International Immunology - Advance Access

Bacillus subtilis-specific poly-{gamma}-glutamic acid regulates development pathways of naive CD4+ T cells through antigen-presenting cell-dependent and -independent mechanisms

2009-06-26

Peripheral naive CD4⁺ T cells selectively differentiate to type 1 T_h, type 2 T_h and IL-17-producing T_h (T_h17) cells, depending on the priming conditions. Since these subsets develop antagonistically to each other to elicit subset-specific adaptive immune responses, balance between these subsets can regulate the susceptibility to diverse immune diseases. The present study was undertaken to determine whether poly--glutamic acid (-PGA), an edible and safe exopolymer that is generated by microorganisms such as Bacillus subtilis, could modulate the development pathways of T_h subsets. The presence of -PGA during priming promoted the development of T_h1 and T_h17 cells but inhibited development of T_h2 cells. -PGA up-regulated the expression of T-bet and ROR-t, the master genes of T_h1 and T_h17 cells, respectively, whereas down-regulating the level of GATA-3, the master gene of T_h2 cells. -PGA induced the expression of IL-12p40, CD80 and CD86 in dendritic cells (DC) and macrophages in a Toll-like receptor-4-dependent manner, and the effect of -PGA on T_h1/T_h2 development was dependent on the presence of antigen-presenting cells (APC). Furthermore, -PGA-stimulated DC favored the polarization of naive CD4⁺ T cells toward T_h1 cells rather than T_h2 cells. In contrast, -PGA affected T_h17 cell development, regardless of the presence or absence of APC. Thus, these data demonstrate that -PGA has the potential to regulate the development pathways of naive CD4⁺ T cells through APC-dependent and -independent mechanisms and to be applicable to treating T_h2-dominated diseases.

Light chain-deficient mice produce novel multimeric heavy-chain-only IgA by faulty class switching

2009-06-26

Recently, we identified that diverse heavy chain (H-chain)-only IgG is spontaneously produced in light chain (L-chain)-deficient mice (L^–/– with silenced and loci) despite a block in B cell development. In murine H-chain IgG, the first C exon, C_H1, is removed after DNA rearrangement and secreted polypeptides are comparable with camelid-type H-chain IgG. Here we show that L^–/– mice generate a novel class of H-chain Ig with covalently linked chains, not identified in any other healthy mammal. Surprisingly, diverse H-chain-only IgA can be released from B cells at levels similar to conventional IgA and is found in serum and sometimes in milk and saliva. Surface IgA without L-chain is expressed in B220⁺ spleen cells, which exhibited a novel B cell receptor, suggesting that associated conventional differentiation events occur. To facilitate the cellular transport and release of H-chain-only IgA, chaperoning via BiP association seems to be prevented as only chains lacking C_H1 are released from the cell. This appears to be accomplished by imprecise class-switch recombination (CSR) from Sµ into the constant region, which removes all or part of the C1 exon at the genomic level.

Gene expression profiling of experimental asthma reveals a possible role of paraoxonase-1 in the disease

2009-06-25

In this study, we aimed to identify novel genes involved in experimental and human asthma, importance of which has not yet been recognized. In an ovalbumin-induced murine model of asthma, we applied microarray gene expression analysis at different time points after allergen challenges. Advanced statistical methods were used to relate gene expression changes to cellular processes and to integrate our results into multiple levels of information available in public databases. At 4 h after the first allergen challenge, gene expression pattern reflected mainly an acute, but non-atopic, inflammatory response and strong chemotactic activity. At 24 h after the third allergen challenge, gene set enrichment analysis revealed significant over-representation of gene sets corresponding to T_h2-type inflammation models. Among the top down-regulated transcripts, an anti-oxidant enzyme, paraoxonase-1 (PON1), was identified. In human asthmatic patients, we found that serum PON1 activity was reduced at exacerbation, but increased parallel with improving asthma symptoms. PON1 gene polymorphisms did not influence the susceptibility to the disease. Our observations suggest that an altered PON1 activity might be involved in the pathogenesis of asthma, and serum PON1 level might be used for following up the effect of therapy.

Apex2 is required for efficient somatic hypermutation but not for class switch recombination of immunoglobulin genes

2009-06-25

The DNA cleavage step in both the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID). However, the detailed mechanisms of the DNA strand cleavage in SHM and CSR are still largely unknown. Recently, the apurinic/apyrimidinic endonucleases, Apex1 and Apex2, were reported to be involved in the DNA cleavage step of CSR. Here, we examined the role of Apex2 in SHM using Apex2-deficient mice and found that the Apex2 deficiency caused a drastic reduction in the frequency of SHM and the number of mutations per mutated clone without affecting the pattern of base substitution. These results suggest that Apex2 may play a critical role in SHM through its 3'–5' exonuclease activity. Unexpectedly, the efficiency of CSR was not reduced in Apex2-deficient B cells. In addition, Apex1 knockdown in CH12F3-2 B lymphoma cells did not affect the CSR frequency, suggesting that neither Apex1 nor Apex2 plays a major role in CSR.

Nucleotide-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice

2009-06-25

Abzymes (Abzs) with different enzymic activities have been detected in the sera of patients with various autoimmune (AI) diseases and in AI mice. In this work, electrophoretically homogeneous IgGs were isolated from the sera of MRL-lpr/lpr mice spontaneously developing lupus-like AI pathology. It was shown for the first time that polyclonal IgGs (pIgGs) and their isolated heavy and light chains hydrolyze different nucleoside-5'-triphosphate (NTPs), nucleoside-5'-diphosphate (NDPs), adenosine monophosphate and deoxiadenosine-5'-monophosphate (dAMP), whereas antibodies from the sera of control healthy mice were catalytically inactive. Monoclonal mouse IgGs also effectively hydrolyze nucleotides. The data demonstrate that nucleotide-hydrolyzing activity is an intrinsic property of isolated mouse pIgG and monoclonal IgG. It was shown that various markers of AI pathologies (proteinuria and antibody titers to native and denatured DNA) demonstrating spontaneous development of AI reactions increased in animals with aging and correlated with an increase in Abz relative activity in hydrolysis of nucleotides. The highest increase in AI reaction markers and in Abz enzymic activity was found in mice immunized with a DNA–protein complex.

Mutational analysis of Cys88 of Toll-like receptor 4 highlights the critical role of MD-2 in cell surface receptor expression

2009-06-25

The role of MD-2 in cell surface expression of Toll-like receptor (TLR) 4 has been controversial. The purposes of this study were to characterize the N-glycan of TLR4 and to investigate the roles of MD-2 in N-linked glycosylation and cell surface expression of TLR4. Lectin blot and cell surface biotinylation revealed that TLR4 exhibited the 110 kDa protein with high mannose type N-glycans and the 130 kDa protein with complex type N-glycans and that only the 130 kDa TLR4 with complex type N-glycans was expressed on the cell surface. The cells transfected with a mutant TLR4^C88A alone expressed only the 110 kDa TLR4 with a high mannose type N-glycan, which did not appear on the cell surface. However, TLR4^C88A acquired complex type N-glycans and was expressed on the cell surface when MD-2 was co-transfected. The amount of the 130 kDa TLR4^C88A with complex type N-glycans expressed on the cell surface depended on that of MD-2 transfected. -Mannosidase II inhibitor blocked the processing N-glycans to complex type, but TLR4 with high mannose type appeared on the cell surface, suggesting that TLR4 is destined to locate on the cell surface before processing N-glycans from a high mannose type to a complex type. From these results, we conclude that MD-2 is critical for cell surface expression of TLR4^C88A. This study provides evidence that MD-2 possesses potential ability to play an essential role in cell surface expression of TLR4.

Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-{alpha} through an ERK1/2-dependent pathway

Espinoza, J. L., Takamatsu, H., Lu, X., Qi, Z., Nakao, S. — 2009-06-25

Antibodies specific to moesin, which are frequently detectable in the serum of patients with aplastic anemia (AA), can induce tumor necrosis factor- (TNF-) secretion from monocytes and a human monocytic leukemia cell line THP-1. We investigated the mechanisms responsible for TNF- secretion from monocytic cells induced by the auto-antibodies that are purified from the sera of AA patients. TNF- induction by anti-moesin antibodies depended on the amount of cell surface moesin expressed by THP-1 cells. F(ab')₂ fragments prepared from the anti-moesin antibodies were able to stimulate THP-1 cells to secrete TNF- and this stimulatory effect was enhanced by cross-linking of moesins with anti-human IgG F(ab')₂ fragment antibodies. Anti-moesin antibodies as well as their F(ab')₂ fragments induced the phosphorylation of ERK1/2 in monocytic cells and this effect was suppressed by the addition of an ERK1/2 inhibitor. Moreover, anti-moesin antibody treatment induced the phosphorylation of moesin proteins in the monocytes and THP-1 cells within 30 min. These results indicate that anti-moesin antibodies induce TNF- secretion from monocytes through the activation of the ERK1/2 pathway provoked by direct binding to moesin on the cells.

Antimicrobial cathelicidin polypeptide CAP11 suppresses the production and release of septic mediators in D-galactosamine-sensitized endotoxin shock mice

2009-06-25

Endotoxin shock is a severe systemic inflammatory response that is caused by the augmented production and release of septic mediators. Among them, inflammatory cytokines such as tumor necrosis factor-, IL-1β and IL-6 play a pivotal role. In addition, anandamide, an endogenous cannabinoid and high-mobility group box-1 (HMGB1), a non-histone chromosomal protein has recently been recognized as members of septic mediators. We previously reported that cationic antibacterial polypeptide of 11-kDa (CAP11), an antimicrobial cathelicidin peptide (originally isolated from guinea pig neutrophils), potently neutralizes the biological activity of LPS and protects mice from lethal endotoxin shock. In this study, to clarify the protective mechanism of CAP11 against endotoxin shock, we evaluated the effects of CAP11 on the production and release of septic mediators in vitro and in vivo using a murine macrophage cell line RAW264.7 and a D-galactosamine-sensitized murine endotoxin shock model. LPS stimulation induced the production of inflammatory cytokines and anandamide and release of HMGB1 from RAW264.7 cells. Importantly, CAP11 suppressed the LPS-induced production and release of these mediators by RAW264.7 cells. Moreover, LPS administration enhanced the serum levels of HMGB1, anandamide and inflammatory cytokines in the endotoxin shock model. Of note, CAP11 suppressed the LPS-induced increase of these mediators in sera, and LPS binding to CD14-positive cells (peritoneal macrophages), accompanied with the increase of survival rates. Together these observations suggest that the protective action of CAP11 on endotoxin shock may be explained by its suppressive effect on the production and release of septic mediators by CD14-positive cells possibly via the inhibition of LPS binding to the targets.

The CD70-CD27 interaction during the stimulation with dendritic cells promotes naive CD4+ T cells to develop into T cells producing a broad array of immunostimulatory cytokines in humans

2009-06-25

CD70 expressed on dendritic cells (DCs) has been shown to play a critical role in inducing effective CD8⁺ T cell responses and a T_h1 response in mice. However, it has not been extensively examined whether human primary DCs express CD70 and whether the CD70–CD27 interaction promotes naive CD4⁺ T cells to acquire the ability to produce effector cytokines during the DC–T cell interaction in humans. Here, we show that human myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells stimulated with CD40 ligand together with pro-inflammatory cytokines or Toll-like receptor ligands express CD70. Thymic stromal lymphopoietin plus prostaglandin E₂ also induced CD70 on mDCs. Naive CD4⁺ T cells stimulated with DCs but not with anti-CD3/CD28 microbeads expressed CD70. Stimulation with CD70 together with anti-CD3/CD28 microbeads imparted the ability to produce T_h1 (IFN-), T_h2 (IL-4, IL-5, IL-13) cytokines, IL-2 and tumor necrosis factor- to naive CD4⁺ T cells. The production of IFN- was associated with the induction of T-bet. Naive CD4⁺ T cells stimulated with mDCs acquired an enhanced ability to produce a broad array of immunostimulatory cytokines in a CD70-dependent manner. These data suggest that human CD70 expressed on mDCs and activated T cells transmits a ‘basal level’ signal, rather than a ‘polarizing’ signal, to naive CD4⁺ T cells, in that CD70 promotes the development of CD4⁺ T cells that produce a variety of effector cytokines including both T_h1 and T_h2 types, thus contributing to the enhancement of a broad spectrum of immune responses.