Lipoteichoic acid improves the capability of mast cells in the host defense system against bacteria

Objectives and design

We investigated the effects of microbial components on the uptake of microbes by mast cells (MCs), and studied the change in cytokine production in MCs after bacterial uptake.

Material or subjects

LAD2 human mast cells, cord-blood and peripheral-blood derived MCs were employed to analyze their surface molecule expression and cytokine generation by flow cytometry. Bacterial internalization in these MCs was observed by confocal microscopy and flow cytometry.

Results

Complement receptor 3 expression was augmented by LTA but not by PGN or 3CpG-oligodeoxynucleotide. LTA also enhanced the uptake of opsonized bacteria (over twofold augmentation). After bacterial uptake, MCs augmented the production of chemoattractant cytokines for neutrophils, while Th1 and Th2 cytokine production showed little or no change.

Conclusions

LTA increases the capability of the MC as a sentinel in the host immune response, and some bacterial components direct human MC function towards innate immunity after pathogen infection.     

Activation of mast cells by double-stranded RNA: evidence for activation through Toll-like receptor 3

BACKGROUND: Although mast cells (MCs) have been clearly implicated in innate immune responses involving bacteria, their ability to respond to viral infection is less clear. OBJECTIVE: Given that MCs increase at sites of inflammation and are located at surfaces where exposure to invading viruses may occur, we explored the ability of MCs to produce cytokines including type I IFNs after exposure to viruses and to polyinosine-polycytidylic acid (polyI:C), a synthetic mimic of viral double-stranded RNA, and characterized the receptors involved, if any. METHODS: Human peripheral blood-derived cultured MCs and 2 MC lines, Laboratory of Allergic Disease MC line and human MC line 1, were stimulated with viruses and polyI:C, and cytokine production, degranulation, and signaling pathway activation were examined. Because polyI:C is a ligand for Toll-like receptor (TLR)-3, human MCs were also analyzed for TLR expression. RESULTS: Viruses and polyI:C induced IFN-alpha and IFN-beta production. PolyI:C did not induce TNF, IL-1beta, IL-5, or GM-CSF production, in contrast with other TLR ligands (LPS, peptidoglycan, CpG-A, or flagellin). IFN-alpha production involved nuclear factor-kappaB, p38, and C-Jun NH2-terminal kinase and mitogen-activated protein kinase. RT-PCR and Western blot analysis confirmed expression of TLR-3 by all MCs. Human cultured MCs also expressed TLR-1, TLR-2, TLR-4, TLR-5, TLR-6, TLR-7 and TLR-9. Antibodies to TLR-3 significantly decreased IFN-alpha production. Bone marrow-derived MCs from TLR-3 knockout mice showed an ablated response to polyI:C. CONCLUSIONS: Murine and human MCs produce type I IFNs after exposure to double-stranded RNA and/or virus, the former via specific interactions with TLR-3. These data suggest that MCs contribute to innate immune responses to viral infection via the production of type I IFNs.     

Hepatic stellate cells primed with cytokines upregulate inflammation in response to peptidoglycan or lipoteichoic acid

Gram-positive bacterial products such as peptidoglycan (PGN) and lipoteichoic acid (LTA) are potent stimulators of innate inflammatory responses. We previously reported that lipopolysaccharide (LPS), a major biologically active agent of gram-negative bacteria, induces a proinflammatory response via the Toll-like receptor (TLR) 4 in hepatic stellate cells (HSCs). Here we investigated the mechanism of proinflammatory action by PGN and LTA in activated human HSCs. Following treatment with either TNF-alpha or IL-1beta, expression of TLR2 and CD14 was determined by real-time PCR and Western blotting. NF-kappaB activation was assessed by NF-kappaB-driven luciferase assay and electrophoretic mobility shift assay. Interleukin-8 (IL-8) from culture supernatant was measured by ELISA. Activated human HSCs express TLR2 and CD14, which are receptors for PGN and LTA signaling. TNF-alpha and IL-1beta significantly upregulated the expression of TLR2 mRNA and protein in HSCs. PGN and LTA induced NF-kappaB activation and stimulated production of IL-8 in HSCs. Pretreatment with TNF-alpha or IL-1beta augmented NF-kappaB activation and IL-8 production in response to PGN or LTA. Both PGN- and LTA-induced NF-kappaB activation and IL-8 secretion were completely inhibited by anti-TLR2 blocking antibody (T2.5). These findings suggest that TNF-alpha or IL-1beta primed HSCs enhance the production of IL-8 in response to PGN and LTA through augmentation of the TLR2 system.     

Altered function of murine mast cells in response to lipopolysaccharide and peptidoglycan

Toll-like receptors (TLRs) recognize and signal the presence of bacterial components such as lipopolysaccharide (LPS) and peptidoglycan (PG) as a part of innate immunity. Our previous studies revealed that mast cells function as effector cells in the protection of mice against lethal enterobacterial infections. In this study, we examined both the gene expression of molecules involved in TLR signaling and the effects of LPS and PG in bone marrow-derived cultured mast cells (BMCMCs). The mRNA expression of TLR2, TLR4 and TLR6 was detected in BMCMCs. CD14, MD-2 and MyD88, which are also involved in TLR pathway, were also expressed. Neither LPS nor PG affected degranulation in BMCMCs, but release of tumor necrosis factor increased slightly in response to LPS and PG. Both LPS and PG enhanced expression of pro-matrix metalloproteinase 9 (pro-MMP-9) in a dose-dependent manner, and DNA fragmentation was induced by LPS, but not by PG. These results suggest that mast cells are the targets of LPS and PG, and that the functions of these molecules produced exclusively by bacteria partly overlap, but are distinct.     

Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells

In the present report we have analyzed whether human normal cord blood-derived mast cells (CBMC) could interact with bacterial products, especially lipopolysaccharide (LPS) from Escherichia coli and peptidoglycan (PGN) from Staphylococcus aureus, known as Toll-like receptor (TLR) 4 and TLR2 agonists, respectively. We found that both LPS and PGN induced significant release of not only tumor necrosis factor-alpha (TNF-alpha), but also IL-5, IL-10 and IL-13 by human mast cells (MC). We also established that the stimulation of CBMC with LPS or with PGN is mediated through interactions with TLR4 or with TLR2, respectively. Thus, our data indicate that activation of either TLR2 or TLR4 pathway may lead to a pro-Th2 immune response. However, the release of TNF-alpha induced by LPS, conversely to PGN, required the priming of CBMC by IL-4 and the presence of serum components, in particular soluble CD14. Of interest, stimulation by PGN, but not by LPS, induced release of histamine by human MC. Altogether, these findings provide the first evidence that human MC differentially respond towards bacterial components, and that their responses depend on TLR pathways and reveal human specificities in the pattern of cytokine production.     

Differential mediator production by dendritic cells upon toll-like receptor stimulation does not impact T cell cytokine expression

Dendritic cells are key components of successful immunological responses bridging innate and adaptive defenses. In this study we wanted to know whether ligation of toll-like receptors (TLR) expressed by dendritic cells would induce differential proinflammatory mediator expression and whether these dendritic cells would differentially impact T cell function. For this purpose bone marrow-derived dendritic cells from OTII mice were used. The dendritic cells showed detectable levels of TLR1, 2, 4, 6, 7, 8 and 9, with TLR2 and TLR4 expressed at the highest levels. To determine whether TLR ligation differentially influenced proinflammatory mediator expression the dendritic cells were stimulated with peptidoglycan (PGN) or lipopolysaccharide (LPS) for TLR2 or TLR4, respectively. Comparisons were made to dendritic cells exposed to TNF-alpha or saline as controls. Whereas, both LPS and PGN were equally effective at inducing CXCL1 and TNF-alpha expression from the dendritic cells, LPS was unique at inducing CCL2 expression, and PGN was unique at inducing IL-1beta expression. Despite these differences, LPS and PGN treated dendritic cells were equally effective at eliciting IFN-gamma expression from T cells in an antigen-specific manner. These data indicate that ligation of TLR by components of Gram+ and Gram- bacteria differentially influence dendritic cell proinflammatory mediator expression, and that differential mediator production by dendritic cells upon TLR stimulation does not impact T cell cytokine production.     

Bacterial components regulate the expression of Toll-like receptor 4 on human mast cells

Objectives and design: The aim of this study was to investigate whether the exposure of mast cells (MCs) to bacterial components affects the expression of Toll-like receptor (TLR) 4, and to elucidate the behavior of MCs during the early response to infection. Materials: Two human MC lines, HMC-1 and LAD2, were employed. Messenger RNA expression was observed by RT and real-time PCR. TLR4 expression was determined by Western blotting. TNF-α secretion was analyzed with ELISA. The degranulation ratio was measured with betahexosaminidase assay. Results: Although bacterial components increased TLR4 mRNA, only lipopolysaccharide (LPS) augmented the TLR4 protein expression. LAD2 pre-treated with LPS for 8 h resulted in 2-fold increased TNF-α secretion on LPS restimulation. Conclusion: These results suggest that the exposure of MCs to LPS may reinforce the innate immune system due to up-regulation of MC TLR4, followed by increased TNF-α release. Received 20 April 2006; returned for revision 14 July 2006; accepted by G. Wallace 11 August 2006     

Critical roles of Toll-like receptors in host defense

Drosophila Toll is involved not only in dorsoventral patterning of embryos but also in immune responses to microbial infection. Several Toll-like receptors (TLRs) have also been identified in mammals. They are expressed on macrophages or dendritic cells (DCs), which are essential sentinels for innate immunity. These cells utilize TLRs as a recognition and signal transducing receptor for microbial molecular components. The most characterized mammalian TLR, TLR4, is a receptor for lipopolysaccharides (LPS). TLR2 recognizes other components, such as peptideglycans (PGN). This recognition, called pattern recognition, is essential for the establishment of innate immunity, which is the basis for host defense. In this article, we review recent findings about this expanding receptor family.     

Regulation of mast cell-mediated innate immunity during early response to bacterial infection

Summary Although the area of research on the role of MCs in innate immunity is relatively new, a number of studies that are reviewed here provide substantial evidence that MCs play a critical role in host immune defense against gram-negative bacteria. The studies show that mast cells have the ability to recognize and engulf bacteria and they release a number of inflammatory mediators including interleukin (IL)-4, IL-6, IL-10, TNFα, and leukotrienes in response to bacterial challenge. MC-derived TNFα and leukotrienes are shown to be important for bacterial clearance and early recruitment of phagocytic help at the site of infection. Studies directed at elucidating the molecular mechanisms associated with mast cell recognition of bacteria and subsequent events leading to mast cell mediator release revealed that GPI anchored CD48 molecule present on the cell surface of mast cells serves as a receptor for the bacterial adhesion molecule, FimH. The ligation of CD48 receptor by FimH-expresing bacteria results in bacterial uptake into caveolar chambers. This distinct mechanism of bacterial uptake promot es bacteral survival inside the cytosol of the mast cells. Although the exact mechanism(s) of how MC-dependent inflammatory responses are regulated is currently not known, recent studies have shown that complement, CD11β/CD18 (Mac-1) and protein tyrosine kinase JAK3, and TLR4 are important for the full expression of MC-dependent innate immunity in mice.     

TLR2和TLR4在人慢性根尖周病组织肥大细胞上的表达

 目的: 观察人不同类型慢性根尖周病组织中肥大细胞(mast cells,MCs)上Toll样受体2(Toll-like receptor 2,TLR2)和TLR4的表达情况,探讨TLR2-类胰蛋白酶(tryptase)和TLR4-tryptase双阳性MCs在慢性根尖周疾病发病机制中的作用。方法: 将60例自愿参与本研究的受试者按根尖周病分类标准分为3组:(1)正常对照组;(2)根尖周肉芽肿组;(3)根尖周囊肿组。将根尖周组织标本置于4%甲醛固定液中浸泡48 h以上,制作连续组织切片。HE染色,光学显微镜下观察各组根尖周标本的组织学变化;免疫荧光双染色后于荧光显微镜下观察TLR2-tryptase和TLR4-tryptase双阳性MCs在根尖周组织中的分布情况。结果: 根尖周病变组TLR2-tryptase和TLR4-tryptase双阳性MCs比正常牙周膜组明显增多(P<0.01);与根尖肉芽肿组相比,根尖囊肿组TLR2-tryptase及TLR4-tryptase双阳性MCs数目明显增高(P<0.01)。结论: TLR2及TLR4在慢性根尖周疾病组织中MCs上表达增加,提示TLR2-tryptase及TLR4-tryptase双阳性MCs可能参与慢性根尖周病发病过程的免疫调节。     

Mast cells play a crucial role in Staphylococcus aureus peptidoglycan-induced diarrhea

Bacterium-induced diarrhea results in 2 to 2.5 million deaths in the world each year. The mechanism needs to be further understood. Staphylococcus aureus infection has a close relation with diarrhea; its cell wall component peptidoglycan (PGN) has strong biological activity on immune cells and possibly plays a role in S. aureus-induced diarrhea. The present study showed that oral PGN-induced diarrhea in mice in a dose-dependent manner. Intestinal epithelial cells absorbed PGN via the intracellular pathway. Intestinal mast cells were activated after PGN gavage. Toll-like receptor (TLR)2 expression was detected in mast cells in the intestine as well as in the murine mast cell line p815 cells. Blocking TLR2 or nucleotide-binding oligomerization domain (NOD)1 with related antibodies or RNA interference abolished PGN-induced p815 cell activation. The mast cell mediator histamine and serotonin had synergistic effects in PGN-induced diarrhea. In summary, oral PGN can induce diarrhea in mice, and TLR2 and NOD1 mediate the PGN-induced mast cell activation that plays a critical role in diarrhea induction. Blockade of TLR2 or NOD1 or treating mice with a mast cell stabilizer can efficiently inhibit PGN-induced-diarrhea, providing potential therapeutic significance.     

Hyperexpression of FcgammaRI and Toll-like receptor 4 in the intestinal mast cells of Crohn's disease patients

We previously reported that human mast cells (MCs) express high affinity IgG receptor (FcgammaRI) and Toll-like receptor 4 (TLR4) in response to interferon (IFN)-gamma in vitro. The number of MCs is known to increase in Crohn's disease (CD) and ulcerative colitis (UC). We aimed to examine the expression and function of the receptors in these diseases by immunohistochemistry of the colonic mucosae and by in vitro experiments. The density of MCs expressing FcgammaRI, TLR4, or both proteins was significantly higher in CD than in UC or control samples. The density of TNF-alpha(+) MCs expressing FcgammaRI or TLR4 was significantly higher in CD than in control samples. LPS and IgG1 cross-linking synergistically induced a high level of TNF-alpha production in IFN-gamma-treated human MCs. Hyperexpression of FcgammaRI and TLR4 on MCs was related to the high frequency of TNF-alpha expression in CD, suggesting the activation of MCs via these receptors in vivo.     

Pulmonary innate immune proteins and receptors that interact with gram-positive bacterial ligands

The two major gram-positive bacterial (GPB) ligands are peptidoglycan (PGN) and lipoteichoic acid (LTA). These polymeric LTA and highly organized PGN contain repeating carbohydrate moieties, which are potential targets for pattern recognition molecules. The major pattern recognition proteins and receptors, which bind GPB, either have a lectin, PGN recognition, collagen or leucine-rich repeat (LRR) domain. The soluble innate immune proteins (IIPs) that bind to PGN and LTA include pulmonary collectins surfactant-associated proteins (SP-) A and D, lectin-like pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP), and sCD14. Membrane-anchored lectin or lectin-like group members include macrophage mannose receptor (MR), complement receptor 3 (CR3, or Mac-1, or integrin CD11b/CD18), scavenger receptor A (SRCL-1), lectin-like oxidized LDL receptor 1 (LOX-1), and GPI-anchored CD14. Although Toll-like receptor (TLR) 2 and 4, and CD14 contain extracellular LRR domains, only TLRs have a cytoplasmic domain for signal transduction. Three of the four recently discovered human PGN recognition proteins (PGRP) have a transmembrane domain, and hence, considered as true receptors for GPB. Since lysozyme is the only known pulmonary enzyme that can lyse bacterial cell wall PGN, other innate immune molecules appear to be responsible for signalling and enhancing the clearance of GPB infection from the lung. Interestingly, pulmonary collectins bind not only to GPB ligands but also to the receptors, CD14 and TLR, and antigen processing cells such as dentritic cells. These complex interactions appear to play major roles in linking innate and adaptive immunity, and maintaining a pathogen-free lung with minimal, or no inflammation.     

Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

To determine how distinct receptors of the immune system can contribute to innate immunity, we investigated the pattern of Toll-like receptor 1 (TLR1) and TLR2 expression in human lymphoid tissue. We found that TLR1 and TLR2 were co-expressed on cells of the innate immune system, including macrophages and dendritic cells. In addition, TLR1 and TLR2 were expressed in mucosa-associated lymphoid tissue on tonsillar crypt epithelium. Of the lymphoid tissue examined, spleen expressed the highest levels of TLR2. Although TLR1- and TLR2-positive cells were in close proximity to T lymphocytes in vivo, lymphocytes themselves were devoid of TLR1 and TLR2 expression. The co-expression of TLR1 and TLR2 on myeloid cells in lymphoid tissue provides the host with the ability to respond to a variety of microbial ligands at sites conducive to the generation of an immune response.     

A20 inhibits toll-like receptor 2- and 4-mediated interleukin-8 synthesis in airway epithelial cells

The zinc finger protein A20 is encoded by an immediate early response gene and acts as an inhibitor of nuclear factor (NF)-kappaB-dependent gene expression induced by different stimuli, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Toll-like receptor 2 (TLR2) and TLR4 have been found to transduce, respectively, peptidoglycan (PGN) and lipopolysaccharide (LPS) signals for the activation of NF-kappaB and the production of inflammatory cytokines. Here, we have examined the role of A20 in TLR-mediated NF-kappaB-dependent gene expression in human airway epithelial cells (AECs). Stimulation with LPS and PGN resulted in a significant increase in the level of A20 mRNA in primary cultured AECs and in NCI-H292 AECs. LPS and PGN induced activation of the IL-8 promoter both in NCI-H292 AECs and in HEK293 cells expressing either TLR2 or TLR4 plus MD-2. Dominant-negative myeloid differentiation protein and a mutant form of IkappaBalpha attenuated this PGN- or LPS-induced activation of the IL-8 promoter. Furthermore, overexpression of A20 inhibited activation of both NF-kappaB and the IL-8 promoter by PGN or LPS in these cells. Taken together, our results suggest that A20 may function as a negative regulator of TLR-mediated inflammatory responses in the airway, thereby protecting the host against harmful overresponses to pathogens.     

A protocol for generating high numbers of mature and functional human mast cells from peripheral blood

BACKGROUND: Mast cells (MCs) are multi-functional effector cells with an essential role in innate immunity and host defence, and under several pathological conditions, such as allergy. Here, we aimed at defining the culture conditions that would allow efficient generation of mature and functional human MCs from their progenitor cells. METHODS: Human peripheral blood-derived CD34(+) progenitor cells were cultured in vitro under serum-free conditions with human stem cell factor for 9 weeks. Growth and differentiation of the cells into MCs were optimized by selected cytokines and a combination of hypoxic and normoxic conditions. MCs were phenotypically characterized by immunocytochemistry, their preformed mediators were quantified, and their functional ability to degranulate and release histamine was tested. RESULTS: On average, 20 x 10(6) mature MCs were generated from 0.5 x 10(6) progenitor cells during 9 weeks of culture, i.e. at least a 40-fold increase in cell number was achieved. The mature MCs had oval-shaped non-lobular nuclei, contained histamine, heparin, tryptase, chymase, and cathepsin G in their secretory granules, and strongly expressed c-kit (CD117) and Fc epsilon receptor I on their surface. Histamine release from the cells could be brought about by IgE-anti-IgE cross-linkage, compound 48/80, substance P, and anaphylatoxin C3a. The MCs remained functional for several weeks after their maturation. CONCLUSION: This study describes an efficient protocol for generating mature MCs from human peripheral blood with a functional phenotype of connective tissue-type MCs. Use of these cultured human MCs will increase our knowledge and understanding about human MC development and biology in human disease.