Inflammation and changes in cytokine levels in neurological feline infectious peritonitis.

Feline infectious peritonitis (FIP) is a progressive, fatal, predominantly Arthus-type immune-mediated disease that is triggered when cats are infected with a mutant enteric coronavirus. The disease presents variably with multiple organ failure, seizures, generalized effusion, or shock. Neurological FIP is clinically and pathologically more homogeneous than systemic 'wet' or 'dry' FIP; thus, comparison of cytokine profiles from cats with neurological FIP, wet FIP, and non-FIP neurological disease may provide insight into some baseline characteristics relating to the immunopathogenesis of neurological FIP. This study characterizes inflammation and changes in cytokines in the brain tissue of FIP-affected cats. Cellular infiltrates in cats with FIP included lymphocytes, plasma cells, neutrophils, macrophages, and eosinophils. IL-1 beta, IL-6, IL-12, IL-18, TNF-alpha, macrophage inhibitory protein (MIP)-1 alpha, and RANTES showed no upregulation in the brains of control cats, moderate upregulation in neurological FIP cats, and very high upregulation in generalized FIP cats. Transcription of IFN-gamma appeared upregulated in cats with systemic FIP and slightly downregulated in neurological FIP. In most cytokines tested, variance was extremely high in generalized FIP and much less in neurological FIP. Principal components analysis was performed in order to find the least number of 'components' that would summarize the cytokine profiles in cats with neurological FIP. A large component of the variance (91.7%) was accounted for by levels of IL-6, MIP-1 alpha, and RANTES. These findings provide new insight into the immunopathogenesis of FIP and suggest targets for immune therapy of this disease

B Cell Adaptor Containing Src Homology 2 Domain (Bash) Links B Cell Receptor Signaling to the Activation of Hematopoietic Progenitor Kinase 1

The B cell adaptor containing src homology 2 domain (BASH; also termed BLNK or SLP-65), is crucial for B cell antigen receptor (BCR)-mediated activation, proliferation, and differentiation of B cells. BCR-mediated tyrosine-phosphorylation of BASH creates binding sites for signaling effectors such as phospholipase Cγ (PLCγ)2 and Vav, while the function of its COOH-terminal src homology 2 domain is unknown. We have now identified hematopoietic progenitor kinase (HPK)1, a STE20-related serine/threonine kinase, as a protein that inducibly interacts with the BASH SH2 domain. BCR ligation induced rapid tyrosine-phosphorylation of HPK1 mainly by Syk and Lyn, resulting in its association with BASH and catalytic activation. BCR-mediated activation of HPK1 was impaired in Syk- or BASH-deficient B cells. The functional SH2 domain of BASH and Tyr-379 within HPK1 which we identified as a Syk-phosphorylation site were both necessary for interaction of both proteins and efficient HPK1 activation after BCR stimulation. Furthermore, HPK1 augmented, whereas its kinase-dead mutant inhibited IκB kinase β (IKKβ) activation by BCR engagement. These results reveal a novel BCR signaling pathway leading to the activation of HPK1 and subsequently IKKβ, in which BASH recruits tyrosine-phosphorylated HPK1 into the BCR signaling complex

Targeting of MIST to Src-family kinases via SKAP55–SLAP-130 adaptor complex in mast cells11The rat SKAP55 cDNA nucleotide sequence has been deposited in DDBJ database under accession number AB092812.

AbstractMIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST–SLAP-130–SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells

New Approach for M-Cell-Specific Molecules Screening by Comprehensive Transcriptome Analysis

A minor population of M cells within the follicle-associated epithelium (FAE) of intestinal Peyer's patches (PPs) serves as a major portal for entry of exogenous antigens. Characterization of the mammalian M cells, including identification of M-cell surface molecules used for bacterial uptake, has been hampered by their relative rarity. In contrast, M cells constitute virtually all of the FAE cells in the avian bursa of Fabricius. We therefore performed comparative gene expression profiling of chicken and murine FAE to identify commonly expressed genes by M cells in both species. The comprehensive transcriptome analysis revealed that 28 genes were commonly up-regulated in FAE from both species. In situ hybridization revealed that annexin A10 (Anxa10) mRNA was scattered in FAE, and co-localized with Ulex europaeus agglutinin-1 binding to M cells. Whole-mount immunostaining also revealed that cellular prion protein (PrPC) was expressed on the luminal side of the apical plasma membrane of M cells, and co-localized with grycoprotein 2 that recognizes only M cells in murine PP. Our findings identify new M-cell-specific molecules through using comprehensive transcriptome analysis. These conserved molecules in M cells of mice and chickens may play essential roles in M-cell function and/or differentiation

CIN85 drives B cell responses by linking BCR signals to the canonical NF-κB pathway

CIN85 transduces B cell receptor signals to IKK-β, and its expression in B cells is essential for T cell–independent type II antibody responses in mice

Antigen sampling CSF1R-expressing epithelial cells are the functional equivalents of mammalian M cells in the avian follicle-associated epithelium

The follicle-associated epithelium (FAE) is a specialized structure that samples luminal antigens and transports them into mucosa-associated lymphoid tissues (MALT). In mammals, transcytosis of antigens across the gut epithelium is performed by a subset of FAE cells known as M cells. Here we show that colony-stimulating factor 1 receptor (CSF1R) is expressed by a subset of cells in the avian bursa of Fabricius FAE. Expression was initially detected using a CSF1R-reporter transgene that also label subsets of bursal macrophages. Immunohistochemical detection using a specific monoclonal antibody confirmed abundant expression of CSF1R on the basolateral membrane of FAE cells. CSF1R-transgene expressing bursal FAE cells were enriched for expression of markers previously reported as putative M cell markers, including annexin A10 and CD44. They were further distinguished from a population of CSF1R-transgene negative epithelial cells within FAE by high apical F-actin expression and differential staining with the lectins jacalin, PHA-L and SNA. Bursal FAE cells that express the CSF1R-reporter transgene were responsible for the bulk of FAE transcytosis of labeled microparticles in the size range 0.02–0.1 µm. Unlike mammalian M cells, they did not readily take up larger bacterial sized microparticles (0.5 µm). Their role in uptake of bacteria was tested using Salmonella, which can enter via M cells in mammals. Labeled Salmonella enterica serovar Typhimurium entered bursal tissue via the FAE. Entry was partially dependent upon Type III secretion system-1. However, the majority of invading bacteria were localized to CSF1R-negative FAE cells and in resident phagocytes that express the phosphatidylserine receptor TIM4. CSF1R-expressing FAE cells in infected follicles showed evidence of cell death and shedding into the bursal lumen. In mammals, CSF1R expression in the gut is restricted to macrophages which only indirectly control M cell differentiation. The novel expression of CSF1R in birds suggests that these functional equivalents to mammalian M cells may have different ontological origins and their development and function are likely to be regulated by different growth factors

The B-cell antigen receptor signals through a preformed transducer module of SLP65 and CIN85

The Slp65 adaptor molecule is important for B-cell receptor signalling. Here, the interactomes of SLP65 in resting and activated B cells are resolved by mass spectrometry. A number of SLP65 interacting partners, including CIN85, continuously associate with SLP65 in a stimulation-independent manner. CIN85 recruits SLP65 to the membrane and it is required for SLP65 phosphorylation