18.

8

J. F., Kastelein, R. A. (2000) Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13, 715–725. Pflanz, S., Timans, J. C., Cheung, J., Rosales, R., Kanzler, H., Gilbert, J., Hibbert, L., Churakova, T., Travis, M., Vaisberg, E., Churakova, T., Liu, M., Gorman, D., Wagner, J., Zurawski, S., Liu, Y., Abrams, J. S., Moore, K. W., Rennick, D., de Waal-Malefyt, R., Hannum, C., Bazan, J. F., Kastelein, R. A. (2002) IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(⫹) T cells. Immunity 16, 779 –790. Granucci, F., Vizzardelli, C., Pavelka, N., Feau, S., Persico, M., Virzi, E., Rescigno, M., Moro, G., Ricciardi-Castagnoli, P. (2001) Inducible IL-2 production by dendritic cells revealed by global gene expression analysis. Nat. Immunol. 2, 882– 888. Granucci, F., Feau, S., Angeli, V., Trottein, F., Ricciardi-Castagnoli, P. (2003) Early IL-2 production by mouse dendritic cells is the result of microbial-induced priming. J. Immunol. 170, 5075–5081. Cunningham, M. W. (2000) Pathogenesis of group A streptococcal infections. Clin. Microbiol. Rev. 13, 470 –511. Schlievert, P. M. (1993) Role of superantigens in human disease. J. Infect. Dis. 167, 997–1002. Norrby-Teglund, A., Lustig, R., Kotb, M. (1997) Differential induction of Th1 versus Th2 cytokines by group A streptococcal toxic shock syndrome isolates. Infect. Immun. 65, 5209 –5215. Miettinen, M., Matikainen, S., Vuopio-Varkila, J., Pirhonen, J., Varkila, K., Kurimoto, M., Julkunen, I. (1998) Lactobacilli and streptococci induce interleukin-12 (IL-12), IL-18, and ␥ interferon production in human peripheral blood mononuclear cells. Infect. Immun. 66, 6058 – 6062. Veckman, V., Miettinen, M., Matikainen, S., Lande, R., Giacomini, E., Coccia, E. M., Julkunen, I. (2003) Lactobacilli and streptococci induce inflammatory chemokine production in human macrophages that stimulates Th1 cell chemotaxis. J. Leukoc. Biol. 74, 395– 402. Cross, M. L., Stevenson, L. M., Gill, H. S. (2001) Anti-allergy properties of fermented foods: an important immunoregulatory mechanism of lactic acid bacteria? Int. Immunopharmacol. 1, 891–901. Kalliomaki, M., Salminen, S., Arvilommi, H., Kero, P., Koskinen, P., Isolauri, E. (2001) Probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial. Lancet 357, 1076 –1079. Pelto, L., Isolauri, E., Lilius, E. M., Nuutila, J., Salminen, S. (1998) Probiotic bacteria down-regulate the milk-induced inflammatory response in milk-hypersensitive subjects but have an immunostimulatory effect in healthy subjects. Clin. Exp. Allergy 28, 1474 –1479. Christensen, H. R., Frokiaer, H., Pestka, J. J. (2002) Lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells. J. Immunol. 168, 171–178. Braat, H., De Jong, E. C., Van Den Brande, J. M., Kapsenberg, M. L., Peppelenbosch, M. P., Van Tol, E. A., Van Deventer, S. J. (2003) Dichotomy between Lactobacillus rhamnosus and Klebsiella pneumoniae on dendritic cell phenotype and function. J. Mol. Med. (E-pub ahead of print). Miettinen, M., Vuopio-Varkila, J., Varkila, K. (1996) Production of human tumor necrosis factor ␣, interleukin-6, and interleukin-10 is induced by lactic acid bacteria. Infect. Immun. 64, 5403–5405. Holm, S. E., F.E. (1967) An antigen free-free medium for cultivation of ␤-hemolytic streptococci. Acta Pathol. Microbiol. Scand. 69, 264 –267.


19. Pirhonen, J., Sareneva, T., Kurimoto, M., Julkunen, I., Matikainen, S. (1999) Virus infection activates IL-1 ␤ and IL-18 production in human macrophages by a caspase-1-dependent pathway. J. Immunol. 162, 7322–7329. 20. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., Rutter, W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294 –5299. 21. Glisin, V., Crkvenjakov, R., Byus, C. (1974) Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13, 2633–2637. 22. Taniguchi, T., Matsui, H., Fujita, T., Takaoka, C., Kashima, N., Yoshimoto, R., Hamuro, J. (1983) Structure and expression of a cloned cDNA for human interleukin-2. Nature 302, 305–310. 23. Pirhonen, J., Matikainen, S., Julkunen, I. (2002) Regulation of virusinduced IL-12 and IL-23 expression in human macrophages. J. Immunol. 169, 5673–5678. 24. Gubler, U., Chua, A. O., Schoenhaut, D. S., Dwyer, C. M., McComas, W., Motyka, R., Nabavi, N., Wolitzky, A. G., Quinn, P. M., Familletti, P. C., et al. (1991) Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor. Proc. Natl. Acad. Sci. USA 88, 4143– 4147. 25. Sallusto, F., Lanzavecchia, A. (1994) Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/ macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor ␣. J. Exp. Med. 179, 1109 –1118. 26. Heufler, C., Koch, F., Stanzl, U., Topar, G., Wysocka, M., Trinchieri, G., Enk, A., Steinman, R. M., Romani, N., Schuler, G. (1996) Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-␥ production by T helper 1 cells. Eur. J. Immunol. 26, 659 – 668. 27. Sallusto, F., Lenig, D., Mackay, C. R., Lanzavecchia, A. (1998) Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J. Exp. Med. 187, 875– 883. 28. Granucci, F., Feau, S., Zanoni, I., Pavelka, N., Vizzardelli, C., Raimondi, G., Ricciardi-Castagnoli, P. (2003) The immune response is initiated by dendritic cells via interaction with microorganisms and interleukin-2 production. J. Infect. Dis. 187 (Suppl. 2), S346 –S350. 29. Serfling, E., Avots, A., Neumann, M. (1995) The architecture of the interleukin-2 promoter: a reflection of T lymphocyte activation. Biochim. Biophys. Acta 1263, 181–200. 30. Rooney, J. W., Sun, Y. L., Glimcher, L. H., Hoey, T. (1995) Novel NFAT sites that mediate activation of the interleukin-2 promoter in response to T-cell receptor stimulation. Mol. Cell. Biol. 15, 6299 – 6310. 31. Bonecchi, R., Bianchi, G., Bordignon, P. P., D’Ambrosio, D., Lang, R., Borsatti, A., Sozzani, S., Allavena, P., Gray, P. A., Mantovani, A., Sinigaglia, F. (1998) Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187, 129 –134. 32. Inngjerdingen, M., Damaj, B., Maghazachi, A. A. (2001) Expression and regulation of chemokine receptors in human natural killer cells. Blood 97, 367–375. 33. Miettinen, M., Lehtonen, A., Julkunen, I., Matikainen, S. (2000) Lactobacilli and Streptococci activate NF-␬ B and STAT signaling pathways in human macrophages. J. Immunol. 164, 3733–3740.
