THE ACCESSORY ROLES OF LIPOPOLYSACCHARIDEACTIVATED MURINE B CELLS IN T CELL POLARIZATION XU HUI (MD, Peking University Health Science Center, PRC) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PAEDIATRICS NATIONAL UNIVERSITY OF SINGAPORE 2008 i Acknowledgements First and foremost, I would like to thank my supervisor, Professor Chua Kaw Yan for her guidance, encouragement and support; especially for the opportunity I was given for the systemic and formal training that will be greatly helpful for my future career development. I would also like to sincerely thank Dr. Teo Boon Wee Jimmy, for his understanding and giving me time to complete my thesis. I express my profound gratitude and blessings to Dr. Liew Lip Nyin, Dr. Lim Lay Hong Renee and Dr. Cheong Nge, who are the great advisors along the way and are generous to provide the fruitful and useful discussions over the past years. I would also like to thank Dr. Huang Chiung-Hui and Dr. Kuo I-Chun for their advice and support. I am very grateful to all my lab mates from Asthma and Allergy Research Laboratory Dr. Seow See Voon, Dr. Yi Fong Cheng, Dr. Tan Li Kiang, Miss Ding Ying, Miss Liew Lee Mei, Mdm Wen Hong Mei and Mr. Soh Gim Hooi, for their support in one way or the other. Last but not least, my deep gratitude goes to my husband, Kai Yu, for his endless inspiration, encouragement and support in the course of my study. I really appreciate him for being so understanding. To my two lovely sons, Ran Ran and Yuan Yuan, they make this hard journey joyful. To my parents and my sister, I am forever grateful and indebted to them for their dedication and trust. Xu Hui September 2008 ii List of Publications Publication derived from this thesis Xu H, Liew LN, Kuo IC, Huang CH, Goh LMD, Chua KY. The modulatory effects of lipopolysaccharide-stimulated B cells on differential T-cell polarizarion. Immunology. 2008; 125: 218-228. Publication in the related field Huang CH, Kuo IC, Xu H, Lee YS, Chua KY. Mite allergen induces allergic dermatitis with concomitant neurogenic inflammation in mouse. J Invest Dermatol. 2003; 121: 289293. iii Table of Contents Title page Acknowledgements Publications Tables of contents List of Figures List of Tables List of Abbreviations Summary i ii iii iv viii x xi xiii Chapter Introduction 1.1 Innate immunity 1.1.1 Dendritic cells (DCs), Toll-like receptors (TLRs) and pathogen-associated molecular patterns (PAMPs) 1.1.2 Lipopolysacharide (LPS) 1.1.2.1 History 1.1.2.2 Definition, components/structure and recognition 1.1.2.3 TLR4, RP105 and accessory molecules 1.1.2.4 LPS receptors association/cluster 1.1.2.5 TLR4 and LPS signaling pathway 1.1.3 Toll-like receptor (TLR) 3 11 13 1.2 Allergy and allergic response 1.2.1 Th1 cells, Th2 cells and Th17 cells 1.2.2 Regulatory T cells (Treg) 1.2.3 Innate control of Th1/Th2 cell differentiation/polarization 1.2.4 Differentiation of CD4+ T-cell subsets by cytokines 1.2.5 House dust mite allergens 1.2.5.1 Group allergens 1.2.5.2 Group allergens 15 17 18 21 22 23 24 25 1.3 Hygiene hypothesis 1.3.1 History 1.3.2 Cellular and molecular mechanisms 1.3.2.1 Dendritic cells (DCs) and Toll-like receptors (TLRs) 1.3.2.2 Endotoxin dose 1.3.2.3 Regulatory T cells (Tregs) 1.3.2.4 Cytokines 1.3.2.4.1 Cytokines that favor Th1 immune response 1.3.2.4.2 Cytokines that favor Th2 immune response 1.3.2.4.3 Cytokines that favor T regulatory immune response 27 27 29 30 32 33 34 34 35 35 1.4 Characteristics and functions of B cells 1.4.1 B cells and subsets 38 38 iv 1.4.2 The Antigen presentation function of B cells 1.4.3 Regulatory B cells (Breg) 43 47 1.5 Rationales and specific aims of the study 1.5.1 Rationales of the study 1.5.2 Specific aims of the study 60 60 63 Chapter Materials and Methods 64 2.1 Mice 2.2 Culture medium, antibodies and lipopolysacharride 2.3 Preparation of single cell suspension 2.4 Splenic B cells purification 2.5 Splenic cell cultures 2.6 B cell proliferation assays 2.6.1 Tritiated thymidine incorporation assays 2.6.2 Staining of splenic B cells with CFSE 2.6.3 BrDU incorporation assay 2.7 B cell surface molecule immunostaining 2.8 Analysis of cells population by flow cytometry 2.9 Detection of cytokine concentration by sandwich ELISA 2.10 Analysis of endocytotic capacity of B cells 2.11 Adoptive B cell transfer experiment 2.12 Assay of Th1/Th2 polarization by pulsed B cells (in vitro) 2.13 Assay of Th1/Th2 polarization by pulsed B cells (in vivo) 2.14 Total RNA extraction 2.15 RT-PCR 2.16 Quantification of cytokine gene expression level by conventional PCR 2.17 Preparation of the spent mite extracts 2.18 Preparation of native Der p by monoclonal antibody affinity purification 2.19 Preparation of recombinant Der p 2.20 Preparation of recombinant glutathione S-transferase (GST) 2.21 LPS concentration measurement 2.22 Removal LPS 2.23 Stain for Intracellular Cytokines 2.24 Data analysis 64 64 66 66 67 67 67 68 68 69 70 70 71 71 72 73 73 74 74 75 75 76 77 77 78 79 79 Chapter Modulatory Effects of Lipopolysaccharide Stimulated B Cells on Differential T Cell Polarization 81 3.1 Introduction 3.2 Results 3.2.1 Activation of B cells by varying doses of LPS 3.2.2 LPS stimulation enhances mannose-receptor mediated endocytosis and pinocytosis in B cells 81 87 87 88 v 3.2.3 Dose-dependent up-regulation of MHC II molecule, CD86, CD40, IgM and ICAM-1 but down-regulation of CD5 and CXCR4 expression on B cells by LPS 89 3.2.4 Dose-dependent effect of LPS on triggering B cell IL-10 production 90 3.2.5 Dose-dependent effect of LPS in conferring differential T cell polarizing capability on B cells 91 3.3 Discussion 115 Chapter Effects of House Dust Mite Allergens on Lipopolysaccharide Stimulated Murine B cells 124 4.1 Introduction 4.2 Materials and methods 124 127 Part I Combined Effects of nDer p and Lipopolysaccharide on Murine B cells 4.3 Results 4.3.1 Activation of B cells by nDer p allergen 4.3.1.1 Enhancement of B cells proliferation by nDer p allergen 4.3.1.2 Combination of nDer p and LPS could enhance B cell proliferation 4.3.1.3 Up-regulation of B cells surface markers by nDer p Allergen 4.3.1.4 B cells cytokine production induced by nDer p allergen 4.3.2 nDer p could enhance endocytosis and pinocytosis in B cells 4.3.3 nDer p 1-activated B cells acquired accessory function to prime antigen specific T cells 4.3.4 nDer p 1-activated B cells skewed Th2 cell polarization Part II Effects of LPS Contaminated rDer p on Lipopolysaccharide Stimulated Murine B cells 4.4 Results 4.4.1 B cells stimulated with LPS contaminated Der p polarized Th2 cells 4.4.2 Effect of clean rDer p on LPS-induced B cell proliferation 4.4.3 Effect of rDer p on LPS-induce B cell surface marker expression 4.4.4 Effect of rDer p on LPS-induce B cell cytokine production 4.4.5 Accessory functions of B cells co-stimulated with low dose of rDer p and low dose of LPS 129 129 129 129 130 131 131 132 132 133 135 135 135 136 137 138 139 4.5 Discussion 178 Chapter Conclusions and Future Prospective 5.1 Conclusion 5.2 Future perspective 190 190 194 vi Chapter References 199 Appendix: Publication 225 vii List of Figures Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 1.6 Figure 1.7 Figure 1.8 Figure 1.9 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Figure 4.10 Figure 4.11 Figure 4.12 Figure 4.13 Structure of Escherichia coli LPS Components of the TLR4–MD2–CD14 receptor complex LPS processing, signaling and clearance Structure of RP105/MD-1 and TLR4/MD-2 Hypothetical model for the immune recognition of bacteria GPCR signaling components regulate TLR signaling The signaling pathways of Toll-like receptor (TLR) 4–MD-2 Differentiation of CD4+ T-cell subsets Sequence alignment of MD-2 homologues and proteins with highest fold recognition score 49 50 51 52 53 54 55 56 Proliferative response of B cells to LPS stimulation Proliferative response of B cells to LPS stimulation Morphological changes of B cells after stimulation with different doses of LPS Effect of LPS stimulation on B cell’s antigen internalization Expression profiles of co-stimulatory and accessory molecules on LPS-stimulated B cells Cytokine production of B cells stimulated with LPS Cytokine profiles of CD4+ DO.11.10 T cell co-cultured with activated B cells 94 95-97 57 98-100 101-3 104-11 112 113-4 nDer p induced B cells to proliferate in a dose-dependent manner 142 Proliferative responses of B cells to nDer p and LPS 143-4 B cells proliferation induced by nDer p 145 Activation of B cells by nDer p 146 Cytokine mRNA expression profile of B cells stimulated with nDer p 147 Effects of nDer p stimulation on antigen internalization of B cells 148 In vivo functional assay of nDer p or rGST-pulsed B cells in BALB/c mice 149-50 In vitro accessory functional assay of nDer p 1-pulsed B cells 151-2 co-cultured with CD4+ T cells from DO11.10 mice In vivo accessory functional assay of nDer p 1-pulsed B cells in DO11.10 mice 153-4 In vitro accessory functional assay of rDer p 2-pulsed B cells co-cultured with CD4+ T cells from DO11.10 mice 155-6 In vivo accessory functional assay of rDer p 2-pulsed B cells in DO11.10 mice 157-8 Proliferative response of B cells to the stimulation by LPS alone or LPS in combination with rDer p 159-61 Expression profiles of costimulatory and accessory molecules on LPS-treated B cells and the combination of rDer p and viii Figure 4.14 Figure 4.15 Figure 4.16 Figure 4.17 Figure 4.18 LPS-treated B cells Cell surface molecule CXCR4 expression of B cells stimulated with LPS alone or in combination with rDer p The cytokine profile of B cells stimulated with LPS alone or in combination with rDer p examined by ELISA The cytokine profile of B cells stimulated with LPS alone or in combination with rDer p examined by intracellular cytokine staining Statistical analysis of the cytokine levels produced by B cells stimulated with LPS alone or in combination with rDer p In vivo accessory functional assay of combined LPS and rDer p -pulsed B cells in DO11.10 mice 162-4 165-6 167-8 169-72 173-5 176-7 ix List of Tables Table 1.1 Table 1.2 TLRs, ligands and expression of TLRs on human immune cells Summary of mouse B cells subsets 58 59 Table 2.1 Primer sequences for cytokine genes 80 Table 4.1 Determination of LPS concentration in nDer p 1, rDer p and rGST 141 x Taylor A, Verhagen J, Blaser K, Akdis M, Akdis CA. 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Chua, Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 117597. Email: paecky@nus.edu.sg Senior author: Kaw Yan Chua Summary Lipopolysaccharide (LPS) is a major component of environmental microbial products. Studies have defined the LPS dose as a critical determining factor in driving differential T-cell polarization but the direct effects of LPS on individual antigen-presenting cells is unknown. Here, we investigated the effects of LPS doses on naive B cells and the subsequent modulatory effects of these LPS-activated B cells on T-cell polarization. The LPS was able to induce a proliferative response starting at a dose of 100 ng/ml and was capable of enhancing antigen internalization at a dose of lg/ml in naive B cells. Following LPS stimulation, up-regulation of the surface markers CD40, CD86, I-Ad, immunoglobulin M, CD54 and interleukin-10 production, accompanied by down-regulation of CD5 and CD184 (CXCR4) were observed in a LPS dose-dependent manner. Low doses (< 10 ng/ml) of LPS-activated B cells drove T helper type polarization whereas high doses (> 0Á1 lg/ml) of LPS-activated B cells resulted in T regulatory type cell polarization. In conclusion, LPS-activated B cells acquire differential modulatory effects on T-cell polarization. Such modulatory effects of B cells are dependent on the stimulation with LPS in a dose-dependent manner. These observations may provide one of the mechanistic explanations for the influence of environmental microbes on the development of allergic diseases. Keywords: accessory function; B cells; lipopolysaccharide; T helper type 2; T regulatory type Introduction Innate immune recognition relies on germ-line encoded receptors known as pattern recognition receptors, which recognize a wide range of molecular structures in groups of microorganisms, commonly known as pathogen-associated molecular patterns (PAMPs). A major component of the environmental microbial products is endotoxin or lipopolysaccharide (LPS). This represents one of the most well-studied PAMPs; it is an immunostimulatory product of Gram-negative bacteria that signals through Toll-like receptor (TLR-4).1,2 Active research focusing on the interactions of LPS and its TLRs expressed by the innate immune response cells such as dendritic cells (DCs), macrophages and B cells have been driven by the wellaccepted ‘hygiene hypothesis’, which proposes that the microbial environment interfaces with the innate immune system and modulates antigen-specific adaptive immune 218 responses in early life.3 Of these, exposure to LPS is an attractive candidate in this hypothesis because it may be the link between microbial exposures and the development of allergic diseases such as asthma and rhinitis through its effects on innate immunity and its subsequent impact on the adaptive immunity. Beside DCs and macrophages, B cells are the largest subset of leucocytes with the capacity to internalize, process and present antigen to the responding T cells. Therefore, B cells also serve as an important link between the innate and adaptive immune systems. In addition, B cells constitute a major lymphocyte subset in neonates and infants, and they are likely to play important roles in driving the development of the immune system in early life.4–8 The important contribution of antigen presentation of B cells to T-cell activation has been demonstrated both in vitro and in vivo. In vitro studies have shown that the capacity of activated B cells to activate naive CD4+ T Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 125, 218–228 LPS-activated B cell modulates T-cell polarization cells is almost as effective as that of DCs.9 In vivo studies using the absence or depletion of B cells in animal models have demonstrated a decrease in antigen-induced T-cell activation, implying that B cells may contribute to the level of T-cell priming.10–12 Lipopolysaccharide is a potent mitogen of B cells and the in vitro responses of B cells to LPS are extremely robust.13,14 Previous studies have shown that like myeloid and lymphoid tissue-resident DCs, B cells exhibit upregulation CD86, CD40, CD54, OX40L, immunoglobulin M (IgM) and major histocompatibility complex (MHC) class II molecules, as well as production of interleukin-10 (IL-10) upon stimulation with LPS.15–18 However, unlike their DC and macrophage counterparts, they not produce the T helper type (Th1) signature cytokine, IL-12.19 This distinct functional feature between B cells and DC subsets hints that B cells as antigen-presenting cells might play a part in initiating or maintaining antigen-specific Th2-skewed immunity. Indeed, studies by Secrist et al. had demonstrated that B-lymphocyteenriched populations preferentially induced antigenspecific CD4+ T cells to produce IL-4.20 Moreover, this notion is further supported by two independent studies showing that LPS-activated splenic B cells are able to initiate antigen-specific Th2-skewed responses in vitro using specific T-cell receptor (TCR) transgenic CD4+ T cells.21,22 Based on these parallels, the default hypothesis for the role of B cells as antigen-presenting cells is proposed in driving Th2-skewed immunity.23 Despite the potential importance of B cells as antigenpresenting and accessory cells for T-cell differentiation and polarization, little attention has been paid to B cells because most of the research work in the field had been focused on DC subsets, which are regarded as the master regulators dictating the T-cell responses. The aim of this study was to elucidate the accessory roles of LPS-stimulated splenic B cells in initiating/modulating T-cell immune responses. To the best of our knowledge, this work represents the first systematic study to examine the modulatory effects of LPS-stimulated murine B cells on differential T-cell polarization. Materials and methods specific pathogen-free conditions at the Satellite Animal Holding Unit, National University of Singapore. All mice used in these experiments were females, aged 6–8 weeks. Animal experiments were performed according to the guidelines set by the Institutional Animal Care and Use Committee, National University of Singapore. Culture medium, antibodies and LPS The culture medium used for all in vitro tissue cultures was RPMI-1640 (HyClone, South Logan, UT) supplemented with mM sodium pyruvate, mM L-glutamine, 100 U/ml penicillin, 100 lg/ml streptomycin (HyClone), 5Á5 · 10)2 mM 2-mercaptoethanol (Gibco BRL Life Technologies, Paisley, UK) and 10% heat-inactivated bovine calf serum (HyClone). The LPS levels in all the medium supplements, RPMI-1640, deionized water and bovine calf serum, were below 0Á09 EU/ml as assayed by Limulus amoebocyte lysate QCL-1000 (50-648U; Cambrex Bio Science Walkersville, MD). Allophycocyanin (APC)-conjugated anti-CD19 (clone 1D3), fluorescein isothiocyanate (FITC)-conjugated antiCD40 (clone 3/23), FITC-conjugated anti-CD54 (Clone 3E2), phycoerythrin (PE)-labelled anti-CD86 (clone GL1), PE-labelled anti-IgM (clone R6-60.2), PE-labelled antiCD184 (CXCR4) (clone 2B11/CXCR4), peridinin chlorophyll protein (PerCP) labelled anti-CD5 (clone 53-7.3), PerCP-labelled anti-I-Ad (clone AMS-32.1), rat antimouse IL-4 (clone BVD4-1D11), rat anti-mouse interferon-c (IFN-c; clone R4-6A2), biotinylated rat antimouse-IL-4 (clone BVD6-24G2) and biotinylated rat antimouse IFN-c (clone XMG1.2) antibodies were purchased from PharMingen (San Diego, CA). An FITC-conjugated anti-CD80 (clone 1G10) monoclonal antibody was obtained from Southern Biotech (Birmingham, AL). Rat anti-mouse tumour necrosis factor-a (TNF-a; clone AF-410-NA), rat anti-mouse IL-10 (clone JES052A5), biotinylated rat anti-mouse TNF-a (clone BAF410) and biotinylated rat anti-mouse IL-10 (clone BAF417) were purchased from R&D Systems (Minneapolis, MN). Lipopolysaccharide (Escherichia coli serotype 0111:B4; Sigma-Aldrich, St Louis, MO) was supplied as lyophilized c-irradiated powder, reconstituted in culture, and further diluted with culture medium to working concentrations. Mice Specific pathogen free BALB/cArc mice were purchased from the Centre for Animal Resources, Singapore and housed at the Animal Holding Unit, National University of Singapore. The BALB/cArc colonies were derived from the Animal Resources Centre, Western Australia. T-cell receptor transgenic C.Cg-Tg (DO11.10)10Dlo/J mice, which bear an I-Ad-restricted, TCR recognizing OVA323–339 peptide, were obtained from The Jackson Laboratories (Bar Harbor, ME) and were bred and maintained under Splenic B-cell purification Splenic B cells from naive BALB/cArc mice were purified by magnetic separation according to the manufacturer’s protocol. Briefly, · 107 splenocytes were resuspended in 90 ll running buffer [phosphate-buffered saline (PBS) containing 0Á5% bovine serum albumin (Sigma Aldrich) and mM ethylenediaminetetraacetic acid, followed by the addition of 10 ll anti-CD19 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 125, 218–228 219 H. Xu et al. incubated for 15–20 at 4°. After washing twice with running buffer, the cells underwent magnetic separation using AutoMACS (Miltenyi Biotec GmbH). The purified splenic B cells (> 95%, as assessed by flow cytometry analysis) were resuspended in culture medium for all in vitro cultures. CFDA-SE labelling of splenocytes Splenocytes were resuspended at · 107/ml in flow cytometry staining buffer (PBS containing 0Á1% bovine serum albumin). A mM stock solution of 5-(and-6)carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Molecular Probes, Eugene, OR) in dimethylsulphoxide was added to a final concentration of lM and incubated at 37° for 10 min. At the end of the incubation period, the cells were immediately washed three times with cold flow cytometry staining buffer. The cells were then resuspended in culture medium for in vitro cultures and LPSstimulated cell proliferation was examined at 24 and 48 hr by flow cytometry analyses using the FACSCalibur flow cytometer with CELLQUEST software (Becton Dickinson, Franklin Lakes, NJ). B-cell surface molecule analyses by flow cytometry Splenocytes (2 · 106) were cultured in ml/well in 24-well plate (Nalge Nunc International, Rochester, NY) in the presence or absence of varying concentrations of LPS (0Á1–10 000 ng/ml) at 37° in 5% humidified CO2 for 24 hr. Subsequently, the splenocytes were stained with APC-conjugated anti-CD19 in combination with either FITC-conjugated anti-CD40, FITC-conjugated anti-CD80, FITC-conjugated anti-CD54, PE-labelled anti-CD86, PE-labelled anti-IgM, PE-labelled anti-CD184 (CXCR4), PerCP-labelled anti-CD5 or PerCP-labelled anti-I-Ad monoclonal antibodies for 30 at 4°. After extensive washing with flow cytometry staining buffer, cells were spun down and resuspended in 0Á5–1 ml fixing buffer (1% paraformaldehyde in PBS) for flow cytometry analyses. Cytokine sandwich enzyme-linked immunosorbent assay The concentrations of IL-4, IFN-c, TNF-a and IL-10 in the culture supernatants were measured by specific solidphase sandwich enzyme-linked immunosorbent assay (ELISA). Mouse recombinant IL-4, IFN-c, TNF-a and IL-10 purchased from Pharmingen were used to generate the standard curves in the assays. The detection limits for IFN-c and TNF-a were 20 pg/ml whereas those for IL-4 and IL-10 were 10 and 40 pg/ml, respectively. The cytokine concentrations of each sample were calculated by converting the raw reading of optical density at 405 nm using the standard curves. 220 Antigen internalization assay Purified splenic B cells (2 · 106) were cultured in ml/ well in 24-well plates in the presence or absence of LPS (1 lg/ml) at 37° in 5% humidified CO2 for 12, 24, 48 or 72 hr. After extensive washing, the B cells were resuspended in culture medium and, · 105 B cells in 0Á1 ml culture medium were incubated with 25 lg/ml of FITCdextrans (Molecular Probes) or mg/ml lucifer yellow (Molecular Probes) at 37° for hr. Subsequently the B cells were washed with flow cytometry staining buffer and fluorescence intensity was measured using flow cytometry analyses. Determination of naive CD4+ DO.11.10 T-cell polarization by LPS-treated splenic B cells Purified splenic B cells (2 · 106) were cultured in ml/ well in 24-well plates in the presence or absence of varying concentrations of LPS (0Á1–10 000 ng/ml) at 37° in a 5% humidified CO2 incubator. After 24 hr, splenic B cells were harvested and washed thoroughly with PBS and resuspended in culture medium. The naive CD4+ T cells (95–98% purity as assessed by flow cytometry) from DO11.10 TCR transgenic mice were purified by positive sorting using anti-CD4 MicroBeads (Miltenyi Biotec) and AutoMACS. The coculture system was modified from the method of Hosken et al. study.21 Briefly, · 105 naive CD4+ DO11.10 T cells in the presence or absence of · 105 LPS-treated splenic B cells were coincubated in 0Á2 ml/well in 96-well, U-bottom plates in the presence or absence of 0Á2 lM OVA323–339 peptide (AnaSpec, Inc., San Jose, CA) at 37° in a 5% humidified CO2 incubator. In another set of experiments, the B cells were treated with 50 lg/ml mitomycin C (Roche Diagnostics GmbH, Mannheim, Germany) at 37° for 20 followed by three washes with PBS before use in the B/T-cell coculture experiment. After 48 hr, culture supernatants were collected for cytokine determination by ELISA. Results Proliferative profile of splenic B cells stimulated with various doses of LPS LPS can stimulate polyclonal mouse B-cell proliferation and is regarded as a mitogen for mouse B cells. To establish the dose–response and proliferative kinetics of LPSstimulated polyclonal B-cell activation, purified splenic B cells were labelled with CFDA-SE and stimulated with various LPS doses (0–10 000 ng/ml). The B-cell proliferation was analysed by flow cytometry. At 24 hr after stimulation LPS-stimulated B-cell proliferation was not observed. At doses of 0Á1–10 ng/ml, LPS-triggered B-cell proliferation was not obvious at 48 hr. However, LPS Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 125, 218–228 LPS-activated B cell modulates T-cell polarization LPS (ng/ml) 0·1 10 100 1000 10 000 104 CD19 24 hr 48 hr 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 98·53 96·51 95·13 74·87 93·23 35·25 44·92 104 1·47 3·49 4·87 6·77 25·13 55·08 64·75 100 10 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CFDA-SE Figure 1. Proliferative response of splenic B cells to lipopolysaccharide (LPS) stimulation. Purified splenic B cells from naive BALB/cArc were labelled with lm 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) for 10 min, washed thoroughly and resuspended in culture medium. The cells (2 · 106 cells) were then cultured in the absence or presence of varying concentrations of LPS (0Á1–10 000 ng/ml) for 24 and 48 hr and assayed for CFDA-SE levels by flow cytometry analyses. The numbers indicate the percentage of cells in the quadrants. These data are representative of two independent experiments. promoted prominent B-cell proliferation at doses above 100 ng/ml and the B-cell proliferative responses increased in a dose-dependent manner at 48 hr (Fig. 1). Dose-dependent up-regulation of MHC II molecule, CD86, CD40, IgM and CD54 but down-regulation of CD5 and CD184 (CXCR4) expression on B cells by LPS Lipopolysaccharide is known to provoke differentiation and maturation of subsets of immature DC with characteristics of high level expression of surface MHC II molecules, CD86, CD80 and CD40 molecules.24,25 Recent in vitro and in vivo studies have shown that the surface density of MHC II and CD86 molecules on DCs may play a key role in regulating the Th1/Th2 polarization.26,27 We analysed the surface expression profiles of MHC class II molecule I-Ad, CD86, CD80, CD40, CD5, CD184 (CXCR4), CD54 and membrane-bound IgM on in vitro LPS-stimulated CD19+ splenic B cells by flow cytometry. As shown in Fig. 2, 24 hr after LPS stimulation, there was marked, dose-dependent, up-regulation of surface expression of CD40, CD86, I-Ad, CD54 and membrane-bound IgM; whereas LPS could dose-dependently down-regulate CD5 and CD184 (CXCR4) expression. Detailed analysis revealed that CD5, CD184 (CXCR4) and membranebound IgM were more sensitive to LPS stimulation; expression levels began to change at a dose of 0Á1 ng/ml LPS. Elevation of surface expression of CD86, I-Ad and CD54 was observed only at a higher dose of LPS (1 ng/ml) and LPS doses of 10 ng/ml or more were required to up-regulate the surface expression of CD40. The expression levels of CD80 remained unaltered on B cells stimulated with various doses of LPS (data not shown). Dose-dependent effect of LPS on triggering B-cell IL-10 production Besides the overall signal strength delivered by the MHC/ TCR cognate interaction and the binding of costimulatory molecules/ligands of APCs and the responding T cells, cytokines such as IL-12, IFN-c, IL-4 and IL-6 produced by the activated antigen-presenting cells, natural killer cells, natural killer T cells, basophils, eosinophils, mast cells or cd T lymphocytes also play an essential role in modulating Th1/Th2 polarization.28–31 We therefore investigated the effects of a variety of LPS doses, ranging from 100 pg/ml to 10 lg/ml, on the splenic B-cell profile of cytokine production. After stimulation with LPS, the B-cell supernatants were analysed for TNF-a, IL-10, IL-6, IL-4, IFN-c and IL-12 using ELISA. Among these cytokines, only IL-10 was consistently produced by B cells following LPS stimulation. As shown in Fig. 3, B cells stimulated with LPS doses between 100 pg/ml and 10 ng/ml secreted low levels of IL-10 (threefold above the background level), while LPS doses of 100 ng/ml or above were able to stimulate B cells to produce enormous levels of IL-10 in a dose-dependent manner (between 10- and 30-fold the background level). The secretion of TNF-a by B cells following stimulation with low doses of LPS was detected inconsistently (data not shown). Cytokines IFN-c, IL-12, IL-6 and IL-4 were consistently undetectable in all the B-cell culture supernatants tested. LPS stimulation enhances mannose-receptor-mediated endocytosis and pinocytosis in B cells To investigate the effect of LPS on augmenting the antigen internalization ability of splenic B cells, uptake of lucifer yellow and FITC-labelled dextrans was employed to monitor the kinetics over a period of 72 hr after stimulation with lg/ml LPS. Twelve hours after LPS stimulation, splenic B cells began to exhibit incremental uptake of lucifer yellow with a mean fluorescence intensity (MFI) of 12Á4 compared with the background value of 10Á7 in non-stimulated splenic B cells. The endocytosis capability on lucifer yellow uptake reached a maximum 48 hr (MFI = 39Á4) after LPS stimulation (Fig. 4a), then decreased to an MFI of 29Á2 at 72 hr. Moreover, uptake of FITC-dextrans showed similar kinetics, which was Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 125, 218–228 221 H. Xu et al. Counts CD40 30 25 20 15 10 Counts LPS (ng/ml) 30 25 20 15 10 0·1 9·2 9·0 10 9·9 100 11·9 18·7 1000 10 000 23·0 24·1 100 101 102 103 104100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CD86 19·7 22·7 43·5 65·3 193·6 325·9 352·4 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Counts 40 I-Ad 30 236·9 246·6 345·7 513·7 1000·3 1135·0 1187·9 20 10 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Counts 40 CD54 30 30·0 31·7 38·5 46·4 68·7 76·7 76·1 20 10 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Counts 20 IgM 15 48·0 58·4 60·7 63·2 95·1 99·8 105·1 10 Counts CD184 Counts 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 30 25 20 15 10 60 50 40 30 20 10 23·7 16·6 13·8 12·8 9·6 9·1 7·3 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103104 100 101 102 103 104 100 101 102 103 104 CD5 49·4% 39·7% 33·0% 28·7% 20·7% 15·4% 13·6% 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 LOG Fluorescence Figure 2. Expression profiles of costimulatory and accessory molecules on lipopolysaccharide (LPS) -stimulated splenic B cells. Splenocytes from naive BALB/cArc were cultured in the absence or presence of varying concentrations of LPS (0Á1–10 000 ng/ml) for 24 hr. Surface expression levels of CD40, CD86, I-Ad, CD54, IgM, CD184 (CXCR4) and CD5 molecules on CD19+ cells were analysed by flow cytometry. The numbers inserted in the CD40, CD86, I-Ad, CD54, IgM and CD184 (CXCR4), histogram plots represent the mean fluorescence intensity (MFI) of that surface molecule. The numbers inserted in the CD5 histogram plots represent the percentages of B cells expressing surface CD5. Filled areas represent the respective isotype controls. These data are representative of three independent experiments. 1500 (a) 12 hr 10·7 12·4 24 hr 100 101 102 103 104 100 101 102 103 104 Counts 120 1200 (b) 39·4 29·2 100 101 102 103 104 100 101 102 103 104 3·5 80 600 4·6 100 101 102 103 104 300 6·7 100 101 102 103 104 10·6 9·3 100 101 102 103 104 100 101 102 103 104 LOG Fluorescence 0 0·1 10 100 LPS [ng/ml] 1000 10 000 Figure 3. Cytokine production of B cells stimulated with lipopolysaccharide (LPS). Purified splenic B cells (2 · 106 cells) from BALB/ cArc mice were cultured in the absence or presence of varying concentrations of LPS (0Á1–10 000 ng/ml) for 48 hr and the culture supernatants were collected for IL-10 detection by enzyme-linked immunosorbent assay. These data are representative of two independent experiments. 222 20·2 72 hr 900 Counts IL-10 [pg/ml] 48 hr Figure 4. Effect of lipopolysaccharide (LPS) stimulation on splenic B-cell antigen internalization. Purified splenic B cells (2 · 106) from BALB/cArc mice were cultured in the presence or absence of lg/ml LPS for 12, 24, 48 or 72 hr. The cells were then incubated with (a) lucifer yellow or (b) fluorescein isothiocyanate (FITC)-dextrans at 37° for hr. Filled area represents the non-stimulated B cells whereas the bold lines represent the LPS-stimulated B cells. The numbers inserted in the histogram plots are the mean fluorescence intensity (MFI) of LPS-stimulated B cells and the numbers shown on the top of the histogram plots are the MFI of unstimulated B cells. These data are representative of four independent experiments. Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 125, 218–228 LPS-activated B cell modulates T-cell polarization detectable at 12 hr after LPS stimulation; and reached a plateau 48 hr after LPS stimulation (MFI = 10Á6) (Fig. 4b). Dose-dependent effect of LPS in conferring differential T-cell polarizing capability on B cells The level of LPS exposure is known to modulate antigenspecific T-cell polarization via the TLR-4 signalling pathway in vivo and has been presumed to be mediated by DCs.32–37 To investigate whether splenic B cells, like DCs, would acquire similar Th-type polarizing capability after receiving a varied strength of LPS stimulus, an in vitro antigen-presenting cell/T-cell coculture system for assessing the T-cell phenotype profile was employed.21 Splenic B cells activated by increasing doses of LPS, ranging from 0Á1 ng/ml to 10 lg/ml, were cocultured with naive CD4+ T cells from DO.11.10 TCR transgenic mice in the presence of 0Á2 lM OVA323–339 peptide for 48 hr. Similar B/ T-cell cocultures without peptide were included as controls. As shown in Fig. 5, splenic B cells activated by 0Á1– 10 ng/ml LPS induced IL-4 production; however, when B cells received LPS stimuli of increasing strength (> 100 ng/ml) an inverse relationship in IL-4 production was observed (Fig. 5a, closed bar). On the other hand, both IFN-c (Fig. 5b, closed bar) and IL-10 (Fig. 5c, closed bar) steadily increased with increasing doses of LPS. In the absence of OVA323–339 peptide, the levels of (d) (c) 160 120 80 40 (e) IFN-γ [pg/ml] 8000 6000 4000 2000 T cells alone 0·1 3000 2500 2000 1500 1000 500 10 100 1000 10 000 LPS [ng/ml] 3000 (f) 1000 2500 800 2000 1500 1000 500 T cells alone 0·1 T cells 0·1 1000 alone LPS [ng/ml] 10 100 1000 10 000 LPS [ng/ml] 10 000 200 0·1 IL-10 [pg/ml] IFN-γ [pg/ml] (b) This study examined the accessory roles of LPS-activated murine splenic B cells in initiating/modulating T-cell 300 250 200 150 100 50 T cells alone IL-10 [pg/ml] Figure 5. Cytokine production of B-cell and CD4+ DO.11.10 T-cell cocultures. Purified splenic B cells were cultured in the absence or presence of varying doses of lipopolysaccharide (LPS; 0Á1–10 000 ng/ml) for 24 hr. The B cells were thoroughly washed and cocultured with naive CD4+ DO.11.10 T cells in the presence (closed bar) or absence (open bar) of 0Á2 lm OVA323–339 peptide for 48 hr (a,–c). In another set of experiments, the LPS-stimulated B cells were treated with mitomycin C before coculture with naive CD4+ DO.11.10 T cells in the presence of 0Á2 lm OVA323–339 peptide for 48 hr (d–f). The culture supernatants were measured for the levels of interleukin-4 (IL-4; a and d), interferon-c (IFN-c; b and e) and IL-10 (c and f) by enzyme-linked immunosorbent assay. T cells alone represent T cells cultured with OVA323–339 peptide without B cells. These data are the representative results of three independent experiments. Discussion IL-4 [pg/ml] IL-4 [pg/ml] (a) IL-10 produced were remarkably attenuated (Fig. 5c, open bar). These results suggested that both peptide-activated T cells and the B cells activated with high-dose LPS were capable of producing IL-10 in an LPS-dose-dependent manner. CD4+ DO11.10 T cells alone produced very low levels of IL-4, IFN-c and IL-10 in the presence of OVA323–339 peptide. To address the question of whether the lower levels of IL-4 in the B/T-cell cocultures could be the result of consumption of IL-4 by the proliferative B cells upon LPS stimulation, we used mitomycin C-treated B cells to perform the B/T-cell coculture experiments. The profile of cytokine production analysed by ELISA showed that in the absence of proliferative B cells, the levels of IL-4 produced by T cells cocultured with high-dose LPS-activated, mitomycin-C-treated B cells remained low, indicating that the low level of IL-4 production was not the result of the consumption of IL-4 by the proliferative B cells (Fig. 5d). The B/T-cell coculture experiments performed with LPSactivated B cells with or without mytomycin C treatment produced similar cytokine profiles, but the levels of cytokine produced were lower when mytocycin-C-treated B cells were used in the coculture experiments (Fig. 5d–f). 10 100 1000 10 000 LPS [ng/ml] Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 125, 218–228 T cells 0·1 1000 alone LPS [ng/ml] 600 400 200 T cells 0·1 1000 alone LPS [ng/ml] 223 H. Xu et al. immune responses. Our results showed that LPS induced up-regulation of CD40, CD86, MHC II, CD54 and IgM as well as down-regulation of CD5 and CD184 (CXCR4) in B cells in a dose-dependent manner (Fig. 2). B cells stimulated with 100–10 000 ng/ml LPS produced significant levels of IL-10, but the B cells stimulated with lowdose LPS (0Á1–10 ng/ml) produced IL-10 at basal levels (Fig. 3). Other cytokines, such as IL-12 and IL-6, were not detected. Importantly, the functional assays revealed that LPS-activated B cells acquired differential modulatory effects on the polarization of T cells. Such modulatory effects of B cells on T cells are dependent on the dose of LPS used for stimulation. Low-dose LPS-stimulated B cells (0Á1–10 ng/ml) were capable of driving Th2 polarization, as demonstrated by the production of high levels of IL-4, accompanied by low levels of IFN-c, by DO11.10 TCR transgenic CD4+ T cells. In contrast, high-dose LPSstimulated B cells (1–10 lg/ml) polarized T cells producing high levels of IFN-c and IL-10 (Fig. 5), resembling the T regulatory type (Tr1) phenotype.38 The surface antigen expression profiles of the LPS-activated B cells revealed down-regulation of CD5 and CD184 (CXCR4) in B cells that was dependent on the LPS dose (Fig. 2). CD5, a 67 000 Da molecular weight transmembrane glycoprotein, has been defined as an inhibitory receptor of B cells.39 CD5 negatively regulated the activation of B1a subsets of B cells. In addition, CD5 is a necessary molecule that maintains tolerance in anergic B cells.40 In this study, the dose-dependent down-regulation of CD5 expression after LPS stimulation may signal the activation of B cells. CD184 (CXCR4), a chemokine receptor, is found on a variety of B-cell subsets including the B-1, B-2 and pro-B cells.41,42 It has been shown that this receptor, mediated via CCL12, plays a role in promoting the migration of B cells to the secondary lymphoid organs.42,43 However, the recent discovery of CD184 (CXCR4) as a member of the putative LPS activation clusters, which include heat-shock protein 70, heat-shock protein 90 and growth differentiation factor in monocytes/epithelial cells, argues for an additional role of CD184 (CXCR4) in operating the TLR-4/LPS signalling pathway.44 To our knowledge, even though studies of monocytes and macrophages have been well illustrated both in vitro and in vivo;45,46 the down-regulation of CD184 (CXCR4) expression in B cells following LPS stimulation has not been reported. Recently Kishore et al. showed that CD184 (CXCR4) can exert local regulatory control over the TLR-4 signalling pathway, implying that there is cross-talk between TLR-4 and CD184 (CXCR4).47 Molecular signalling studies will be required to determine whether such cross-talk between G-protein-coupled receptor-mediated and TLR-mediated pathways exist to coregulate the activation and mediate the bimodal effects of the LPS-stimulated B cells on T cells as seen in our study.48 224 Interleukin-10 is a pleiotropic cytokine that modulates the interaction between innate and adaptive immune responses, but its role on T-cell responses remain ambiguous as reflected by conflicting findings of several studies.49 Indeed, the roles of IL-10 produced by DCs in innate immunity remain unclear despite extensive studies. Langenkamp et al. demonstrated that LPS-stimulated human monocyte-derived DCs produced IL-10 from hr post-stimulation and this production was sustained at least up to 24 hr.50 This IL-10 production preceded IL-12 production. Animal studies showed that IL-10 secreted by DCs has been associated with Th2 T cells.51,52 However, IL-10-secreting DCs were shown to be associated with the T regulatory phenotype in studies using a Bordetella pertussis infection model. The filamentous haemagglutinin of B. pertussis inhibited IL-12 production and stimulated IL-10 production by DCs, and then induced polarization of IL-10-producing T regulatory cells.53 Furthermore, it has been reported that signalling through TLR4 in response to B. pertussis activates IL-10 production by DCs and macrophages, which promotes IL-10-producing T cells.54 However, a recent study demonstrated that DCderived IL-10 is not required for the induction of Th1 or Th2 in vivo, but IL-10 could regulate the outcomes of a DC-driven immune response.55 In the B-cell scenario, the notion that antigen presentation by B cells preferentially drives the development of Th2 cells has been suggested by several studies.20,56 It was further suggested that the production of IL-10, but not of IL-12, by B cells is the critical determining factor for Th2 induction and polarization. Both CD5+ and CD5) naive murine B cells have been shown to have the capacity to produce IL-10 in response to LPS stimulation.15,18 However, the effects of LPS dose on these naive B cells and the subsequent modulatory effects of the LPS-induced activated B cells on T-cell polarization have not been fully elucidated. Our data derived from the LPS dosage studies showed that B cells stimulated with low-dose LPS (0Á1–10 ng/ml) produced only basal levels of IL-10 (Fig. 3) and these LPS-stimulated B cells were shown to drive Th2 polarization (Fig. 5). Whereas B cells stimulated with high-dose LPS (1–10 lg/ml) polarized T cells to produce high levels of IFN-c and IL-10 (Fig. 5), resembling the Tr1 phenotype.38 In previous studies on the role of B cells in regulating T helper cell differentiation,21,22 splenic B cells stimulated by high doses of LPS were used for coculture experiments with naive transgenic CD4+ T cells bearing TCRs specific for OVA323–339 or C5 peptide. Interestingly, both groups were able to demonstrate the differentiation of naive CD4+ T cells to Th2 cells by producing high levels of IL-4, the Th2 signature cytokine. In contrast, our data showed the acquisition of Th1-like T regulatory phenotype polarizing capacity by B cells following stimulation with high doses of LPS, which therefore challenged their hypothesis that a default pathway Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 125, 218–228 LPS-activated B cell modulates T-cell polarization was operated by B cells as antigen-presenting cells to initiate Th2 polarization.23 Our results suggested that LPS can exert a dose-dependent bimodal effect on splenic B cells, in which B cells stimulated with low-dose LPS were capable of driving Th2-skewed T-cell polarization whereas high-dose LPS-stimulated B cells drove Th1-like T regulatory cell polarization. Interferon-c-producing and IL-10-producing Th1-like T regulatory cells have been observed in a T-cell line specific for Borrelia burgdorferi57 and in animals infected with Toxoplasma gondii.58 Stock et al.59 reported that antigen-specific IFN-c- and IL-10-producing Th1-like regulatory cells induced by CD8a+ DCs could potently inhibit the development of airway hypersensitivity. More recently, Anderson et al.60 reported that the immune suppression seen in chronic cutaneous leishmaniasis was mediated by IL-10 produced by CD4+ CD25) Foxp3) Th1 cells. The roles of IL-10 produced by the Th1-like cells have been extensively reviewed by O’Garra and Vieira,61 but the molecular mechanisms that regulate the production of IL-10 by these Th1-like cells remain unclear. For example, Gerosa et al.62 reported that IL-12 could prime human T-cell clones to produce high levels of both IL-10 and IFN-c; but data derived from a T. gondii infection model demonstrated that production of IL10 by Th1 cells was independent of IL-12.58 Our unique finding that high-dose LPS-activated B cells were capable of polarizing IL-10- and IFN-c-producing Th1-like cells in the absence of IL-12 is interesting, but the cellular signals and molecular mechanisms involved remain to be elucidated. It is unlikely that such observations were the result of trace contamination by splenic residual DCs during B-cell sorting leading to the production of IL-12 in the B/T-cell coculture because the B cells used in this study were positively isolated by anti-CD19 MicroBeads with purity greater than 95%. It is also unlikely that contamination by CD4+ CD8) splenic DCs during positive isolation of DO11.10 CD4+ T cells contributed to such observations, because CD4+ CD8) splenic DCs are poor producers of IL-12.63 This notion is further supported by the evidence that only background levels of IFN-c or IL-4 were produced by CD4+ DO11.10 T cells in the presence of OVA323–339 peptide. In addition, although we cannot rule out the possible trace contamination of CD19+ DCs during B-cell sorting, these CD19+ DCs not express TLR-4 and hence are unable to respond to LPS stimulation.64,65 It is highly possible that additional signal(s) delivered by unknown cytokine(s) or accessory molecules from high-dose LPS-activated B cells might play an instrumental role in orchestrating the polarization of Tr1 cells. Nevertheless, like the current scenario seen in DCs, the cellular signals and molecular mechanisms that induced Tr1 cells by LPS-activated B cells, as observed in our study, remain to be elucidated. Future study will also include blocking experiments using anti-IL-10 neutralization antibody to determine whether the differential modulatory effects on T-cell polarization are mediated solely by the relative amounts of IL-10 produced by the LPS-stimulated B cells. The results from the current study may have some significant implications in physiological settings. In the context of systemic infection, the activation of antigenpresenting cells by pathogens is an essential step towards the generation of effective immune responses to ensure rapid clearance of pathogens by the host. As a counterregulatory mechanism to prevent the detrimental effects of an excessive inflammatory response linked to potent immune activation, the production of anti-inflammatory cytokines, such as IL-10, by antigen-presenting cells and T cells is essential.66 In this regard, the Th1-like Tr1 cells driven by B cells stimulated with high-dose LPS may play a significant role in maintaining a balance between protective immunity and detrimental immunopathology. The amount of LPS encountered by systemically infected individuals is difficult to quantify and, to the best of our knowledge, there is no such report has been published. It is also noteworthy that exposure to microbes can occur in the absence of infection. For example, endotoxin (LPS) is one of most commonly found non-viable microbial products in the living environment and is likely to play an important role in modulating immune responses to environmental allergens.67 Additionally, it is also well established that LPS frequently coexists with the major inhalant allergens.68 Allergen exposure is a major triggering factor associated with the development and persistence of Th2-mediated allergic diseases; however, the mechanisms for the initiation and development of such Th2 responses remain ambiguous. Animal and human studies have been carried out to elucidate the role(s) of LPS in initiating/maintaining allergen-specific T-cell immune responses. A study using a mouse model of allergic sensitization showed that low-dose endotoxin promotes Th2 responses to allergen.32 Furthermore, Alexis et al.69 reported that airways challenged with low doses of endotoxin resulted in a Th2-skewed airway inflammatory response in humans. Additionally, epidemiological studies have shown that the levels of exposure to LPS are inversely correlated to atopy and related allergic diseases. High-level exposure to environmental endotoxin in early life might protect against the development of atopy and allergic diseases,70 but the underlying mechanisms for such protective immunity against atopy remain unclear. Taken together, these studies imply that in the context of allergen sensitization and development of allergic diseases, the effect of LPS on the immune response depends on the level of exposure (low levels directing Th2 responses and high levels dampening these responses). The mechanism of this paradox of differential adjuvant activities associated with high- and low-dose LPS has yet to be fully resolved. We would like to propose that our observation Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 125, 218–228 225 H. Xu et al. that B cells activated by low doses of LPS drive T helper type (Th2) polarization, while those activated by high doses of LPS result in Tr1 cell polarization, may offer a possible mechanistic explanation for the opposing influences of low and high levels of environmental LPS on the development of allergic diseases. This notion is further supported by the observation that B cells represent a dominant population of antigen-presenting cells coexisting with DCs in the respiratory tract71 and that these respiratory tract antigen-presenting cells play an important immunomodulatory role in controlling the balance between tolerance and immunity in response to environmental antigens, such as pathogens or allergens, in the respiratory tract. In conclusion, our data demonstrate that LPS-activated B cells acquire differential modulatory effects on T-cell polarization. Such modulatory effects of B cells on T cells are dependent on the stimulation with LPS in a dosedependent manner. Low-dose LPS (< 10 ng/ml) stimulation of B cells results in Th2 polarization whereas high-dose LPS (> 0Á1 lg/ml) stimulation of B cells results in Tr1 polarization. Further studies are necessary to evaluate the cellular and molecular mechanisms of this phenomenon. Knowledge gained from such studies will shed new light on our understanding of the effects of LPS on the underlying responses of innate and adaptive immunity driving and modulating the development of the Th2-mediated allergic responses. Understanding of the signalling pathways involved in the interaction between B cells and LPS can potentially provide useful information for developing novel strategies to prevent allergy onset or to treat allergic diseases by therapeutic manipulation of the innate immune system. Acknowledgements We would like to thank Dr Bee Wah Lee for the critical reading of this manuscript. This study was supported by grant (NMRC/0364/1999) from the National Medical Research Council, Republic of Singapore. 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MHC restricted (Marrack P et al., 1986; Grey HM et al., 1989) Traditionally, Th cells can further differentiate into two types of < /b> effectors T cells, named Th1 and Th2 cells, based on their functional capabilities and lymphokine profiles Since the < /b> original findings of < /b> Th1/Th2 CD4+ T cells subsets by Mosmann TR et al., (Mosmann TR et al., 1986) the < /b> study of < /b> the < /b> Th1/Th2 CD4+ T cells dichotomy has become... not yet fully defined, it is clear that cytokines present during the < /b> initial proliferative phase of < /b> T cell activation have profound influence Naїve CD4+ T cells initially stimulated in the < /b> presence of < /b> IL-12 and IFN-γ tend to develop into Th1 cells, in part because IFN-γ inhibits the < /b> proliferation of < /b> Th2 cells (Maggi E et al 1992; Hsieh CS et al., 1993) By contrast, CD4+ T cells activated < /b> in the < /b> presence... shown to interact with the < /b> environments, modulating the < /b> protective effect of < /b> allergy (Eder W et al., 200 4b) In the < /b> recent report, Barr TA and colleagues assayed the < /b> distinct cytokines profiles from TLR-mediated stimulation of < /b> two types of < /b> APCs: B cells and DCs They highlighted the < /b> potentially unique nature of < /b> immune modulation when B cells acted as APCs (Barr TA, et al., 2007) Other TLRs such as TLR9,... exposure to antigen in a distinct immunological context (Bluestone JA et al., 2003) Depending on the < /b> method and mode of < /b> generation, aTreg can be classified as either T regulatory type 1 (Tr1) or Th3 cells (Chen Y et al., 1994; Groux H et al., 1996) Tr1 cells can be generated by activation in the < /b> presence of < /b> the < /b> immunomodulating cytokine IL-10 Tr1 cells secrete a pattern of < /b> cytokines distinct from that of < /b> Th1... T- cell subsets or nTreg The < /b> level of < /b> expression of < /b> CD25 by Chapter 1 Introduction 19 aTreg is variable, depending on the < /b> disease setting and the < /b> site of < /b> regulatory activity Of < /b> note, aTreg functions in vivo in a cytokine-dependent manner So, it is proposed that aTreg is distinguished from nTreg not by their origin (the < /b> thymus), but rather by the < /b> requirement for further differentiation as a consequence of.< /b> .. responding T cells or APCs (Shevach EM 2002; Bluestone JA et al., 2003) 1.2.3 Innate control of < /b> Th1/Th2 cell differentiation /polarization Factors that are responsible for the < /b> differentiation of < /b> naїve CD4+ T cells into a Th1 or Th2 polarization profile have been extensively investigated Strong evidence suggests that Th1 and Th2 cells do not derive from distinct lineage, but rather from the < /b> same T helper cell. .. O’Neill LA et al., 2000) In the < /b> signaling pathway downstream of < /b> the < /b> TIR domain, a TIR domain-containing adaptor, MyD88, is first characterized to play a crucial role MyD88 consists of < /b> a TIR domain in the < /b> C-terminal portion, and a death domain in the < /b> N-terminal portion MyD88 associates with the < /b> TIR domain of < /b> TLRs TIR domain-containing adaptor protein Chapter 1 Introduction 11 (TIRAP) is another adaptor molecule... presence of < /b> IL-4, especially when IL-6 is also present, tend to differentiate into Th2 cells This is because IL-4 and IL-6 promote the < /b> differentiation of < /b> Th2 cells IL-4 is the < /b> most dominant factor in determining the < /b> Th2 polarization in cultured cells (Maggi E et al., 1992) IL-4 or IL-10, either alone or in combination, can also inhibit the < /b> generation of < /b> Th1 cells (Manetti R et al., 1994; Rincon M et al.,... and it induces production of < /b> the < /b> neutrophil chemoattractant IL‑8 (CXCL8) by human airway smooth muscle cells (Dragon S et al., 2007) 1.2.2 Regulatory T cells (Treg) Having been long debated, the < /b> notion of < /b> suppressor T cells — renamed regulatory T cells (Treg) — is back on the < /b> map, but many questions remain regarding the < /b> nature of < /b> these regulatory cells It is now generally acceptable that Treg cells . and B cells. Most previous studies focused on DCs. The main objective of this study is to address the question from the B cell perspective. The first part of the study focused on the investigation. the innate arm of the immune system leads to the release of cytokines, resulting in a state of inflammation. For most infections, the innate immune system is sufficient to clear the infection;. classical protein toxin. His experiments led him to formulate the concept that V. cholerae harbored a heat-stable toxic substance that was associated with the insoluble part of the bacterial cell.