Jeremy K. Brown

Leukotriene B4, an activation product of mast cells, is a chemoattractant for their progenitors

Mast cells are tissue-resident cells with important functions in allergy and inflammation. Pluripotential hematopoietic stem cells in the bone marrow give rise to committed mast cell progenitors that transit via the blood to tissues throughout the body, where they mature. Knowledge is limited about the factors that release mast cell progenitors from the bone marrow or recruit them to remote tissues. Mouse femoral bone marrow cells were cultured with IL-3 for 2 wk and a range of chemotactic agents were tested on the c-kit+ population. Cells were remarkably refractory and no chemotaxis was induced by any chemokines tested. However, supernatants from activated mature mast cells induced pronounced chemotaxis, with the active principle identified as leukotriene (LT) B4. Other activation products were inactive. LTB4 was highly chemotactic for 2-wk-old cells, but not mature cells, correlating with a loss of mRNA for the LTB4 receptor, BLT1. Immature cells also accumulated in vivo in response to intradermally injected LTB4. Furthermore, LTB4 was highly potent in attracting mast cell progenitors from freshly isolated bone marrow cell suspensions. Finally, LTB4 was a potent chemoattractant for human cord blood–derived immature, but not mature, mast cells. These results suggest an autocrine role for LTB4 in regulating tissue mast cell numbers.

The role of the peritoneum in the pathogenesis of endometriosis

BACKGROUND Endometriosis affects 6-10% of women of reproductive age and is associated with chronic pelvic pain, dysmenorrhoea, dyspareunia and infertility. Endometriosis is defined by the presence of endometrial tissue outside the uterus, most commonly attached to the pelvic peritoneum. The endometrium in women with endometriosis is reported to be altered and there is increasing evidence that the phenotype of the pelvic peritoneum may also play a role in the establishment and maintenance of the disease. The aim of this review is to discuss the putative role of the pelvic peritoneum in the pathophysiology of peritoneal endometriosis. METHODS A review was undertaken of the published literature on (i) the anatomy and physiology of the peritoneum and (ii) the potential roles played by peritoneal cells in the establishment and maintenance of peritoneal endometriosis. The current understanding of the biology of peritoneal endometriosis is summarized and the potential interaction of the peritoneum with ectopic endometrial cells in endometriosis is highlighted. RESULTS Several studies indicate that differential expression of peritoneal mesothelial adhesion factors occurs in women with endometriosis, providing potential ectopic endometrial cell attachment sites for the establishment of endometriosis lesions. Changes in the peritoneal mesothelial cell phenotype, including loss of tight junctions, may allow ectopic cells to bind to, or early lesions to invade into, the extracellular matrix. Epithelial-to-mesenchymal transition of peritoneal mesothelial cells may also lead to an increase in lesion invasion and formation of fibrotic tissue in and around the lesion. There is evidence that the peritoneal mesothelium may also play a role in the invasion potential of ectopic cells by production of MMPs increasing local tissue remodelling. Peritoneal immune scavenging function may be lowered in women with endometriosis; for example there is a notable increase in macrophage-derived secretion products in women with endometriosis associated with increases in cell proliferation, cell adhesion and neovascularization. CONCLUSIONS The pelvic peritoneum appears to play a key role in the development and maintenance of endometriosis.

Innate immune response mechanisms in the intestinal epithelium: potential roles for mast cells and goblet cells in the expulsion of adult Trichinella spiralis

SUMMARYGastrointestinal infection with the nematode Trichinella spiralis is accompanied by a rapid and reversible expansion of the mucosal mast cell and goblet cell populations in the intestinal epithelium, which is associated with the release of their mediators into the gut lumen. Both goblet cell and mast cell hyperplasia are highly dependent on mucosal T-cells and augmented by the cytokines IL-4 and IL-13. However, the contribution of both mast and goblet cells, and the mediators they produce, to the expulsion of the adults of T. spiralis is only beginning to be elucidated through studies predominantly employing T. spiralis-mouse models. In the present article, we review the factors proposed to control T. spiralis-induced mucosal mast cell (MMC) and goblet cell differentiation in the small intestine, and focus on some key MMC and goblet cell effector molecules which may contribute to the expulsion of adult worms and/or inhibition of larval development.

Enteric Expression of the Integrin αvβ6 Is Essential for Nematode-Induced Mucosal Mast Cell Hyperplasia and Expression of the Granule Chymase, Mouse Mast Cell Protease-1

The immunoregulatory cytokine transforming growth factor (TGF)-β1 is secreted as a biologically inactive complex with latency-associated peptide, which must be modified by local factors to expose the functionally active cytokine. The epithelial integrin αvβ6 mediates local activation of TGF-β1 in the lung and β6−/− mice exhibit exaggerated pulmonary inflammation, but their response to inflammatory stimuli in the gut has not been investigated. We found that both β6 and TGF-β1 are constitutively expressed in the jejunal epithelial compartment in uninfected mice and during infection with the intestinal nematode Nippostrongylus brasiliensis. We also present data showing that β6−/− mice are seriously compromised in their ability to mount a mucosal mast cell response after infection, and there is a significant reduction in the expression and systemic release of the granule chymase, mouse mast cell protease-1. Because in vitro expression of this chymase is regulated by TGF-β1, these data indicate that in the absence of αvβ6 epithelially expressed TGF-β1 may not be activated, with a consequent absence of expression of mouse mast cell protease-1 and down-regulation of the mucosal mast cell response.

Proteomic identification of interactions between histones and plasma proteins: Implications for cytoprotection

Extracellular histones released from cells during acute inflammation contribute to organ failure and death in a mouse model of sepsis, and histones are known to exert in vitro cytotoxicity in the absence of serum. Since addition of histones to serum and plasma is known to induce protein aggregation, we reasoned that plasma proteins may afford protection from cytotoxicity. We found that MODE‐K mouse small intestinal epithelial cells were protected from histone‐induced toxicity in the presence of 10% FCS. Therefore, the main aim of this study was to identify histone‐interacting plasma proteins that might be involved in cytoprotection. The precipitate formed following addition of calf thymus histones to human EDTA plasma was characterised by shotgun proteomics, identifying a total of 36 protein subunits, including complement components, coagulation factors, protease inhibitors and apolipoproteins. The highly sulphated glycosaminoglycan heparin inhibited histone‐induced plasma protein aggregation. Moreover, histones bound to heparin agarose were capable of pulling down plasma proteins from solution, indicating their effective cross‐linking properties. It was particularly notable that inter‐α‐trypsin inhibitor was prominent among the histone‐precipitated proteins, since it contains a chondroitin sulphate glycan chain, and suggests a potential role for this protein in histone sequestration during acute inflammation in vivo.

Purification and characterization of mouse mast cell proteinase-2 and the differential expression and release of mouse mast cell proteinase-1 and -2 in vivo.

Background Gastrointestinal nematode infection is associated with mucosal mast cell (MMC) hyperplasia. In the mouse, this is accompanied by the release of substantial quantities of the chymase mouse mast cell proteinase‐1 (mMCP‐1) into the gut lumen and peripheral bloodstream. Expression of mMCP‐1 is largely restricted to intraepithelial MMC and is thought to play a role in the regulation of epithelial permeability. MMCs also express mouse mast cell proteinase‐2 (mMCP‐2), but less is known about the expression or biological function of this proteinase.

The Peritoneum Is Both a Source and Target of TGF-β in Women with Endometriosis

Transforming growth factor-β (TGF-β) is believed to play a major role in the aetiology of peritoneal endometriosis. We aimed to determine if the peritoneum is a source of TGF-β and if peritoneal TGF-β expression, reception or target genes are altered in women with endometriosis. Peritoneal fluid, peritoneal bushings and peritoneal biopsies were collected from women with and without endometriosis. TGF-β1, 2 and 3 protein concentrations were measured in the peritoneal fluid. TGF-β1 was measured in mesothelial cell conditioned media. Control peritoneum and peritoneum prone to endometriosis (within Pouch of Douglas) from women without disease (n = 16) and peritoneum distal and adjacent to endometriosis lesions in women with endometriosis (n = 15) and were analysed for TGF-β expression, reception and signalling by immunohistochemistry, qRT-PCR and a TGF-β signalling PCR array. TGF-β1 was increased in the peritoneal fluid of women with endometriosis compared to those without disease (P<0.05) and peritoneal mesothelial cells secrete TGF-β1 in-vitro. In women with endometriosis, peritoneum from sites adjacent to endometriosis lesions expressed higher levels of TGFB1 mRNA when compared to distal sites (P<0.05). The TGF-β-stimulated Smad 2/3 signalling pathway was active in the peritoneum and there were significant increases (P<0.05) in expression of genes associated with tumorigenesis (MAPK8, CDC6), epithelial-mesenchymal transition (NOTCH1), angiogenesis (ID1, ID3) and neurogenesis (CREB1) in the peritoneum of women with endometriosis. In conclusion, the peritoneum, and in particular, the peritoneal mesothelium, is a source of TGF-β1 and this is enhanced around endometriosis lesions. The expression of TGF-β-regulated genes is altered in the peritoneum of women with endometriosis and this may promote an environment favorable to lesion formation.

Role of Alveolar Macrophages in Respiratory Transmission of Visna/Maedi Virus

ABSTRACT A major route of transmission of Visna/maedi virus (VMV), an ovine lentivirus, is thought to be via the respiratory tract, by inhalation of either cell-free or cell-associated virus. In previous studies, we have shown that infection via the lower respiratory tract is much more efficient than via upper respiratory tissues (T. N. McNeilly, P. Tennant, L. Lujan, M. Perez, and G. D. Harkiss, J. Gen. Virol. 88:670-679, 2007). Alveolar macrophages (AMs) are prime candidates for the initial uptake of virus in the lower lung, given their in vivo tropism for VMV, abundant numbers, location within the airways, and role in VMV-induced inflammation. Furthermore, AMs are the most likely cell type involved in the transmission of cell-associated virus. In this study, we use an experimental in vivo infection model that allowed the infection of specific segments of the ovine lung. We demonstrate that resident AMs are capable of VMV uptake in vivo and that this infection is associated with a specific up-regulation of AM granulocyte-macrophage colony-stimulating factor mRNA expression (P < 0.05) and an increase in bronchoalveolar lymphocyte numbers (P < 0.05), but not a generalized inflammatory response 7 days postinfection. We also demonstrate that both autologous and heterologous VMV-infected AMs are capable of transmitting virus after lower, but not upper, respiratory tract instillation and that this transfer of virus appears not to involve the direct migration of virus-infected AMs from the airspace. These results suggest that virus is transferred from AMs into the body via an intermediate route. The results also suggest that the inhalation of infected AMs represents an additional mechanism of virus transmission.

Constitutive secretion of the granule chymase mouse mast cell protease‐1 and the chemokine, CCL2, by mucosal mast cell homologues

Background The mucosal mast cell (MMC) granule‐specific β‐chymase, mouse mast cell protease‐1 (mMCP‐1), is released systemically into the bloodstream early in nematode infection before parasite‐specific IgE responses develop and TGF‐β1 induces constitutive release of mMCP‐1 by homologues of MMC in vitro. Intraepithelial MMC may also express the chemokine CCL2 (monocyte chemotactic protein‐1) during nematode infection but the expression of this chemokine by MMC homologues has not been investigated.

Expression of Integrin-αE by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-β1-Activating Integrin αvβ6

Peak intestinal mucosal mast cell (MMC) recruitment coincides with expulsion of Trichinella spiralis, at a time when the majority of the MMCs are located within the epithelium in BALB/c mice. Although expression of integrin-αEβ7 by MMCs has not been formally demonstrated, it has been proposed as a potential mechanism to account for the predominantly intraepithelial location of MMCs during nematode infection. Co-expression of integrin-αEβ7 and the MMC chymase mouse mast cell protease-1, by mouse bone marrow-derived mast cells, is strictly regulated by transforming growth factor (TGF)-β1. However, TGF-β1 is secreted as part of a latent complex in vivo and subsequent extracellular modification is required to render it biologically active. We now show, for the first time, that intraepithelial MMCs express integrin-αEβ7 in Trichinella-infected BALB/c and S129 mice. In S129 mice that lack the gene for the integrin-β6 subunit and, as consequence, do not express the epithelial integrin-αvβ6, integrin-αE expression is virtually abolished and recruitment of MMCs into the intestinal epithelium is dramatically reduced despite significant overall augmentation of the MMC population. Because a major function of integrin-αvβ6 is to activate latent TGF-β1, these findings strongly support a role for TGF-β1 in both the recruitment and differentiation of murine MMCs during nematode infection.