Mast Cell Reconstitution Research Articles

Mast cells: innate attractors recruiting protective CD8 T cells to sites of cytomegalovirus infection.

Reactivation of latent cytomegalovirus (CMV) in the transient immunocompromised state after hematoablative treatment is a major concern in patients undergoing hematopoietic cell transplantation (HCT) as a therapy of hematopoietic malignancies. Timely reconstitution of antiviral CD8 T cells and their efficient recruitment to the lungs is crucial for preventing interstitial pneumonia, the most severe disease manifestation of CMV in HCT recipients. Here, we review recent work in a murine model, implicating mast cells (MC) in the control of pulmonary infection. Murine CMV (mCMV) productively infects MC in vivo and triggers their degranulation, resulting in the release of the CC chemokine ligand 5 (CCL5) that attracts CD8 T cells to infiltrate infected tissues. Comparing infection of MC-sufficient C57BL/6 mice and congenic MC-deficient Kit (W-sh/W-sh) "sash" mutants revealed an inverse relation between the number of lung-infiltrating CD8 T cells and viral burden in the lungs. Specifically, reduced lung infiltration by CD8 T cells in "sash" mutants was associated with an impaired infection control. The causal, though indirect, involvement of MC in antiviral control was confirmed by reversion of the deficiency phenotype in "sash" mutants reconstituted with MC. These recent findings predict that efficient MC reconstitution facilitates the control of CMV infection also in immunocompromised HCT recipients.

Mast cells expedite control of pulmonary murine cytomegalovirus infection by enhancing the recruitment of protective CD8 T cells to the lungs.

The lungs are a noted predilection site of acute, latent, and reactivated cytomegalovirus (CMV) infections. Interstitial pneumonia is the most dreaded manifestation of CMV disease in the immunocompromised host, whereas in the immunocompetent host lung-infiltrating CD8 T cells confine the infection in nodular inflammatory foci and prevent viral pathology. By using murine CMV infection as a model, we provide evidence for a critical role of mast cells (MC) in the recruitment of protective CD8 T cells to the lungs. Systemic infection triggered degranulation selectively in infected MC. The viral activation of MC was associated with a wave of CC chemokine ligand 5 (CCL5) in the serum of C57BL/6 mice that was MC-derived as verified by infection of MC-deficient KitW-sh/W-sh “sash” mutants. In these mutants, CD8 T cells were recruited less efficiently to the lungs, correlating with enhanced viral replication and delayed virus clearance. A causative role for MC was verified by MC reconstitution of “sash” mice restoring both, efficient CD8 T-cell recruitment and infection control. These results reveal a novel crosstalk axis between innate and adaptive immune defense against CMV, and identify MC as a hitherto unconsidered player in the immune surveillance at a relevant site of CMV disease.

Inflammation induced by mast cell deficiency rather than the loss of interstitial cells of Cajal causes smooth muscle dysfunction in W/Wv mice

The initial hypothesis suggested that the interstitial cells of Cajal (ICC) played an essential role in mediating enteric neuronal input to smooth muscle cells. Much information for this hypothesis came from studies in W/Wv mice lacking ICC. However, mast cells, which play critical roles in regulating inflammation in their microenvironment, are also absent in W/Wv mice. We tested the hypothesis that the depletion of mast cells in W/Wv mice generates inflammation in fundus muscularis externa (ME) that impairs smooth muscle reactivity to Ach, independent of the depletion of ICC. We performed experiments on the fundus ME from wild type (WT) and W/Wv mice before and after reconstitution of mast cells by bone marrow transplant. We found that mast cell deficiency in W/Wv mice significantly increased COX-2 and iNOS expression and decreased smooth muscle reactivity to Ach. Mast cell reconstitution or concurrent blockade of COX-2 and iNOS restored smooth muscle contractility without affecting the suppression of c-kit in W/Wv mice. The expression of nNOS and ChAT were suppressed in W/Wv mice; mast cell reconstitution did not restore them. We conclude that innate inflammation induced by mast cell deficiency in W/Wv mice impairs smooth muscle contractility independent of ICC deficiency. The impairment of smooth muscle contractility and the suppression of the enzymes regulating the synthesis of Ach and NO in W/Wv mice need to be considered in evaluating the role of ICC in regulating smooth muscle and enteric neuronal function in W/Wv mice.

Roles of mast cells in the pathogenesis of inflammatory myopathy

IntroductionIn addition to the pivotal roles of mast cells in allergic diseases, recent data suggest that mast cells play crucial roles in a variety of autoimmune responses. However, their roles in the pathogenesis of autoimmune skeletal muscle diseases have not been clarified despite their distribution in skeletal muscle. Therefore, the objective of this study is to determine the roles of mast cells in the development of autoimmune skeletal muscle diseases.MethodsThe number of mast cells in the affected muscle was examined in patients with dermatomyositis (DM) or polymyositis (PM). The susceptibility of mast cell-deficient WBB6F1-KitW/KitWv mice (W/Wv mice) to a murine model of polymyositis, C protein-induced myositis (CIM), was compared with that of wild-type (WT) mice. The effect of mast cell reconstitution with bone marrow-derived mast cells (BMMCs) on the susceptibility of W/Wv mice to CIM was also evaluated.ResultsThe number of mast cells in the affected muscle increased in patients with PM as compared with patients with DM. W/Wv mice exhibited significantly reduced disease incidence and histological scores of CIM as compared with WT mice. The number of CD8+ T cells and macrophages in the skeletal muscles of CIM decreased in W/Wv mice compared with WT mice. Engraftment of BMMCs restored the incidence and histological scores of CIM in W/Wv mice. Vascular permeability in the skeletal muscle was elevated in WT mice but not in W/Wv mice upon CIM induction.ConclusionMast cells are involved in the pathogenesis of inflammatory myopathy.

Mast cell deficiency results in the accumulation of preadipocytes in adipose tissue in both obese and non-obese mice

Mast cells have been suggested to play key roles in adipogenesis. We herein show that the expression of preadipocyte, but not adipocyte, marker genes increases in the white adipose tissue of mast cell-deficient (KitW-sh/W-sh) mice under both obese and non-obese conditions. In vitro culturing with adipogenic factors revealed increased adipocytes differentiated from the KitW-sh/W-sh stromal vascular fraction, suggesting the accumulation of preadipocytes. Moreover, the increased expression of preadipocyte genes was restored by mast cell reconstitution in the KitW-sh/W-sh mice. These results suggest positive effects of mast cells on the preadipocyte to adipocyte transition under both physiological and pathological conditions.

Staphylococcus δ-toxin induces allergic skin disease by activating mast cells

Atopic dermatitis (AD) is a chronic inflammatory skin disease that affects 15 to 30% of children and ~5% of adults in industrialized countries1. Although the pathogenesis of AD is not fully understood, the disease is mediated by an abnormal immunoglobulin E (IgE) immune response in the setting of skin barrier dysfunction2. Mast cells (MCs) contribute to IgE-mediated allergic disorders including AD3. Upon activation, MCs release their membrane-bound cytosolic granules leading to the release of multiple molecules that are important in the pathogenesis of AD and host defense4. More than 90% of AD patients are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbor the pathogen5. Several Staphylococcal exotoxins (SEs) can act as superantigens and/or antigens in models of AD6. However, the role of these SEs in disease pathogenesis remains unclear. Here, we report that culture supernatants of S. aureus contain potent MC degranulation activity. Biochemical analysis identified δ-toxin as the MC degranulation-inducing factor produced by S. aureus. MC degranulation induced by δ-toxin depended on phosphoinositide 3-kinase (PI3K) and calcium (Ca2+) influx, but unlike that mediated by IgE crosslinking, it did not require the spleen tyrosine kinase (Syk). In addition, IgE enhanced δ-toxin-induced MC degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from AD patients produced high levels of δ-toxin. Importantly, skin colonization with S. aureus, but not a mutant deficient in δ-toxin, promoted IgE and IL-4 production, as well as inflammatory skin disease. Furthermore, enhancement of IgE production and dermatitis by δ-toxin was abrogated in KitW-sh/W-sh MC-deficient mice and restored by MC reconstitution. These studies identify δ-toxin as a potent inducer of MC degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.

Mast Cells in Abdominal Aortic Aneurysms

Mast cells (MCs) are proinflammatory cells that play important roles in allergic responses, tumor growth, obesity, diabetes, atherosclerosis, and abdominal aortic aneurysm (AAA). Although the presence and function of MCs in atherosclerotic lesions have been thoroughly studied in human specimens, in primary cultured vascular cells, and in atherosclerosis in animals, their role in AAA was recognized only recently. Via multiple activation pathways, MCs release a spectrum of mediators — including histamine, inflammatory cytokines, chemokines, growth factors, proteoglycans, and proteases — to activate neighboring cells, degrade extracellular matrix proteins, process latent bioactive molecules, promote angiogenesis, recruit additional inflammatory cells, and stimulate vascular cell apoptosis. These activities associate closely with medial elastica breakdown, medial smooth-muscle cell loss and thinning, outer media and adventitia inflammation, aortic wall expansion, endothelium erosion, and eventual rupture and thrombosis. Experimental animal AAA models and MC reconstitution technique allowed examination of a direct role of MCs in AAA pathogenesis, and identification of the exact role of each MC-derived molecule. Genetic deficiency of MCs, pharmacological inhibition of MC degranulation, absence of MC-specific chymase and tryptase, or inhibition of these proteases, all effectively attenuated or abolished experimental AAA growth. The role of MCs have been reproduced in humans in several case-control studies, and two cohort studies showing the systemic level of MC specific chymase and tryptase is associated with aneurysmal growth rate, need for later aneurysmal repair and even overall mortality. These observations offer new opportunities to prevent or slow AAA growth in humans, and specific antimastcell drugs inhibiting degranulation of MCs have been used for especially allergic diseases for decades. It clearly calls for randomized clinical trials as no medical treatment to inhibit AAA progression so far have shown to be efficient.

Mast cells contribute to double-stranded RNA-induced augmentation of airway eosinophilia in a murine model of asthma.

BackgroundClinical studies showed the contribution of viral infection to the development of asthma. Although mast cells have multiple roles in the pathogenesis of allergic asthma, their role of in the virus-associated pathogenesis of asthma remains unknown. Most respiratory viruses generate double-stranded (ds) RNA during their replication. dsRNA provokes innate immune responses. We recently showed that an administration of polyinocinic polycytidilic acid (poly IC), a mimetic of viral dsRNA, during allergen sensitization augments airway eosinophilia and hyperresponsiveness in mice via enhanced production of IL-13.MethodsThe effect of poly IC on allergen-induced airway eosinophilia was investigated for mast cell-conserved Kit+/+ mice and -deficient KitW/KitW-v mice. The outcome of mast cell reconstitution was further investigated.ResultsAirway eosinophilia and IL-13 production were augmented by poly IC in Kit+/+ mice but not in KitW/KitW-v mice. When KitW/KitW-v mice were reconstituted with bone marrow-derived mast cells (BMMCs), the augmentation was restored. The augmentation was not induced in the mice systemically deficient for TIR domain-containing adaptor-inducing IFN-β (TRIF) or interferon regulatory factor (IRF)-3, both mediate dsRNA-triggered innate immune responses. The augmentation was, however, restored in KitW/KitW-v mice reconstituted with TRIF-deficient or IRF-3-deficient BMMCs. Although leukotriene B4 and prostaglandin D2 are major lipid mediators released from activated mast cells, no their contribution was shown to the dsRNA-induced augmentation of airway eosinophilia.ConclusionsWe conclude that mast cells contribute to dsRNA-induced augmentation of allergic airway inflammation without requiring direct activation of mast cells with dsRNA or involvement of leukotriene B4 or prostaglandin D2.

Perivascular Mast Cells Dynamically Probe Cutaneous Blood Vessels to Capture Immunoglobulin E

Mast cells are tissue-resident immune cells that play a central role in allergic disease. These contributions are largely dependent on the acquisition of antigen-specific immunoglobulin E (IgE). Despite this requirement, studies of mast cell and IgE interactions have overlooked the mechanism by which mast cells acquire IgE from the blood. To address this gap, we developed reporter IgE molecules and employed imaging techniques to study mast cell function insitu. Our data demonstrate that skin mast cells exhibit selective uptake of IgE based on perivascular positioning. Furthermore, perivascular mast cells acquire IgE by extending cell processes across the vessel wall to capture luminal IgE. These data demonstrate how tissue mast cells acquire IgE andreveal a strategy by which extravascular cells monitor blood contents to capture molecules central to cellular function.

Mast cells and IgE activation do not alter the development of oral tolerance in a murine model

In addition to their well-known role as potent effector cells in patients with allergic disease, mast cells have important immunomodulatory roles regulating tolerance in allograft rejection models. The roles of mast cells in oral tolerance development have not previously been examined. We sought to evaluate the importance of mast cells, IgE-mediated mast cell activation, and histamine receptor 1 or 2 blockade on oral tolerance development in mice. Oral tolerance was assessed in 2 mast cell-deficient murine strains (Kit(W-sh/W-sh) and Kit(W/W-v) mice) and control mice. Mice were fed ovalbumin (OVA) or peanut butter for 1 week and then immunized and boosted with relevant protein antigens. Antibody responses were assessed by using ELISA. The oral antihistamines pyrilamine and ranitidine were administered during tolerance induction to OVA. IgE-mediated mast cell activation was initiated during oral tolerance induction or OVA immunization. OVA-specific regulatory Tcells were assessed in the Peyer patches, mesenteric lymph nodes, and spleens by using flow cytometry after adoptive transfer. Oral tolerance was successfully induced to OVA and peanut butter in mast cell-deficient mice. Kit(W-sh/W-sh) mice had higher proportions of antigen-specific regulatory T cells in the mesenteric lymph nodes than mast cell-containing control mice. However, mast cell reconstitution studies suggested this effect was mast cell independent. Oral antihistamine treatments with pyrilamine or ranitidine did not impair tolerance and neither did IgE-mediated activation. Mast cells are not necessary for the induction of oral tolerance, and allergic activation of mast cells does not impair tolerance to OVA. Oral antihistamine treatments do not disrupt the development of oral tolerance.

Abstract 317: Mast Cells Do Not Contribute to a Reduction in Vein Graft Neointima in the C-kit Mutant KitW-sh/w-sh Mice

Background: Recent evidence suggests mast cells are involved in the development of vascular diseases such as atherosclerosis. The presence of mast cells in vein grafts led us to hypothesise that mast cells may regulate the extent of vein graft neointima hyperplasia. In this study, we employed the Kit W-sh/W-sh mouse strain, a widely accepted mast cell deficient mouse model induced by a spontaneous inversion mutation in the transcriptional regulatory elements of the c-kit gene. Methods: The vein graft surgery was performed by inserting the inferior thoracic vena cava from the donor mouse into the common carotid artery of the littermate recipient. Vein grafts were harvested at 4 weeks and fixed with 4% formaldehyde for histology. In some groups, Kit W-sh/W-sh mice were reconstituted with cultured mast cells either systemically or locally 9 weeks before vein graft surgery. For primary cell culture studies, aortic endothelial cells and smooth muscle cells were isolated by collagenase digestion method and cultured in standard conditions. Results: Kit w-sh/w-sh vein grafts had a significant reduction (33%; p<0.05) in neointima area, compared to the wild type controls. The cellular composition of the Kit w-sh/w-sh neointima was similar to the wild type controls, most cells being smooth muscle alpha-actin positive. Neither the systemic nor local reconstitution of mast cells restored the neointima area to the wild type level. In the primary cell culture, Kit w-sh/w-sh vascular cells showed less migrating activity in the scratch wound healing test, which may account for the decrease in neointima formation. Conlusion: This study suggests that it is not the deficiency of mast cells in the Kit W-sh/W-sh mice which accounts for the reduction in neointima formation in vein grafts. Other cell types affected by the c-kit mutation may be responsible for the altered neointima formation in Kit W-sh/W-sh mice.

Crucial roles of mast cells in the development of a murine model of polymyositis (171.20)

Abstract Objective: In addition to a pivotal role of mast cells in allergic diseases, recent data suggest that mast cells play a crucial role in a variety of immune responses. However, their role in the pathogenesis of autoimmune skeletal muscle diseases has not been clarified despite their distribution in skeletal muscle. Therefore, the objective of this study is to determine the role of mast cells in the development of a murine model of polymyositis, C protein-induced myositis (CIM). Methods: The susceptibility of mast cell-deficient WBB6F1-KitW/KitWv mice (W/Wv mice) to CIM was compared with that of wild-type (WT) mice. The effect of mast cell reconstitution with bone marrow-derived mast cells (BMMCs) on the susceptibility of W/Wv mice to CIM was also evaluated. Results: W/Wv mice exhibited significantly reduced disease incidence and histological scores of CIM as compared with WT mice. The infiltration of macrophages but not of CD8+ T cells in the skeletal muscle was attenuated in CIM of W/Wv mice. The expression of monocyte chemotactic protein-1 (MCP-1) in skeletal muscle was reduced in W/Wv mice. Vascular permeability in skeletal muscle was elevated in WT mice but not in W/Wv mice upon CIM induction. Engraftment of BMMCs restored the incidence and histological scores of CIM in W/Wv mice. Conclusion: Mast cells play critical roles in the development of CIM.

Cre-Mediated Cell Ablation Contests Mast Cell Contribution in Models of Antibody- and T Cell-Mediated Autoimmunity

Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy.

The pivotal role of fibrocytes and mast cells in mediating fibrotic reactions to biomaterials

Almost all biomaterial implants are surrounded by a fibrotic capsule, although the mechanism of biomaterial-mediated fibrotic reactions is mostly unclear. To search for the types of cells responsible for triggering the tissue responses, we used poly-L glycolic acid polymers capable of releasing various reagents. We first identified that CD45 +/Collagen 1 + fibrocytes are recruited and resided within the fibrotic capsule at the implant interface. Interestingly, we found that the recruitment of fibrocytes and the extent of fibrotic tissue formation (collagen type I production) were substantially enhanced and reduced by the localized release of compound 48/80 and cromolyn, respectively. Since it is well established that compound 48/80 and cromolyn alter mast cell reactions, we hypothesized that mast cells are responsible for triggering fibrocyte recruitment and subsequent fibrotic capsule formation surrounding biomaterial implants. To directly test this hypothesis, similar studies were carried out using mast cell deficient mice, WBB6F1/J- Kit W / Kit W-v /, and their congenic controls. Indeed, mast cell deficient mice prompted substantially less fibrocyte and myofibroblast responses in comparison to C57 wild type mice controls. Most interestingly, subcutaneous mast cell reconstitution of WBB6F1/J- Kit W / Kit W-v /J mice almost completely restored the fibrocyte response in comparison to the C57 wild type response. These results indicate that the initial biomaterial interaction resulting in the stimulation of mast cells and degranulation byproducts not only stimulates the inflammatory cascade but significantly alters the downstream fibrocyte response and degree of fibrosis.

Abstract 3655: Mast cells promote immunosuppression and pancreatic ductal adenocarcinoma development

Abstract Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a mostly uniformly lethal disease and is notoriously resistant to most therapies. Immunotherapy may be a fourth strategy for treating PDAC, supplementing surgery, chemotherapy, and radiation therapy. Immunosuppression is one of the largest bottlenecks in cancer immunotherapy development. In our preliminary studies, we analyzed a panel of immunocytes in human PDAC tissue samples and found that mast cells infiltrated at the tumor margin. This high infiltration of human mast cells into the tumor microenvironment was associated with poor survival. An early influx of mast cells into the tumor microenvironment combined with the IL-10 and receptor upregulation we found in a transgenic spontaneous PDAC mouse model, was associated with malignant progression. In addition, mast cells were found to be essential to PDAC tumorigenesis in a mast cell-deficient Kitw-sh/w-sh mouse model (Kit-/- mice) compared to wild-type C57BL/6 control mice (WT mice). However, how mast cells regulate immunologic effects in the tumor microenvironment and contribute to PDAC tumorigenesis are unknown. We hypothesized that mast cells mediate the immunosuppression that promotes PDAC development. Methods: We quantified mast cells in 16 normal human pancreas and 22 pancreatitis patients’ tissue samples. Panc-02 PDAC cells were orthotopically implanted into the pancreases of Kit-/-, mast cell-reconstituted, and WT mice. In the mast cell reconstitution model, Kit-/- mice received bone marrow-derived mast cells (BMMCs) from IL-10-/-, PGE2-/-, and WT mice. To determine whether targeting mast cells has a therapeutic effect in PDAC, we treated mice PDAC with cromolyn, a mast cell stabilizer. Saline and gemcitabine were used as controls. Tumor sizes were measured by bioluminescence imaging. Results: Mast cell infiltration to human pancreatic disease was associated with inflammation and malignancy progression in PDAC (P<0.0001), which was consistent with our previous observation in the spontaneous PDAC mouse model. The growth of orthotopically implanted PDAC tumor was significantly depressed in mice reconstituted with IL-10-/- BMMCs compared with that in Kit-/- mice reconstituted with normal BMMCs (P = 0.025). Most PDAC tumors that developed in Kit-/- mice reconstituted with IL-10-/- BMMCs were limited to the pancreas, with little peritoneal involvement. No tumors were visible in the pancreas after we treated tumor-bearing mice with cromolyn, which was more effective at eliminating tumors than was low-dose gemcitabine (P < 0.001). Conclusions: Mast cells promote inflammatory progression and immune suppression in PDAC tumorigenesis. Mast cell-targeted therapy resulted in a higher treatment response rate in mouse PDAC than did classic chemotherapy. Therefore, therapy targeting on mast cells may overcome immune suppression and improve PDAC immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3655. doi:10.1158/1538-7445.AM2011-3655

Mast Cells Are an Essential Component of Human Radiation Proctitis and Contribute to Experimental Colorectal Damage in Mice

Radiation proctitis is characterized by mucosal inflammation followed by adverse chronic tissue remodeling and is associated with substantial morbidity and mortality. Mast cell hyperplasia has been associated with diseases characterized by pathological tissue remodeling and fibrosis. Rectal tissue from patients treated with radiotherapy shows mast cell hyperplasia and activation, suggesting that these cells play a role in the development of radiation-induced sequelae. To investigate the role of mast cells in radiation damage, experimental radiation proctitis was induced in a mast cell-deficient (W(sh)/W(sh)) mouse model. The colon and rectum of W(sh)/W(sh) and wild-type mice were exposed to 27-Gy single-dose irradiation and studied after 2 and 14 weeks. Irradiated rodent rectum showed mast cell hyperplasia. W(sh)/W(sh) mice developed less acute and chronic rectal radiation damage than their control littermates. Tissue protection was associated with increased tissue neutrophil influx and expression of several inflammatory mediators immediately after radiation exposure. It was further demonstrated that mast cell chymase, tryptase, and histamine could change human muscularis propria smooth muscle cells into a migrating/proliferating and proinflammatory phenotype. These data show that mast cells have deleterious effects on both acute and chronic radiation proctitis, possibly by limiting acute tissue neutrophil influx and by favoring phenotypic orientation of smooth muscle cells, thus making them active participants in the radiation-induced inflammatory process and dystrophy of the rectal wall.

Tissue Eosinophilia in a Mouse Model of Colitis Is Highly Dependent on TLR2 and Independent of Mast Cells

The mechanisms initiating eosinophil influx into sites of inflammation have been well studied in allergic disease but are poorly understood in other settings. This study examined the roles of TLR2 and mast cells in eosinophil accumulation during a nonallergic model of eosinophilia-associated colitis. TLR2-deficient mice (TLR2(-/-)) developed a more severe colitis than wild-type mice in the dextran sodium sulfate (DSS) model. However, they had significantly fewer eosinophils in the submucosa of the cecum (P < 0.01) and mid-colon (P < 0.01) than did wild-type mice after DSS treatment. Decreased eosinophilia in TLR2(-/-) mice was associated with lower levels of cecal CCL11 (P < 0.01). Peritoneal eosinophils did not express TLR2 protein, but TLR2 ligand injection into the peritoneal cavity induced local eosinophil recruitment, indicating that TLR2 activation of other cell types can mediate eosinophil recruitment. After DSS treatment, mast cell-deficient (Kit(W-sh/W-sh)) mice had similar levels of intestinal tissue eosinophilia were observed as those in wild-type mice. However, mast cell-deficient mice were partially protected from DSS-induced weight loss, an effect that was reversed by mast cell reconstitution. Overall, this study indicates a critical role for indirect TLR2-dependent pathways, but not mast cells, in the generation of eosinophilia in the large intestine during experimental colitis and has important implications for the regulation of eosinophils at mucosal inflammatory sites.

A Critical Role for Mast Cells and Mast Cell-Derived IL-6 in TLR2-Mediated Inhibition of Tumor Growth

Several TLR agonists are effective in tumor immunotherapy, but their early innate mechanisms of action, particularly those of TLR2 agonists, are unclear. Mast cells are abundant surrounding solid tumors where they are often protumorigenic and enhance tumor angiogenesis. However, antitumor roles for mast cells have also been documented. The impact of mast cells may be dependent on their activation status and mediator release in different tumors. Using an orthotopic melanoma model in wild-type C57BL/6 and mast cell-deficient Kit(W-sh/W-sh) mice and a complementary Matrigel-tumor model in C57BL/6 mice, mast cells were shown to be crucial for TLR2 agonist (Pam(3)CSK(4))-induced tumor inhibition. Activation of TLR2 on mast cells reversed their well-documented protumorigenic role. Tumor growth inhibition after peritumoral administration of Pam(3)CSK(4) was restored in Kit(W-sh/W-sh) mice by local reconstitution with wild-type, but not TLR2-deficient, mast cells. Mast cells secrete multiple mediators after Pam(3)CSK(4) activation, and in vivo mast cell reconstitution studies also revealed that tumor growth inhibition required mast cell-derived IL-6, but not TNF. Mast cell-mediated anticancer properties were multifaceted. Direct antitumor effects in vitro and decreased angiogenesis and recruitment of NK and T cells in vivo were observed. TLR2-activated mast cells also inhibited the growth of lung cancer cells in vivo. Unlike other immune cells, mast cells are relatively radioresistant making them attractive candidates for combined treatment modalities. This study has important implications for the design of immunotherapeutic strategies and reveals, to our knowledge, a novel mechanism of action for TLR2 agonists in vivo.

Abstract 3822: Immunosuppressive role of mast cells is essential for the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC)

Abstract Objectives: Pancreatic ductal adenocarcinoma (PDAC), which is characterized by a dense tumor microenvironment containing a cell rich population, is a relatively common malignancy with a dismal prognosis. Our previous studies using a transgenic spontaneous PDAC mouse model (K-rasG12V mice) revealed an early influx of mast cells into the tumor microenvironment. Further, we found decreased tumor growth when PDAC was implanted into mast cell-deficient Kitw-sh/w-sh mice (Kit−/− mice) compared to wild type C57BL/6 control mice (WT mice). To further assess the role of mast cells in the tumorigenesis of human PDAC, we correlated mast cells infiltration with clinical outcome. Using PDAC mouse models, we explored the immunosuppressive mechanisms of mast cells on PDAC in vivo. Methods: To assess the clinical significance of mast cells, we quantified it in a tissue array of 66 human PDAC samples and correlated the quantities with clinical outcome. Pancreas tissues were obtained from K-rasG12V mice and grouped by stages: chronic pancreatitis (CP, N = 3); pancreatic intraepithelial neoplasia (PanIN, N = 3); and PDAC (N = 3). Normal pancreas tissues (NP, N = 3) were obtained from K-ras wild-type mice. RNA expression of IL-10 and its receptors were determined with a cDNA microarray on an Illumina whole-genome-expression array containing 45 281 probes and confirmed by real-time RT-PCR. Panc-02 PDAC cells were orthotopically implanted into the pancreas of Kit−/− mice and WT mice. In the mast cell reconstitution model, Kit−/− mice received intraperitoneal injections of bone marrow-derived mast cells from of IL-10−/−, PGE2−/−, WT mice (BMMC–&amp;gt;Kit−/− mice), and six weeks later were orthotopically implanted with Panc-02 cells. Tumor sizes were measured at day 28 and survival times were monitored. Pancreatic tissues were studied by immunohistochemistry and flow cytometry to analyze immunocytes. Results: A relatively high infiltration of human mast cells into the tumor microenvironment was predictive of poor patient survival (P = 0.008). An early influx of mouse mast cells and upregulation of IL-10 and receptors were consistently found in the microenvironment during PDAC development in K-rasG12V mice. By day 36, none of PDAC tumor bearing WT mice survived, 77.5% of tumor bearing Kit−/− mice survived, and 53.3% of tumor bearing BMMC–&amp;gt;Kit−/− mice survived (P &amp;lt; 0.001). There were significantly more immature neutrophils (CD45+ F4/80− CD11b+ Gr-1low) in the tumor microenvironment of WT mice than in Kit−/− mice (P &amp;lt; 0.001). Preliminary test showed tumor sizes were smaller in IL-10−/− BMMC–&amp;gt;Kit−/− mice than PGE2−/− BMMC–&amp;gt;Kit−/− mice (P = 0.017). Conclusions: Mast cells migrate to the tumor microenvironment and mediate the local immune suppression that promotes the growth and development of PDAC. These data suggest that targeting mast cell immunosuppressive function may be a promising novel therapy for PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3822.

Unique populations of lung mast cells are required for antigen-mediated bronchoconstriction (86.11)

Abstract Several studies implicate mast cells as important effector cells of anaphylaxis. Much of our understanding of the functions of mast cells emanates from the study of mice lacking theses cells, particularly mice carrying mutations in c-Kit. Definitive evidence for the role of mast cells in these mice requires the normalization of altered immune responses by reconstitution with cultured mast cells (BMMC). While many niches can be restored with BMMC, this has not been demonstrated for mast cells present in the airways of the lung, cells poised to mediate bronchoconstriction during allergic responses. To determine if mast cell reconstitution is an appropriate model for the study of bronchoconstriction associated with allergic responses, KitWsh/Wsh mice were engrafted with either whole bone marrow (WBM) or BMMC and responses to IgE-mediated anaphylaxis determined. Engraftment of KitWsh/Wsh mice with WBM or BMMC results in reconstitution of the central airways with mast cells. While the treatment of both groups results similar systemic changes in a model of passive anaphylaxis, bronchoconstriction was observed only in animals which received WBM. While BMMC can populate the lung, they cannot restore IgE/Ag-mediated bronchoconstriction to mast cell-deficient animals. This suggests the mast cell population mediating this function may be unique, and to fill this niche in the lung cells must undergo a specific developmental program that is no longer available to cultured mast cells.