Ei Wakamatsu

Conservation and divergence in the transcriptional programs of the human and mouse immune systems

Much of the knowledge about cell differentiation and function in the immune system has come from studies in mice, but the relevance to human immunology, diseases, and therapy has been challenged, perhaps more from anecdotal than comprehensive evidence. To this end, we compare two large compendia of transcriptional profiles of human and mouse immune cell types. Global transcription profiles are conserved between corresponding cell lineages. The expression patterns of most orthologous genes are conserved, particularly for lineage-specific genes. However, several hundred genes show clearly divergent expression across the examined cell lineages, and among them, 169 genes did so even with highly stringent criteria. Finally, regulatory mechanisms—reflected by regulators’ differential expression or enriched cis-elements—are conserved between the species but to a lower degree, suggesting that distinct regulation may underlie some of the conserved transcriptional responses.

Pathogenic role of immune response to M3 muscarinic acetylcholine receptor in Sjögren's syndrome-like sialoadenitis

The aim of this study was to clarify the role of the immune response to muscarinic type 3 receptor (M3R) in the pathogenesis of Sjögrens syndrome (SS). M3R(-/-) mice were immunized with murine M3R peptides and their splenocytes were inoculated into Rag1(-/-) (M3R(-/-)→Rag1(-/-)) mice. M3R(-/-)→Rag1(-/-) mice had high serum levels of anti-M3R antibodies and low saliva volume. Histological examination showed marked infiltration of mononuclear cells in the salivary glands and immunohistochemistry demonstrated that the majority of these cells were CD4(+) T cells with a few B cells and several IFN-γ- and IL-17-producing cells. Apoptotic cells were present in the salivary glands of M3R(-/-)→Rag1(-/-) mice. Moreover, transfer of only CD3(+) T cells from M3R(-/-) immunized with M3R peptides into Rag1(-/-) mice resulted in cell infiltration and destruction of epithelial cells in the salivary glands, suggesting that M3R reactive CD3(+) T cells play a pathogenic role in the development of autoimmune sialoadenitis. Our findings support the notion that the immune response to M3R plays a crucial role in the pathogenesis of SS-like autoimmune sialoadenitis.

New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome

M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögrens syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti‐M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human‐M3R including the N‐terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme‐linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti‐M3R antibody‐positive SS, ‐negative SS and controls for 12 h. After loading with Fluo‐3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca2+ concentrations [(Ca2+)i] were measured. Antibodies to the N‐terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS‐IgG inhibited the increase of (Ca2+)i induced by cevimeline hydrochloride. Antibodies to the N‐terminal positive SS‐IgG and antibodies to the first loop positive SS‐IgG enhanced it, while antibodies to the third loop positive SS‐IgG showed no effect on (Ca2+)i as well as anti‐M3R antibody‐negative SS‐IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti‐M3R antibodies on salivary secretion might differ based on these epitopes.

Convergent and divergent effects of costimulatory molecules in conventional and regulatory CD4+ T cells

Costimulatory molecules of the CD28 family on T lymphocytes integrate cues from innate immune system sensors and modulate activation responses in conventional CD4+ T cells (Tconv) and their FoxP3+ regulatory counterparts (Treg). To better understand how costimulatory and coinhibitory signals might be integrated, we profiled the changes in gene expression elicited in the hours and days after engagement of Treg and Tconv by anti-CD3 and either anti-CD28, -CTLA4, -ICOS, -PD1, -BLA, or -CD80. In Tconv, a shared “main response” was induced by CD28, ICOS, and, surprisingly, BTLA and CD80, with very limited CD28-specific (primarily Il2) or ICOS-specific elements (including Th1 and Th2 but not the follicular T signature). CTLA4 and PD1 had a very subtle impact in this system, similarly inhibiting the response to anti-CD3. Treg responded to the same costimulatory hierarchy and to the same extent as Tconv, but inducing different clusters of genes. In this reductionist system, costimulatory or coinhibitory engagement mainly elicits generic responses, suggesting that the variability of their effects in vivo result from temporal or anatomical differences in their engagement, rather than from inherently different wiring.

High prevalence of autoantibodies to muscarinic-3 acetylcholine receptor in patients with juvenile-onset Sjögren syndrome

Sjogren syndrome (SS) is an autoimmune disease characterised pathologically by lymphocytic infiltration into the lacrimal and salivary glands, and clinically by dry eyes and mouth. Lymphocytic infiltration is also found in the kidneys, lungs, thyroid, and liver. Immunohistochemical studies have shown that most infiltrating lymphocytes around the labial salivary and lacrimal glands and the kidneys are CD4-positive αβT cells.1 Candidate autoantigens recognised by T cells that infiltrate the labial salivary glands of SS have been analysed and Ro/SS-A 52 kDa,2 α-amylase, heat shock protein, and TCR BV63 have been identified, although Ro/SS-A 52 kDa reactive T cells were not increased in peripheral blood. …

DNA microarray analysis of labial salivary glands of patients with Sjögren’s syndrome

Sjogren’s syndrome (SS) is a chronic autoimmune disease characterised by dry eyes, dry mouth and focal lymphocytic infiltration in lacrimal and salivary glands. The infiltrating lymphocytes are mainly CD4 α/β T cells,1 especially T helper 1 (Th1) type T cells, because they produce both interferon (IFN)γ and interleukin 2.2,3 To understand the pathogenesis of SS, several molecules in labial salivary glands (LSGs) have been screened by microarray analysis in human SS. Hjelmervik et al 4 and Gottenberg et al 5 reported that the upregulated genes in SS salivary glands were IFN-inducible genes, such as IFN-stimulated transcription factor 3, IFN-regulatory factor 1 and B cell-activation factor of the TNF family. …

Altered peptide ligands regulate muscarinic acetylcholine receptor reactive T cells of patients with Sjögren’s syndrome

In the generation of Sjogren’s syndrome (SS), CD4 positive αβ T cells have a crucial role. Previous studies have provided evidence about the T cell receptor (TCR) Vβ and Vα genes on these T cells, and sequence analysis of the CDR3 region indicates the presence of some conserved amino acid motifs, supporting the notion that infiltrating T cells recognise relatively few epitopes on autoantigens.1 Candidate autoantigens recognised by T cells that infiltrate the labial salivary glands of patients with SS have been analysed, and Ro/SSA 52 kDa,2 α-amylase, heat shock protein, and TCR BV6 have been identified, although Ro/SSA 52 kDa reactive T cells were not increased in peripheral blood.3 Gordon et al indicated that anti-M3R autoantibodies occurred in SS and were associated with the sicca symptoms.4 Recently, we provided evidence for the presence of autoantibodies against the second extracellular domain of muscarinic acetylcholine receptor (M3R) in a subgroup of patients with SS.5 The M3R is an interesting molecule, because this portion has an important role in intracellular signalling,5 although the function of anti-M3R autoantibodies remains unknown. The mechanism through which a peptide is recognised by a TCR is flexible. If the amino acid residue of the peptide ligands for TCR is substituted by a different amino acid and can still …

Overexpression of T‐bet gene regulates murine autoimmune arthritis

OBJECTIVE To clarify the role of T-bet in the pathogenesis of collagen-induced arthritis (CIA). METHODS T-bet-transgenic (Tg) mice under the control of the CD2 promoter were generated. CIA was induced in T-bet-Tg mice and wild-type C57BL/6 (B6) mice. Levels of type II collagen (CII)-reactive T-bet and retinoic acid receptor-related orphan nuclear receptor γt (RORγt) messenger RNA expression were analyzed by real-time polymerase chain reaction. Criss-cross experiments using CD4+ T cells from B6 and T-bet-Tg mice, as well as CD11c+ splenic dendritic cells (DCs) from B6 and T-bet-Tg mice with CII were performed, and interleukin-17 (IL-17) and interferon-γ (IFNγ) in the supernatants were measured by enzyme-linked immunosorbent assay. CD4+ T cells from B6, T-bet-Tg, or T-bet-Tg/IFNγ-/- mice were cultured for Th17 cell differentiation, then the proportions of cells producing IFNγ and IL-17 were analyzed by fluorescence-activated cell sorting. RESULTS Unlike the B6 mice, the T-bet-Tg mice did not develop CIA. T-bet-Tg mice showed overexpression of Tbx21 and down-regulation of Rorc in CII-reactive T cells. Criss-cross experiments with CD4+ T cells and splenic DCs showed a significant reduction in IL-17 production by CII-reactive CD4+ T cells in T-bet-Tg mice, even upon coculture with DCs from B6 mice, indicating dysfunction of IL-17-producing CD4+ T cells. Inhibition of Th17 cell differentiation under an in vitro condition favoring Th17 cell differentiation was observed in both T-bet-Tg mice and T-bet-Tg/IFNγ-/- mice. CONCLUSION Overexpression of T-bet in T cells suppressed the development of autoimmune arthritis. The regulatory mechanism of arthritis might involve dysfunction of CII-reactive Th17 cell differentiation by overexpression of T-bet via IFNγ-independent pathways.

Importance of serine727 phosphorylated STAT1 in IFNγ-induced signaling and apoptosis of human salivary gland cells.

Aim:  It is reported that in salivary glands of Sjögren’s syndrome (SS), interferon gamma (IFNγ) and IFNγ‐inducible genes containing signal transducers and activators of transcription 1 (STAT1) are upregulated and play a crucial role in the pathogenesis of SS. The aim of this study is to clarify which phosphorylation of STAT1, serine727 (Ser727) or tyrosine701 (Tyr701) of STAT1, is important for IFNγ signaling and IFNγ‐induced apoptosis in salivary gland cells.

AUTOANTIBODIES AGAINST MUSCARINIC ACETYLCHOLINE RECEPTOR IN PATIENTS WITH SJÖGREN'S SYNDROME

ABSTRACT The M3Rs are expressed on salivary and lachrymal glands, thus they should be key receptors involved in the production of saliva and tear following stimulation of acetylcholine (ACh). Thence, autoantibodies (Abs) against M3 muscarinic acetylcholine receptors (M3R) could interfere with the production of saliva and tear. To test this hypothesis, we analysed in the present study the prevalence of anti-M3R Abs in patients with Sjogrens syndrome (SS). We observed evidence that autoantibodies against the 25mer synthetic amino acids encoding the second extracellular domain of M3R were detected specifically in both primary (9%) and secondary (14%) SS. Moreover, anti-SS-B Abs are strongly associated with anti-M3R Abs. Therefore, we suggest that anti-M3R Abs could be used as a new diagnostic marker for SS. Further experiments on the functional analysis of anti-M3R Abs in SS using M3R transfectant cell lines should shed light on the relationship between the presence of anti-M3R autoantibodies and pathogenesis of SS.