Equine Airway Mast Cells are Sensitive to Cell Death Induced by Lysosomotropic Agents.

Mast cells are known for their detrimental effects in various inflammatory conditions. Regimens that induce selective mast cell apoptosis may therefore be of therapeutic significance. Earlier studies have demonstrated that murine- and human-cultured mast cells are highly sensitive to apoptosis induced by the lysosomotropic agent LeuLeuOMe (LLME). However, the efficacy of lysosomotropic agents for inducing apoptosis of in vivo-derived airway mast cells and the impact on mast cells in other species have not been assessed. Here we addressed whether lysosomotropic agents can induce cell death of equine in vivo-derived mast cells. Bronchoalveolar lavage (BAL) fluids from horses were incubated with LLME at 15-100 µm for up to 48 h. The overall cell viability was unaffected by 15 µm LLME up to 48 h, whereas a relatively modest drop in total cell counts (~30%) was seen at the highest LLME dose used. In contrast to the relatively low effect on total cell counts, LLME efficiently and dose dependently reduced the number of mast cells in BAL fluids, with an almost complete depletion (96%) of mast cells after 24 h of incubation with 100 µm LLME. A significant but less dramatic reduction (up to ~45%) of lymphocytes was also seen, whereas macrophages and neutrophils were essentially resistant. The appearance of apoptotic bodies suggested a mechanism involving apoptosis rather than necrosis. These findings suggest that equine airway mast cells are highly sensitive to lysosomotropic agents. Possibly, lysosomotropic agents could be of therapeutic value to treat disorders involving harmful accumulation of mast cells in the airways.

Metabolic and Hormonal Response to a Feed-challenge Test in Lean and Overweight Dogs.

BACKGROUND: Obese dogs risk poor life quality, creating a need for increased knowledge of metabolism in overweight dogs. OBJECTIVES: Investigate postprandial metabolic and hormonal responses to a high-fat mixed-meal in dogs and responses of lean versus overweight dogs. ANIMALS: Twenty-eight healthy intact male Labrador Retrievers were included. METHODS: Prospective observational study. Twelve dogs were grouped as lean (body condition score (BCS 4-5), 10 as slightly overweight (BCS 6), and 6 as overweight (BCS 6.5-8) on a 9-point scale. After an overnight fast, urine and blood samples were collected. Dogs were then fed a high-fat mixed-meal, and blood was collected hourly for 4 hours and urine after 3 hours. RESULTS: Postprandial concentrations of insulin and glucagon were increased at 1 hour (both P < 0.0001), triglycerides at 2 hours (P < 0.0001), and glucose at 3 hours (P = 0.004); and all remained increased throughout the feed-challenge in all dogs. Postprandial urine cortisol/creatinine ratio was higher than fasting values (P = 0.001). Comparing between groups, there was an overall higher triglyceride response in overweight compared to lean (P = 0.001) and slightly overweight (P = 0.015) dogs. Overweight dogs also had higher fasting cortisol/creatinine ratio compared to lean dogs (P = 0.024). CONCLUSIONS AND CLINICAL IMPORTANCE: Postprandial responses of dogs to a high-fat mixed-meal were similar to those previously reported in people. The higher postprandial triglyceride response and fasting cortisol/creatinine ratio in the overweight dogs could be early signs of metabolic imbalance. Thus, although overweight dogs often appear healthy, metabolic alterations might be present.

Increased concentrations of C-reactive protein but not high-mobility group box 1 in dogs with naturally occurring sepsis.

Sepsis is difficult to diagnose and remains a common mortality cause worldwide in both humans and animals. The uterine infection pyometra causes sepsis in more than half of affected dogs and therefore allows the natural physiological development of sepsis to be studied. To find a sepsis-specific biochemical marker that could be combined with conventional clinical criteria for a more robust and quick diagnosis of sepsis, we measured systemic concentrations of high-mobility group box 1 (HMGB1) in 23 healthy control dogs and in 27 dogs with pyometra, 74% of which had sepsis. We also measured concentrations of the major acute phase protein C-reactive protein (CRP) and an indicator for endotoxaemia, prostaglandin F2α metabolite (PGM) to assess the relative contribution of HMGB1 to the detection of systemic inflammation and endotoxaemia. We found that HMGB1 concentrations, in line with concentrations of CRP and PGM, were significantly increased in dogs with pyometra, and that concentrations of CRP, but not HMGB1, were significantly higher in dogs with sepsis compared to dogs without sepsis. Although serum HMGB1 did not differ between dogs with or without sepsis and was not correlated with either CRP or PGM concentrations, HMGB1 was correlated with the total white blood cell counts, suggesting an independent regulation and involvement in inflammation.

Mast cell chymase modulates IL-33 levels and controls allergic sensitization in dust-mite induced airway inflammation.

Mast cells (MCs) are major effector cells contributing to allergic conditions. When activated, they can release large amounts of active proteases, including chymase from their secretory granules. Here we assessed the role of the chymase mouse mast cell protease 4 (mMCP-4) in allergic airway inflammation induced by house-dust mite (HDM) extract. mMCP-4-/-) mice demonstrated elevated airway reactivity and eosinophilia compared with wild-type (WT) animals, suggesting a protective role for mMCP-4 during the late inflammatory phase of the disease. However, mMCP-4 also contributed to the sensitization phase, as indicated by higher levels of serum immunoglobulin E in mMCP-4(-/-) vs. WT mice and higher levels of cytokines secreted by HDM-restimulated mMCP-4(-/-) vs. WT splenocytes. In line with a contribution of mMCP-4 in the early stages of disease, HDM extract directly induced chymase secretion from MCs. The elevated airway and inflammatory responses of mMCP-4(-/-) mice were associated with a profound increase in the levels of interleukin (IL)-33 in the lung tissue. Moreover, WT MCs degraded IL-33 more efficiently than did MCs lacking mMCP-4. Together, our findings identify a protective role of a MC chymase in a physiologically relevant model for airway inflammation and suggest that chymase-mediated regulation of IL-33 can account for this protective function.

Cytokines as immunological markers for systemic inflammation in dogs with pyometra.

Pyometra is a disease in dogs caused by bacterial infection of the uterus and resulting in SIRS (systemic inflammatory response syndrome) in nearly 6 of 10 cases. Clinical diagnostic criteria for SIRS are relatively unspecific, and biomarkers for the diagnosis of pyometra and SIRS in dogs are needed. Serum samples from 32 dogs were used in this study and grouped into dogs with pyometra and SIRS, dogs with pyometra without SIRS and healthy controls. The serum concentrations of IFN-γ, IL-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18 and TNF-α were measured using multiplex analyses. The serum concentrations of CRP (C-reactive protein) were determined using sandwich ELISA. IL-7, IL-8, IL-15, IL-18 and TNF-α were detected in >94% of samples. IL-10 was detected in 28% of samples, and IL-4, IL-6 and IFN-γ were undetectable. Higher serum concentrations of IL-7 (p < 0.05) were detected in SIRS-positive dogs with pyometra (n = 13) as compared with healthy controls (n = 11). The concentrations of IL-8 were higher in SIRS-positive dogs with pyometra compared to the SIRS-negative group (n = 8; p < 0.05). Positive correlations of IL-15 with IL-18 (p < 0.0001) and with the concentrations of IL-7 (p < 0.0001 for both) were found, although there was no significant difference between groups. Furthermore, IL-15 correlated with concentrations of CRP (p < 0.05), which were higher in dogs with pyometra compared to controls (p < 0.0001). Our data suggest a role of several cytokines in the development of a systemic disease in dogs with pyometra and a possible diagnostic value for serum CRP, IL-7, IL-15 and IL-18 in canine SIRS caused by pyometra.

Lysosomal membrane permeabilization induces cell death in human mast cells.

Mast cells (MC) have pathogenic roles in numerous disorders, and strategies that stabilize MC or induce MC apoptosis are therefore emerging as possible therapeutic regimens. A typical feature of MC is their high content of secretory lysosomes (granules), containing numerous components such as biogenic amines, cytokines, serglycin proteoglycan and proteases. Damage to the secretory lysosomes will thus lead to leakage of these compounds, including the proteases, into the cytosol, and this could potentially trigger apoptosis. Here, we evaluated whether MC are sensitive to cell death induced by secretory lysosome destabilization, induced by the lysosomotropic agent Leu-Leu-OMe (LLME). Human MC were sensitive to LLME-induced cell death. In contrast, fibroblasts and HEK-293 cells were largely resistant. As judged by Annexin V/propidium iodide staining, LLME caused apoptotic cell death, and this was supported by induction of caspase-3-like activity, detection of activated caspase-3 by immunoblot analysis and reduced cell death in the presence of a caspase inhibitor. In support of a role for serglycin in regulating LLME-induced cell death, the survival rate of various cell types correlated negatively with the level of serglycin expression. In summary, this study introduces the concept of using lysosomotropic agents to induce cell death of human MC.

Isolation of transcriptionally active umbilical cord blood-derived basophils expressing Fc epsilon RI, HLA-DR and CD203c.

BACKGROUND: Basophils are inflammatory cells associated with allergy and parasite infections. Investigation of their true biological function has long been hampered by the difficulty in obtaining sufficient amounts of pure basophils and by the lack of phenotypic markers. Moreover, it has been very difficult to clone and identify basophil-specific granule proteins, partially because of an almost complete lack of mRNA in mature circulating basophils. METHODS: To obtain transcriptionally active immature basophils, umbilical cord blood cells were cultured in the presence of interleukin (IL)-3. The cells were analysed by flow cytometry and by histological staining. RESULTS: The continuous presence of IL-3 in cord blood cultures resulted in the expansion of basophil precursors co-expressing FcepsilonRI and the recently described mast cell/basophil marker, 97A6 (CD203c). Several nonbasophil markers (i.e. CD3, CD14, CD15, CD16, CD19 and CD21) were absent on the cultured basophils. However, we show that in early cultures, almost 60% of the CD203c+ cells co-express human leukocyte antigen (HLA)-DR, a marker that is absent on mature circulating basophils. The presence of HLA-DR on basophil precursors may explain the low recovery (24+/-5.2%) obtained after isolation of cultured basophils, when using a conventional basophil isolation kit that removes HLA-DR+ cells. A novel purification method was developed, including a two-step cocktail of antibodies against selected markers, which resulted in both high purity (95+/-0.5%) and recovery (59+/-1.5%) of cultured basophils. CONCLUSIONS: We here establish cord blood cultures as a source from which transcriptionally active basophil precursors can be isolated in reasonable quantities for thorough biochemical characterization.

Immune complex-mediated enhancement of antibody responses without induction of delayed-type hypersensitivity.

Immunoglobulin (Ig)G and IgE antibodies enhance the humoral response in vivo to soluble antigens with which they form complexes. In vitro, antigen is targeted to B cells by IgE antibodies and to macrophages and dendritic cells (DCs) by IgG, thus leading to increased antigen presentation to specific T cells. Possibly these mechanisms are also responsible for antibody-mediated enhancement in vivo. We now address the question of whether IgG- and/or IgE-antigen complexes can prime for delayed-type hypersensitivity (DTH), a reaction known to require primed T helper (Th)1 cells. Mice were immunized with IgG-anti-2,4,6-trinitrophenyl (TNP)/BSA-TNP or IgE-anti-TNP/BSA-TNP. Mice given BSA-TNP alone or BSA-TNP in complete Freund's adjuvans (CFA) were used as controls. DTH and IgG-anti-BSA levels were measured after subsequent challenge with BSA. A potent BSA-specific antibody response was induced by IgE- or IgG-complexed antigen as well as by CFA/antigen but DTH-reactions were only observed in mice immunized with CFA/antigen. Both IgE and IgG enhanced the production of BSA-specific IgG1, IgG2a and IgG2b, although the most pronounced enhancement was seen in the production of IgG1. These findings suggest that Th2 cells rather than Th1 cells are involved in the immune response to IgG- and IgE-immune complexes.

Restoration of the antibody response to IgE/antigen complexes in CD23-deficient mice by CD23+ spleen or bone marrow cells.

Mice immunized with IgE/Ag complexes produce significantly more Ag-specific Abs than mice immunized with Ag alone. The enhancement is mediated via the low-affinity receptor for IgE (FcepsilonRII or CD23), as shown by its complete absence in mice pretreated with mAbs specific for CD23 and in CD23-deficient mice. Because the constitutive expression of murine CD23 is limited to B cells and follicular dendritic cells (FDCs), one of these cell types is likely to be involved. One of the suggested modes of action of IgE/CD23 is to increase the ability of B cells to present Ag to T cells, as demonstrated to take place in vitro. Another possibility is that FDCs capture the IgE/Ag complexes and present these directly to B cells. The purpose of the present study was to determine whether CD23+ B cells or FDCs are responsible for the IgE/CD23-mediated enhancement of specific Ab responses in vivo. We show that the enhancement is completely restored in irradiated CD23-deficient mice reconstituted with CD23+ spleen or bone marrow cells. In these mice, the B cells are CD23+ and the FDCs are presumably CD23- because the FDCs are radiation resistant and are reported not to be replaced by donor cells after this type of cell transfer. In contrast, enhancement was not restored in irradiated wild-type mice reconstituted with CD23- cells. These results indicate that CD23+ B cells, and not FDCs, are the cells that capture IgE/Ag complexes and induce enhancement of Ab responses in vivo.

IgG-mediated enhancement of antibody responses is low in Fc receptor gamma chain-deficient mice and increased in Fc gamma RII-deficient mice.

Immunization with IgG/Ag or IgE/Ag complexes leads to a higher production of specific Abs than immunization with Ag alone. The enhancing effect of IgE is exclusively dependent upon the low-affinity receptor for IgE, Fc epsilon RII, whereas the mechanism behind IgG-mediated enhancement is unknown. We have investigated whether receptors for the Fc part of IgG are required for responses to IgG/Ag. Mice lacking the gamma subunit of Fc receptors (FcRs) (FcR gamma-/-), Fc gamma RII (Fc gamma RII-/-), or Fc gamma RIII (Fc gamma RIII-/-) were immunized with BSA-2,4,6-trinitrophenyl (TNP) alone or BSA-TNP complexed to monoclonal TNP-specific IgG1, IgG2a, or IgG2b. As expected, all subclasses enhanced the Ab-response to BSA in wild-type mice. Enhancement was in the same order of magnitude in Fc gamma RIII-/- mice (

Efficient IgG-mediated suppression of primary antibody responses in Fcgamma receptor-deficient mice.

IgG antibodies can suppress more than 99% of the antibody response against the antigen to which they bind. This is used clinically to prevent rhesus-negative (Rh-) women from becoming immunized against Rh+ erythrocytes from their fetuses. The suppressive mechanism is poorly understood, but it has been proposed that IgG/erythrocyte complexes bind to the inhibitory Fc receptor for IgG (FcgammaRIIB) on the B cell surface, thereby triggering negative signals that turn off the B cell. We show that IgG induces the same degree of suppression of the response to sheep erythrocytes in animals lacking the known IgG-binding receptors FcgammaRIIB, FcgammaRI + III, FcgammaRI + IIB + III, and FcRn (the neonatal Fc receptor) as in wild-type animals. Reinvestigation of the ability of F(ab')2 fragments to suppress antibody responses demonstrated that they were nearly as efficient as intact IgG. In addition, monoclonal IgE also was shown to be suppressive. These findings suggest that IgG inhibits antibody responses through Fc-independent mechanisms, most likely by masking of antigenic epitopes, thereby preventing B cells from binding and responding to antigen. In agreement with this, we show that T cell priming is not abolished by passively administered IgG. The results have implications for the understanding of in vivo regulation of antibody responses and Rh prophylaxis.