Murine Peritoneal Cavity Research Articles

A Technique to Isolate Primary Mast Cells from a Murine Peritoneal Cavity

A Technique to Isolate Primary Mast Cells from a Murine Peritoneal Cavity

A Technique to Isolate Primary Mast Cells from a Murine Peritoneal Cavity

A Technique to Isolate Primary Mast Cells from a Murine Peritoneal Cavity

The synthetic antimicrobial peptide LTX21 induces inflammatory responses in a human whole blood model and a murine peritoneum model.

The global spread of antimicrobial resistance and the increasing number of immune-compromised patients are major challenges in modern medicine. Targeting bacterial virulence or the human host immune system to increase host defence are important strategies in the search for novel antimicrobial drugs. We investigated the inflammatory response of the synthetic short antimicrobial peptide LTX21 in two model systems: a human whole blood exvivo model and a murine invivo peritoneum model - both reflecting early innate immune response. In the whole blood model, LTX21 increased the secretion of a range of different cytokines, decreased the level of tumour necrosis factor (TNF) and activated the complement system. In a haemolysis assay, we found 2.5% haemolysis at a LTX21 concentration of 500mg/L. In the murine model, increased influx of white blood cells (WBCs) and polymorphonuclear neutrophils (PMNs) in the murine peritoneal cavity was observed after treatment with LTX21. In addition, LTX21 increased monocyte chemoattractant protein-1 (MCP-1). In conclusion, LTX21 affected the inflammatory response; the increase in cytokine secretion, complement activation and WBC influx indicates an activated inflammatory response. The present results indicate the impact of LTX21 on the host-pathogen interplay. Whether this will also affect the course of infection has to be investigated.

Loss of humoral immunity in a mouse model of ovarian cancer

Abstract Although a low incidence cancer, ovarian carcinoma (OvCa) has a high mortality rate due to late detection. To study the humoral immune response to OvCa we transplant ID8 cells (mouse epithelial carcinoma) into the murine peritoneal cavity (PerC). We found that as the OvCa developed PerC B cells, particularly B-1 B cells, were depleted. To assess the systemic impact of this depletion we assessed humoral immunity. We found a reduction in “natural” IgM and IgG3 production and increased IgA and IgG1. Following immunization, there was loss of the TI-2 response (FITC-dextran, FITC-Ficoll); the TI-1 (FITC-LPS) response was intact until very late stage disease. Since B-1 cells also serve a housekeeping role in apoptotic corpse clearance, we used a FACS assay and a cell-based ELISA to monitor antibodies directed at apoptotic ID8 cells. Mice with OvCa had lower titers of sera that bound apoptotic ID8 cells. Collectively these data validate the systemic loss of B-1 B cells in mice with OvCa. These results could serve to inform strategies designed for early detection of OvCa.

Development and evaluation of a modified agglutination test for diagnosis of Toxoplasma infection using tachyzoites cultivated in cell culture

Toxoplasma gondii is a zoonotic protozoan parasite that can cause significant disease in most warm-blooded animals, including humans. Surveillance testing of human and animal populations is essential to estimate disease prevalence, assess food safety risks and establish control programmes. Serological tests are the most practical methods to detect the prevalence of infection in a broad range of host populations. The modified agglutination test (MAT) is a sensitive serological method to detect Toxoplasma IgG antibodies in livestock and wild animals. An in-house MAT was developed using tachyzoites produced by in vitro cultivation instead of traditional propagation in murine peritoneal cavity. The assay was evaluated using samples of serum and/or meat juice from pigs and cats experimentally infected with T. gondii. Samples were also tested by a commercially available MAT kit. Comparative analysis of test results from serum and meat juice samples showed excellent agreement between the in-house MAT and the commercial MAT. Serum and/or blood samples from naturally infected cats, sheep, and 20 wildlife host species were also tested by the in-house MAT, with overall results comparable to those obtained using the commercial MAT kit. Therefore, this new MAT is an efficient and convenient method for testing a variety of terrestrial and aquatic domestic or wild host species for T. gondii.

Calprotectin Increases the Activity of the SaeRS Two Component System and Murine Mortality during Staphylococcus aureus Infections.

Calprotectin, the most abundant cytoplasmic protein in neutrophils, suppresses the growth of Staphylococcus aureus by sequestering the nutrient metal ions Zn and Mn. Here we show that calprotectin can also enhance the activity of the SaeRS two component system (TCS), a signaling system essential for production of over 20 virulence factors in S. aureus. The activity of the SaeRS TCS is repressed by certain divalent ions found in blood or neutrophil granules; however, the Zn bound-form of calprotectin relieves this repression. During staphylococcal encounter with murine neutrophils or staphylococcal infection of the murine peritoneal cavity, calprotectin increases the activity of the SaeRS TCS as well as the production of proinflammatory cytokines such as IL-1β and TNF-α, resulting in higher murine mortality. These results suggest that, under certain conditions, calprotectin can be exploited by S. aureus to increase bacterial virulence and host mortality.

Isolation of murine peritoneal macrophages to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer's thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequentlygene expression was evaluated.

882: Early development of CD49d-high Th1 memory phenotype CD4+T cells in the murine peritoneal cavity

882: Early development of CD49d-high Th1 memory phenotype CD4+T cells in the murine peritoneal cavity

Role of main neuroendocrine pathways activated by swim stress on mast cell-dependent peritoneal TNF production after LPS administration in mice

To characterize the effects of swim stress on the early mast cell (MC)-dependent peritoneal production of TNF in response to lipopolysaccharide (LPS) administration in mice, identifying the neuroendocrine mediators involved. Ten to twelve-week-old Swiss Webster, C57BL/6J or c-Kit (Wsh/Wsh) mice were used. Animals were intraperitoneally challenged with LPS at different times after forced swimming (FS) and peak TNF production was determined in peritoneal washes at optimal time after LPS administration. Selective blockage of main neuroendocrine pathways was performed before swim stress. TNF concentrations were determined by ELISA. FS provoked an immediate and transient inhibition of LPS-elicited, MC-dependent TNF accumulation in peritoneum, which lasted around 30min. Suppresive effects of FS were absent on MC-deficient c-Kit (Wsh/Wsh) mice but were recovered after reconstitution with MC. Adrenalectomy or DSP4 administration increased basal ip TNF levels and enhanced LPS-induced TNF release without any effect on stress-induced inhibitory effects, mifepristone did not produce any change on stress-induced inhibition, whereas mecamylamine administration increased basals and attenuated stress effects. Swim stress transiently inhibits the canonical MC-dependent response of TNF production in response to LPS in murine peritoneal cavity with the main participation of the cholinergic anti-inflammatory reflex.

Morphine Prevents Lipopolysaccharide-Induced TNF Secretion in Mast Cells Blocking IκB Kinase Activation and SNAP-23 Phosphorylation: Correlation with the Formation of a β-Arrestin/TRAF6 Complex

We have previously shown that morphine pretreatment inhibits mast cell-dependent TNF production after LPS injection in the murine peritoneal cavity. In this study, we used bone marrow-derived mast cells (BMMCs) to investigate the molecular mechanisms of that inhibition. We found that morphine prevented LPS-induced TNF secretion in these cells. The observed inhibition was not due to morphine-induced TLR4 internalization and it was related to the blockage of preformed TNF secretion. LPS-induced TNF exocytosis in BMMCs was dependent on tetanus toxin-insensitive vesicle-associated membrane proteins and calcium mobilization, as well as PI3K, MAPK, and IκB kinase (IKK) activation. TNF secretion was also associated to the phosphorylation of synaptosomal-associated protein 23 (SNAP-23), which was found forming a complex with IKK in LPS-activated BMMCs. Morphine pretreatment prevented TLR4-dependent ERK and IKK phosphorylation. Analyzing the signaling events upstream of IKK activation, we found diminished TGF-β-activated kinase 1 (TAK1) phosphorylation and TNFR-associated factor (TRAF) 6 ubiquitination in BMMCs pretreated with morphine and stimulated with LPS. Morphine pretreatment provoked a marked increase in the formation of a molecular complex composed of TRAF6 and β-arrestin-2. Naloxone and a combination of μ and δ opioid receptor antagonists prevented morphine inhibitory actions. In conclusion, our results show that activation of μ and δ opioid receptors with morphine suppresses TLR4-induced TNF release in mast cells, preventing the IKK-dependent phosphorylation of SNAP-23, which is necessary for TNF exocytosis, and this inhibition correlates with the formation of a β-arrestin-2/TRAF6 complex. To our knowledge, these findings constitute the first evidence of molecular crosstalk between opioid receptors and the TLR4 signal transduction system in mast cells.

A distinct subset of podoplanin (gp38) expressing F4/80+ macrophages mediate phagocytosis and are induced following zymosan peritonitis

Macrophages are important tissue resident cells that regulate the dynamics of inflammation. However, they are strikingly heterogeneous. During studies looking at podoplanin (gp38) expression on stromal cells in the murine spleen and peritoneal cavity we unexpectedly discovered that podoplanin was expressed on a subset of F4/80 + macrophages; a subset which we have termed fibroblastic macrophages (FM). These cells function as phagocytes in vitro as measured by bead mediated phagocytosis assays. FM also exist at high frequency in the peritoneal cavity and in zymosan induced peritonitis in vivo. These FM represent a unique subgroup of F4/80 + macrophages and their presence in the inflamed peritoneum suggests that they play a role in zymosan induced peritonitis.

Erythropoietin increases macrophage-mediated suppression in a model of the tumor microenvironment (100.1)

Abstract Erythropoietin (EPO), a glycoprotein that increases RBC production, is used to reverse chemotherapy-induced anemia in cancer patients. Unfortunately some patients have experienced accelerated tumor growth and decreased survival rates. We have been modeling the in vivo tumor microenvironment by culturing murine peritoneal cavity (PerC) cells in vitro. Many tumors have significant leucocyte composition including increased representation of myeloid suppressor cells. When PerC T cells are activated in a macrophage-rich environment such as this their proliferation is suppressed. Interferon-gamma (IFN-g)-triggered inducible nitric oxide synthase (iNOS) expression by macrophages drives T cell suppression via arginine catabolism. Addition of 1-methyl arginine (1-MA), a competitive inhibitor of iNOS, recovers the T cell response. We have studied EPO’s impact upon T cell activation in the PerC macrophage model. Exogenous EPO increased T cell suppression by C57BL/6J (wildtype) macrophages and this effect could be blocked by the addition of 1-MA. EPO’s enhanced suppressive effect was not evident with macrophages from iNOS or IFN-g receptor knockout mice. Studies of BALB/c PerC T cells, which were less susceptible to iNOS-mediated suppression, revealed that EPO had no effect upon T cell activation. These results suggest that exogenous EPO can increase T cell suppression and does so by acting through an IFN-g/iNOS-triggered myeloid cell pathway. Supported by NIH AREA R15-CA136901-01.

Gelatinase B/MMP-9 as an inflammatory marker enzyme in mouse zymosan peritonitis: Comparison of phase-specific and cell-specific production by mast cells, macrophages and neutrophils

Neutrophil infiltration during zymosan peritonitis depends on matrix metalloproteinase-9 (MMP-9) activity as it is impaired both in MMP-9 −/− and gelatinase inhibitor-treated animals. The producer cells of MMP-9 and their relative contribution are not known. The aim of this study was to identify and compare the cellular sources, timing and intensity of MMP-9 induction by zymosan in the murine peritoneal cavity. We detected MMP-9 mRNA in unstimulated peritoneal leukocytes and its levels increased after zymosan administration. To detect MMP-9 by flow cytometry, we selected and compared two specific monoclonal antibodies. We show that MMP-9 protein was absent in control peritoneal macrophages, whereas already at 30 min of peritonitis almost all macrophages were producing the enzyme. Conversely, MMP-9 was constitutively present in unstimulated mast cells. Macrophages turned out to be prevalent MMP-9 producers in the early phase of peritonitis. During later stages macrophages kept the high expression of MMP-9 for at least 6 h of inflammation. In contrast, the early phase expression of MMP-9 by neutrophils was limited albeit the highest percentage of MMP-9 + neutrophils was observed at 2 h but absolute numbers of the MMP-9 carrying neutrophils were low at that time. In contrast, during the late phase of peritonitis neutrophils became major producers of MMP-9 as they numerously infiltrated peritoneum. In conclusion, the study reports detection of MMP-9 at the single-cell level during peritonitis, demonstrates unexpectedly fast MMP-9 expression in macrophages and reveals quantitatively phase-specific contribution of mast cells, macrophages and neutrophils.

The nonopsonic allogeneic cell phagocytosis of macrophages detected by flow cytometry and two photon fluorescence microscope

Phagocytosis, one of the apparent functions for macrophages, represents an early and crucial event in triggering host defenses against invading pathogens as well as allo- or xenogeneic rejection. Now, some methods have been used in detecting the opsonic phagocytosis of macrophages in xenogeneic settings. Efficient nonopsonic phagocytosis analysis method has not been established yet. In the present studies, allogeneic lymphocytes pre-labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) or derived from green fluorescent protein transgeneic B6 mice (GFP-B6 mice) were co-incubated with primary murine peritoneal macrophages (PEMs) for 1–2 h or were injected into murine peritoneal cavity for 30 to 240 min. Assays by flow cytometry (FCM) and two photon laser scanning microscope (TPM) showed an efficient uptake of both allogeneic lymphocytes and xenogeneic chicken red blood cells. The continuing process of nonopsonic phagocytosis of allogeneic lymphocytes by PEMs was recorded by TPM. Furthermore, the phenotype differences of PEMs with or without phagocytosis of allogeneic cells were determined by three-color FCMs. Significantly upregulated expressions of CD11b, CD44, TLR2 and TLR4 on PEMs were observed as early as 6 h after phagocytosis of allogeneic cells. Our present data indicated that the FCM and TPM combined method is a practical approach to detect macrophage nonopsonic phagocytosis of allogeneic lymphocytes and to identify the phenotype alteration of macrophages after phagocytosis.

D-Trp8]-γ-Melanocyte-Stimulating Hormone Exhibits Anti-Inflammatory Efficacy in Mice Bearing a Nonfunctional MC1R (Recessive Yellow e/e Mouse)

Two melanocortin receptors (MC1 and MC3R) have been identified as main transducers of the anti-inflammatory effects of natural and synthetic melanocortins. In this study, we have taken advantage of the recent description of the selective MC3R agonist [d-Trp(8)]-gamma-melanocyte-stimulating hormone (MSH) and of the recessive yellow (e/e) mouse, bearing a nonfunctional MC1R, thereby incrementing our knowledge on this topic. Culturing peritoneal macrophages of recessive yellow (e/e) mice with [d-Trp(8)]-gamma-MSH led to accumulation of cAMP, indicating MC3R receptor functionality: this effect was blocked by a neutralizing antibody against MC3R. Likewise, release of the chemokine KC by urate crystals was attenuated by [d-Trp(8)]-gamma-MSH, and this effect was prevented by synthetic [Ac-Nle(4)-c[Asp(5)-2'-Nal(7),Lys(10)]alpha-MSH(4-10)-NH(2) (SHU9119)] and natural [agouti-related protein (AGRP)] MC3R antagonists but not by the MC4R antagonist Ac-Cys-Nle-Arg-His-d-2-Nal-Arg-Trp-Cys-NH(2) (HS024). Systemic treatment of mice with [d-Trp(8)]-gamma-MSH inhibited KC release and polymorphonuclear cell accumulation elicited by urate crystals in the murine peritoneal cavity. SHU9119 and AGRP prevented the inhibitory actions of [d-Trp(8)]-gamma-MSH, whereas HS024 was inactive. We also demonstrate here that [d-Trp(8)]-gamma-MSH displays a dual mechanism of action by inducing the anti-inflammatory protein heme-oxygenase 1 (HO-1). Treatment with the HO-1 inhibitor zinc protoporphyrin IX exacerbated the inflammatory response elicited by urate crystals and abrogated the anti-inflammatory effects of [d-Trp(8)]-gamma-MSH. In conclusion, these data support the development of the selective MC3R agonist [d-Trp(8)]-gamma-MSH for the treatment of inflammatory pathologies, based on a dual mechanism of cytokine/chemokine inhibition and induction of the anti-inflammatory protein HO-1.

Efficient delivery of small interfering RNA for inhibition of IL-12p40 expression in vivo

BackgroundRNA interference is an evolutionary conserved immune response mechanism that can be used as a tool to provide novel insights into gene function and structure. The ability to efficiently deliver small interfering RNA to modulate gene expression in vivo may provide new therapeutic approaches to currently intractable diseases.MethodsIn vitro, siRNA targeting IL-12p40 was delivered to the murine macrophage cell line (J774A.1) encapsulated in a liposome with an IL-12 inducing agent (LPS/IFN-γ) over a number of time points. Controls included a variety of non-target specific siRNA reagents. Supernatants were analyzed for cytokine production while the cells were removed for mRNA profiling.In vivo, siRNA-targeting IL-12p40 was delivered to the murine peritoneal cavity in a therapeutic fashion, after endotoxin (LPS) challenge. Cells from the peritoneal cavity were removed by lavage and analyzed by flow cytometry. Levels of IL-12 present in lavage and in serum were also examined by ELISA.ResultsIn this report, we show that IL-12p40 siRNA can specifically silence macrophage expression of IL-12p40 mRNA and IL-12p70 protein in vitro. We extend this finding to demonstrate that delivery of liposome encapsulated siRNA targeting IL-12p40 to the murine peritoneal cavity can modulate an inflammatory stimulus in vivo. Furthermore, specific siRNA can be used therapeutically after endotoxin challenge to reduce both the local and systemic inflammatory response. Thus, the delivery of siRNA can be used to elicit specific non-permanent inhibition of endogenous protein expression.ConclusionIn vitro silencing of IL-12p40 using siRNA at selected doses leads to specific knockdown of IL-12p70 protein production without inducing type I interferons. Furthermore, siRNA targeting murine IL-12p40 can be used therapeutically to counter an inflammatory response in vivo.

Macrophage distinguishes Vibrio cholerae hemolysin from its protease insensitive oligomer by time dependent and selective expression of CD80–CD86

The monomeric and oligomeric forms of Vibrio cholerae hemolysin (HlyA), a membrane damaging toxin that forms transmembrane pentameric diffusion channels in target eukaryotic membrane, show a pronounced difference in protease susceptibility, presumably due to masking of sensitive peptide bonds during oligomerization. In this work, we examined if resistance of a protein to proteolytic processing affects the expression of costimulatory molecules, CD80 and CD86, on macrophage exposed to the same antigen. The murine peritoneal cavity macrophages expressed both CD80 and CD86 after 24 h of incubation with HlyA monomer but failed to express the costimulatory molecules when treated with the HlyA oligomer. The expression of CD80 molecule on macrophage after 48 h by the HlyA oligomer that failed to express the costimulatory molecules after 24 h indicates that proteolytic processing plays a decisive role in expression of CD80 and CD86 on cell surface.

Redundancy of a functional melanocortin 1 receptor in the anti-inflammatory actions of melanocortin peptides: studies in the recessive yellow (e/e) mouse suggest an important role for melanocortin 3 receptor.

The issue of which melanocortin receptor (MC-R) is responsible for the anti-inflammatory effects of melanocortin peptides is still a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of MC3-R mRNA and protein in peritoneal macrophages (M phi) collected from recessive yellow (e/e) mice and wild-type mice. This receptor was functional as Mphi incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro M phi activation, determined as release of the CXC chemokine KC and IL-1 beta, was inhibited by the more selective MC3-R agonist gamma(2)-melanocyte stimulating hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited IL-1 beta release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the inflammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of gamma(2)-melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R expressed on peritoneal M phi leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e) mice.

Primed peritoneal B lymphocytes are sufficient to transfer protection against Brugia pahangi infection in mice.

Lymphatic filariasis is a tropical disease caused by the nematode parasites Wuchereria bancrofti and Brugia malayi. Whereas the protective potential of T lymphocytes in filarial infection is well documented, investigation of the role of B lymphocytes in antifilarial immunity has been neglected. In this communication, we examine the role of B lymphocytes in antifilarial immunity, using Brugia pahangi infections in the murine peritoneal cavity as a model. We find that B lymphocytes are required for clearance of primary and challenge infections with B. pahangi third-stage larvae (L3). We assessed the protective potential of peritoneal B lymphocytes by adoptive transfer experiments. Primed but not naïve peritoneal B cells from wild-type mice that had been immunized with B. pahangi L3 protected athymic recipients from challenge infection. We evaluated possible mechanisms by which B cells mediate protection. Comparisons of cytokine mRNA expression between B-lymphocyte-deficient and immunocompetent mice following B. pahangi infection suggest that B cells are required for the early production of Th2-type cytokines by peritoneal cells. In addition, B-cell-deficient mice demonstrate a defect in inflammatory cell recruitment to the peritoneal cavity following B. pahangi infection. The data demonstrate a critical role of B lymphocytes in antifilarial immunity in naïve mice and in the memory response in primed mice.

Cytokine-dependent inflammatory cell recruitment patterns in the peritoneal cavity of mice exposed to the parasitic nematode Brugia malayi.

Mice exposed intraperitoneally to either adult or first larval stage (microfilaria) of the human nematode parasite Brugia malayi display polarized cytokine responses. We have used this model to investigate the impact of altered cytokine profiles on inflammatory cell recruitment patterns in vivo. Here we demonstrate that Th2-inducing adult parasites drive the recruitment of eosinophils and macrophages after implant into the murine peritoneal cavity whereas Th1-inducing microfilaria do not. The underlying mechanism of recruitment was further defined by use of mice deficient in the key Th2 cytokines IL-4 or IL5 and mice that lack T cells (nude mice). Recruitment dynamics differed in IL-4 and IL-5 deficient mice, showing reduced or absent eosinophilia. These data emphasize the pivotal role of these cytokines in shaping the cellular profile of inflammatory responses. Surprisingly, the absence of T cells failed to influence inflammatory cell recruitment indicating that recruitment signals are provided by other cell types.