Congenic Littermates Research Articles

Neutrophil-specific deletion of Syk results in recruitment-independent stabilization of the barrier and a long-term improvement in cognitive function after traumatic injury to the developing brain

While traumatic brain injury (TBI) is the leading cause of death and disability in children, we have yet to identify those pathogenic events that determine the extent of recovery. Neutrophils are best known as “first responders” to sites of infection and trauma where they become fully activated, killing pathogens via proteases that are released during degranulation. However, this activational state may generate substantial toxicity in the young brain after TBI that is partially due to developmentally regulated inadequate antioxidant reserves. Neutrophil degranulation is triggered via a downstream signaling pathway that is dependent on spleen tyrosine kinase (Syk). To test the hypothesis that the activational state of neutrophils is a determinant of early pathogenesis and long-term recovery, we compared young, brain-injured conditional knockouts of Syk (sykf/fMRP8-cre+) to congenic littermates (sykf/f). Based upon flow cytometry, there was an extended recruitment of distinct leukocyte subsets, including Ly6G+/Ly6C− and Ly6G+/Ly6Cint, over the first several weeks post-injury which was similar between genotypes. Subsequent assessment of the acutely injured brain revealed a reduction in blood-brain barrier disruption to both high and low molecular weight dextrans and reactive oxygen species in sykf/fMRP8-cre+ mice compared to congenic littermates, and this was associated with greater preservation of claudin 5 and neuronal integrity, as determined by Western blot analyses. At adulthood, motor learning was less affected in brain-injured sykf/fMRP8-cre+ mice as compared to sykf/f mice. Performance in the Morris Water Maze revealed a robust improvement in hippocampal-dependent acquisition and short and long-term spatial memory retention in sykf/fMRP8-cre+ mice. Subsequent analyses of swim path lengths during hidden platform training and probe trials showed greater thigmotaxis in brain-injured sykf/f mice than sham sykf/f mice and injured sykf/fMRP8-cre+ mice. Our results establish the first mechanistic link between the activation state of neutrophils and long-term functional recovery after traumatic injury to the developing brain. These results also highlight Syk kinase as a novel therapeutic target that could be further developed for the brain-injured child.

Retinal proteome alterations in a mouse model of type 2 diabetes

Aims/hypothesisDiabetic retinopathy is a major complication of type 2 diabetes and the leading cause of blindness in adults of working age. Neuronal defects are known to occur early in disease, but the source of this dysfunction is unknown. The aim of this study was to examine differences in the retinal membrane proteome among non-diabetic mice and mouse models of diabetes either with or without metformin treatment.MethodsAlterations in the retinal membrane proteome of 10-week-old diabetic db/db mice, diabetic db/db mice orally treated with the anti-hyperglycaemic metformin, and congenic wild-type littermates were examined using label-free mass spectrometry. Pathway enrichment analysis was completed with Genomatix and Ingenuity. Alterations in Slc17a7 mRNA and vesicular glutamate transporter 1 (VGLUT1) protein expression were evaluated using real-time quantitative PCR and immunofluorescence.ResultsA total of 98 proteins were significantly differentially abundant between db/db and wild-type animals. Pathway enrichment analysis indicated decreases in levels of proteins related to synaptic transmission and cell signalling. Metformin treatment produced 63 differentially abundant proteins compared with untreated db/db mice, of which only 43 proteins were found to occur in both datasets, suggesting that treatment only partially normalises the alterations induced by diabetes. VGLUT1, which is responsible for loading glutamate into synaptic vesicles, was found to be differentially abundant in db/db mice and was not normalised by metformin. The decrease in Slc17a7/VGLUT1 was confirmed by transcriptomic and immunocytochemical analysis.Conclusions/interpretationThese findings expand the knowledge of the protein changes in diabetic retinopathy and suggest that membrane-associated signalling proteins are susceptible to changes that are partially ameliorated by treatment.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-013-3070-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Expression of human CEACAM1 in transgenic mice limits the Opa-specific immune response against meningococcal outer membrane vesicles

Outer membrane vesicles (OMVs) have been extensively investigated as meningococcal vaccine candidates. Among their major components are the opacity (Opa) proteins, a family of surface-exposed outer membrane proteins important for bacterial adherence and entry into host cells. Many Opa-dependent interactions are mediated through the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family of receptors. Importantly, binding of Opa to CEACAM1 has been reported to suppress human CD4 T cell proliferation in vitro in response to OMV preparations. This raises the question whether OMV vaccines should contain Opa proteins at all. Until now it has been difficult to answer this question, as the proposed immunosuppressive effect was only demonstrated with human cells in vitro, while immunization experiments in mice are not informative because the Opa interaction is specific for human CEACAM1. In the present study we have used Opa+ and Opa− OMVs for immunization experiments in a human CEACAM1 transgenic mouse model. OMVs were prepared from a meningococcal strain H44/76 variant expressing the CEACAM1-binding OpaJ protein, and from an isogenic variant in which all opa genes have been inactivated. Both the CEACAM1 expressing transgenic mice and their congenic littermates lacking it were immunized twice with the OMV preparations, and the sera were analyzed for bactericidal activity and ELISA antibody titres. Total IgG antibodies against the OMVs were similar in both mouse strains. Yet the titres for IgG antibodies specific for purified OpaJ protein were significantly lower in the mice expressing human CEACAM1 than in the nontransgenic mice. No significant differences were found in bactericidal titres among the four groups. Overall, these data indicate that expression of human CEACAM1 confers a reduced Opa-specific antibody response in vivo without affecting the overall immune response against other OMV antigens.

Increased GABAA channel subunits expression in CD8+ but not in CD4+ T cells in BB rats developing diabetes compared to their congenic littermates

GABA (γ-aminobutyric acid), the main inhibitory neurotransmitter in the central nervous system is also present in the pancreatic islet β cells where it may function as a paracrine molecule and perhaps as an immunomodulator of lymphocytes infiltrating the pancreatic islet. We examined CD4+ and CD8+ T cells from diabetes prone (DRlyp/lyp) or resistant (DR+/+) congenic biobreeding (BB) rats for expression of GABAA channels. Our results show that BB rat CD4+ and CD8+ T cells express α1, α2, α3, α4, α6, β3, γ1, δ, ρ1 and ρ2 GABAA channel subunits. In CD8+ T cells from DRlyp/lyp animals the subunits were significantly upregulated relative to expression levels in the CD8+ T cells from DR+/+ rats as well as from CD4+ T cells from both DRlyp/lyp and DR+/+ rats. Functional channels were formed in the T cells and physiological concentrations of GABA (100nM) decreased T cell proliferation. Our results are consistent with the hypothesis that GABA in the islets of Langerhans may diminish inflammation by inhibition of activated T lymphocytes.

Confirmation of multiple seizure susceptibility QTLs on chromosome 15 in C57BL/6J and DBA/2J inbred mice

To confirm seizure susceptibility (SZS) quantitative trait loci (QTLs) on chromosome (chr) 15 identified previously using C57BL/6J (B6) and DBA/2J (D2) mice and to refine their genomic map position, we studied a set of three congenic strains in which overlapping segments of chr 15 from D2 were transferred onto the B6 background. We measured thresholds for generalized electroshock seizure (GEST) and maximal electroshock seizure (MEST) in congenic strains and B6-like littermates and also tested their responses to kainic acid (KA) and pentylenetetrazol (PTZ). Results document that MEST is significantly lower in strains 15M and 15D, which harbor medial and distal (telomeric) segments of chr 15 (respectively) from D2, compared with strain 15P, which harbors the proximal (acromeric) segment of chr 15 from D2, and with control littermates. Congenic strains 15P and 15M exhibited greater KA SZS compared with strain 15D and B6-like controls. All congenic strains were similar to controls with regard to PTZ SZS. Taken together, results suggest there are multiple SZS QTLs on chr 15 and that two QTLs harbor gene variants that affect MEST and KA SZS independently. The MEST QTL is refined to a 19 Mb region flanked by rs13482630 and D15Mit159. This interval contains 350 genes, 183 of which reside in areas where the polymorphism rate between B6 and D2 is high. The KA QTL interval spans a 65 Mb region flanked by markers D15Mit13 and rs31271969. It harbors 83 genes in highly polymorphic areas, 310 genes in all. Complete dissection of these loci will lead to identification of genetic variants that influence SZS in mice and provide a better understanding of seizure biology.

Mast Cells Regulate the Magnitude and the Cytokine Microenvironment of the Contact Hypersensitivity Response

The role that mast cells play during contact hypersensitivity (CS) response is unclear because some studies have shown that mast cell-deficient mice have relatively intact CS responses whereas others have shown opposing results. Mast cells secrete a wide range of immunomodulatory mediators and can potentially influence the type of immune response generated in the skin during CS. Therefore, we examined the type of microenvironment generated during CS in both W/Wv mast cell-deficient and wild-type mice in response to different immunizing doses of hapten (oxazolone). The CS response elicited after low-dose oxazolone was significantly diminished in W/Wv mice compared with wild-type mice. Unexpectedly, the CS response elicited in W/Wv mice immunized with high-dose oxazolone was more severe compared with wild-type mice. In addition, after immunization with high-dose oxazolone, the granulocyte infiltrate in W/Wv mice was increased by twofold compared with wild-type mice. A shift in the cytokine milieu toward the expression of type-1 cytokines as well as a significant increase in the local adhesion of neutrophils and CD4 T cells in the microvasculature of the skin was observed after hapten challenge in W/Wv mice immunized with high-dose oxazolone compared with wild-type mice. These results suggest that mast cells can act as regulators and inducers of the inflammatory response depending on immunizing stimulus strength.

The Potential of Bone Marrow Stem Cells to Correct Liver Dysfunction in a Mouse Model of Wilson's Disease

Metabolic liver diseases are excellent targets for correction using novel stem cell, hepatocyte, and gene therapies. In this study, the use of bone marrow stem cell transplantation to correct liver disease in the toxic milk (tx) mouse, a murine model for Wilson's disease, was evaluated. Preconditioning with sublethal irradiation, dietary copper loading, and the influence of cell transplantation sites were assessed. Recipient tx mice were sublethally irradiated (4 Gy) prior to transplantation with bone marrow stem cells harvested from normal congenic (DL) littermates. Of 46 transplanted tx mice, 11 demonstrated genotypic repopulation in the liver. Sublethal irradiation was found to be essential for donor cell engraftment and liver repopulation. Dietary copper loading did not improve cell engraftment and repopulation results. Both intravenously and intrasplenically transplanted cells produced similar repopulation successes. Direct evidence of functionality and disease correction following liver repopulation was observed in the 11 mice where liver copper levels were significantly reduced when compared with mice with no liver repopulation. The reversal of copper loading with bone marrow cells is similar to the level of correction seen when normal congenic liver cells are used. Transplantation of bone marrow cells partially corrects the metabolic phenotype in a mouse model for Wilson's disease.

Increased tissue survival in experimental skin flaps in mast cell-deficient rats.

The role of mast cells and their principal mediator, histamine, in surgical skin flap survival was investigated using mast cell-deficient (Ws/Ws); their congenic littermates wild-type (+/+), and Wistar rats. A standardized dorsal skin flap was raised and sutured back into position, and 6 days later the percentage of flap survival was assessed. Moreover, endogenous histamine concentration in the dorsal skin during the surgical preparation was determined using in vivo microdialysis technique together with high performance liquid chromatography-fluorometry. Accumulation of skin flap myeloperoxidase (MPO) (reflecting leucocyte recruitment) was determined spectrophotometrically. The experimental skin flaps in genetically mast cell-deficient rats exhibited increased tissue survival and showed little accumulation of MPO and rather low and stable level of histamine output in comparison with skin flaps in the wild-type (+/+) littermates or normal Wistar rats. Antihistamine treatment inhibited but did not prevent leucocyte recruitment in the skin flaps post-surgery in +/+ and Wistar rats. It is suggested that mast cell derived histamine plays an important role in leucocyte recruitment in skin flaps. However, mast cell-independent factors should be taken into consideration and needs further investigation as even in mast cell-deficient animals there was some accumulation of leucocytes and tissue necrosis in the skin flaps post-surgery.

Histamine deficiency in gene-targeted mice strongly reduces antigen-induced airway hyper-responsiveness, eosinophilia and allergen-specific IgE.

Histamine is an important mediator released from activated mast cells provoked by allergen and has a substantial role in the pathophysiology of asthma. However, several lines of evidence indicate that histamine could also have important functions in the regulation of basic cell biological processes. We have used histidine decarboxylase gene-targeted (HDC-KO) mice, lacking histamine, to investigate the effect of histamine deficiency in an animal model of asthma. Our previous investigations revealed that HDC-KO mice had fewer mast cells with reduced granular content and defective degranulation characteristics. Ovalbumin (OVA)-sensitized and challenged HDC-KO mice had significantly reduced airway hyper-responsiveness, lung inflammation, bronchoalveolar lavage eosinophilia, and OVA-specific IgE compared with congenic wild-type littermates treated in the same way. Comparing the expression profiles of cytokines, the levels of IL-1alpha, IL-1beta, IL-4, IL-5, IL-6 and IFN-gamma were significantly lower in the HDC-KO mice in asthmatic late phase, indicating a significantly altered immune response to OVA provocation and challenge. Evaluation of chemokine gene expression revealed that OVA treatment caused elevation of both T(h)1- and T(h)2-type chemokines in wild-type mice, while the chemokine expression was polarized toward a T(h)1 response in HDC-KO mice. According to our results we can suggest that the possible causes of the reduced asthma symptoms in the HDC-KO mice may be the imperfect mast and eosinophil cell system, and an altered immune response to OVA provocation and challenge.

Enhanced Th1 response to Staphylococcus aureus infection in human lactoferrin-transgenic mice.

Lactoferrin (Lf) is an iron-binding protein of external secretions and neutrophil secondary granules with antimicrobial and immunomodulatory activities. To further define these properties of Lf, we have investigated the response to Staphylococcus aureus infection in transgenic mice carrying a functional human Lf gene. The transgenic mice cleared bacteria significantly better than congenic littermates, associated with a trend to reduced incidence of arthritis, septicemia, and mortality. We identified two pathways by which S. aureus clearance was enhanced. First, human Lf directly inhibited the growth of S. aureus LS-1 in vitro. Second, S. aureus-infected transgenic mice exhibited enhanced Th1 immune polarization. Thus, spleen cells from infected transgenic mice produced higher levels of TNF-alpha and IFN-gamma and less IL-5 and IL-10 upon stimulation ex vivo with the exotoxin toxic shock syndrome toxin-1 compared with congenic controls. To confirm that these effects of Lf transgene expression could occur in the absence of live bacterial infection, we also showed that Lf-transgenic DBA/1 mice exhibited enhanced severity of collagen-induced arthritis, an established model of Th1-induced articular inflammation. Higher levels of stainable iron in the spleens of transgenic mice correlated with human Lf distribution, but all other parameters of iron metabolism did not differ between transgenic mice and wild-type littermates. These results demonstrate that human Lf can mediate both antimicrobial and immunomodulatory activities with downstream effects on the outcome of immune pathology in infectious and inflammatory disease.

A NO-releasing derivative of acetaminophen spares the liver by acting at several checkpoints in the Fas pathway.

NCX-701 is a nitric oxide (NO)-releasing acetaminophen (APAP) derivative. In the present study we demonstrated that NCX-701 is as effective as APAP in controlling body temperature in a rat model of endotoxin-induced fever. Liver toxicity is a major complication of APAP overdosing. To investigate whether NCX-701 is hepatotoxic, BALB/C mice were injected with 100 - 500 mg kg(-1) APAP or NCX-701 alone or in combination (i.e. 500 mg kg(-1) of both compounds). Our results demonstrated that although APAP caused a dose-dependent liver injury, NCX-701 was completely devoid of liver toxicity. At the dose of 500 mg kg(-1) APAP caused an approximately 40 fold increase of AST plasma levels and extensive centrilobular necrosis. APAP and NCX-701 share the same metabolic pathway as demonstrated by the time-course of APAP-glucuronide concentrations in plasma and liver. NCX-701 was safe in mice with pre-existing chronic liver disease. Indeed, while C57BL6 transgenic mice expressing the hepatitis B virus (HBV) at the age of 8 months were significantly more susceptible to liver damage induced by APAP (500 mg kg(-1)) than their congenic littermates, treating HBV-transgenic mice with NCX-701, 500 mg kg(-1), caused no damage. Co-administration of NCX-701 at the dose 500 mg kg(-1) to mice treated with APAP, 500 mg kg(-1), completely protected against liver damage induced by APAP. APAP, but not NCX-701, upregulated liver Fas and Fas Ligand mRNA expression in vivo. Incubating mouse hepatocytes with APAP, but not with NCX-701, increased cell surface Fas expression and sensitized hepatocytes to death induced by challenge with a Fas-agonistic antibody. Collectively, these observations suggest that APAP toxicity is Fas mediated and that NCX-701 spares the liver by acting at several checkpoints in the Fas pathway.

Transient contribution of mast cells to pulmonary eosinophilia but not to hyper-responsiveness.

We have recently demonstrated that the transfer of interleukin (IL)-5-producing CD4+ T cell clones into unprimed mice is sufficient for the development ofeosinophilic inflammation in the bronchial mucosa upon antigen inhalation. The aim of this study was to elucidate the possible contribution of mast cells in eosinophilic inflammation and bronchial hyper-responsiveness (BHR), and to discriminate between the roles of CD4+ T cells and mast cells. Mast cell-deficient mice (WBB6F1-W/Wv) and their congenic normal littermates (WBB6F1-+/+) were immunized with ovalbumin and challenged by inhalation with the relevant antigen. Airway eosinophilia was induced with equivalent intensity in +/+ and W/Wv mice 6, 24, 96 and 216 h after antigen inhalation. In contrast, 48 h after antigen challenge, eosinophilic infiltration into the bronchial mucosa was significantly less pronounced in W/Wv mice than in +/+ mice. Anti-CD4 monoclonal antibody (mAb), anti-IL-5 mAb, and cyclosporin A were administered next, demonstrating that the airway eosinophilia of W/Wv mice induced 48 h after antigen challenge was almost completely inhibited by each of these three treatments, but that of +/+ mice was significantly less susceptible. Bronchial responsiveness to acetylcholine was increased 48 h after antigen challenge and was not significantly different between +/+ and W/Wv mice. Administration of anti-IL-5 mAb completely inhibited the development of BHR in both +/+ and W/Wv mice. These results indicate that, in mice, mast cells do have a supplemental role in the development of pulmonary eosinophilia but not BHR. CD4+ T cells totally regulate these responses by producing IL-5.

Differential dietary effects on colonic and small bowel neoplasia in C57BL/6J Apc Min/+ mice.

The effects of fiber on colon cancer are controversial. Twenty 5-week old C57BL/6J Apc Min/+ mice were fed for 60 days with a commercial mouse diet (Teklad LM-485) and eight semidefined diets containing 5-10% various fibers and 20% soybean oil. Ten additional C57BL/6J congenic litter-mates were fed each diet to assay colonic SCFA. SCFA, stool bulk, and colonic tumor incidence differed only slightly among the semidefined diets despite variations in fiber content and source. However, food consumption, caloric intake, stool bulk, and SCFA were substantially increased by the Teklad diet compared with all other groups. The Teklad diet significantly increased the number of mice with colonic tumors, average number of tumors/mouse, total tumor burden, colonic atypical hyperplasia, and small bowel tumors. Mice fed high-fat, no-fiber diets had more small bowel tumors (29.8 +/- 3.1) than mice fed diets with fiber (8.2 +/- 2.1) or with low fat and no fiber (18.1 +/- 3.4) (P < 0.05 for each group). These studies suggest that fat predisposes to and fiber protects against small bowel tumors but not colon tumors in these mice. Thus, diets high in fiber or yielding high colonic luminal SCFA may not necessarily protect against colonic cancer. Furthermore, the effects of dietary fiber in Teklad appear overshadowed by some other biologically active factors in this animal model.

Spontaneous leukocyte rolling in rat and mouse microvessels is independent of mast cell activity

The role of mast cells in spontaneous leukocyte rolling in venules of the mouse cremaster muscle and rat mesentery was investigated. The experiments were carried out using mast cell-deficient rats (Ws/Ws), WBB6F1 mice (W/Wv), and their congenic littermates (wild type). Administration of compound 48/80 intraperitoneally (50 microg) in rats and intrascrotally (5 microg) in mice, 4 h prior to the experiments. Intravital microscopy of the terminal vascular beds in mouse cremaster muscle and rat mesentery. The level of spontaneous leukocyte rolling and the rolling velocity in venules of mast cell-deficient animals exactly matched that seen in wild-type animals. Challenge with compound 48/80 markedly increased leukocyte adhesion and emigration in venules of wild-type animals. In contrast, the number of adherent and extravascular leukocytes was very low in compound 48/80-challenged animals lacking mast cells and did not differ from that seen in control animals treated with phosphate-buffered saline. The presence or activation of mast cells has no bearing on spontaneous leukocyte rolling, at least not in rat and mouse microvessels.

An essential role of mast cells in the development of airway hyperresponsiveness in a murine asthma model.

Immunization of BALB/c mice with alum-adsorbed OVA, followed by three bronchoprovocations with aerosolized OVA, resulted in the development of airway hyperresponsiveness (AHR) and allergic inflammation in the lung accompanied by severe infiltration of eosinophils into airways. In this murine asthma model, administration of monoclonal anti-IL-5 Ab before each Ag challenge markedly inhibited airway eosinophilia, but the treatment did not affect the development of AHR. Immunization and aerosol challenges with OVA following the same protocol failed to induce AHR in the mast cell-deficient W/Wv mice, but induced AHR in their congenic littermates, i.e., WBB6F1 (+/+) mice. No significant difference was found between the W/Wv mice and +/+ mice with respect to the IgE and IgG1 anti-OVA Ab responses and to the airway eosinophilia after Ag provocations. It was also found that reconstitution of W/Wv mice with bone marrow-derived mast cells cultured from normal littermates restored the capacity of developing Ag-induced AHR, indicating that lack of mast cells was responsible for the failure of W/Wv mice to develop Ag-induced AHR under the experimental conditions. However, the OVA-immunized W/Wv mice developed AHR by increasing the frequency and Ag dose of bronchoprovocations. The results suggested that AHR could be developed by two distinct cellular mechanisms. One would go through mast cell activation and the other is IgE/mast cell independent but an eosinophil/IL-5-dependent mechanism.

Mast cells are essential for early onset and severe disease in a murine model of multiple sclerosis.

In addition to their well characterized role in allergic inflammation, recent data confirm that mast cells play a more extensive role in a variety of immune responses. However, their contribution to autoimmune and neurologic disease processes has not been investigated. Experimental allergic encephalomyelitis (EAE) and its human disease counterpart, multiple sclerosis, are considered to be CD4(+) T cell-mediated autoimmune diseases affecting the central nervous system. Several lines of indirect evidence suggest that mast cells could also play a role in the pathogenesis of both the human and murine disease. Using a myelin oligodendrocyte glycoprotein (MOG)-induced model of acute EAE, we show that mast cell-deficient W/W(v) mice exhibit significantly reduced disease incidence, delayed disease onset, and decreased mean clinical scores when compared with their wild-type congenic littermates. No differences were observed in MOG-specific T and B cell responses between the two groups, indicating that a global T or B cell defect is not present in W/W(v) animals. Reconstitution of the mast cell population in W/W(v) mice restores induction of early and severe disease to wild-type levels, suggesting that mast cells are critical for the full manifestation of disease. These data provide a new mechanism for immune destruction in EAE and indicate that mast cells play a broader role in neurologic inflammation.

Primary Role of CD4+ T Cells and Supplemental Role of Mast Cells in Allergic Pulmonary Eosinophilia

Background: We have recently demonstrated that allergic eosinophilic inflammation is transferred to unprimed mice by infusing IL-5-producing CD4+ T cells. The contribution of mast cells to the development of eosinophilic inflammation is controversial. Methods: To clarify the possible different roles of CD4+ T cells and mast cells in eosinophilic inflammation, we compared antigen-induced airway eosinophilia between mast-cell-deficient mice (WBB6F1-W/W^v) and their congenic normal littermates (WBB6F1-+/+). Results: The time course study indicated that equivalent numbers of eosinophils were recruited into the airway of both +/+ and W/W^v mice 6, 24, 96, and 216 h after antigen challenge, whereas the number of eosinophils 48 h after antigen challenge was significantly lower in W/W^v compared to +/+ mice. Administration of either anti-CD4 or anti-IL-5 monoclonal antibody almost completely inhibited antigen-induced eosinophil recruitment in W/W^v mice 48 h after antigen challenge. In contrast, the inhibitory effect of these antibodies in +/+ mice were partial (∼50% inhibition). Anti-CD4 and anti-IL-5 antibodies equally suppressed airway eosinophilia in both +/+ and W/W^v mice 96 h after antigen challenge. Conclusion: Our study indicates that CD4+ T cells are crucially involved in the development of allergic eosinophilic inflammation, while mast cells may play a supplemental role depending on the kinetics of the response.

Role of IgE in the development of allergic airway inflammation and airway hyperresponsiveness--a murine model.

The importance of IgE in airway inflammation and development of AHR in allergen-sensitized mice has been compared and contrasted in different models of sensitization and challenge. Using different modes of sensitization in normal and genetically manipulated mice after anti-IgE treatment, we have been able to distinguish the role of IgE under these different conditions. Striking differences in the three sensitization protocols were delineated in terms of the role of allergen-specific IgE, extent of eosinophilic airway inflammation, and development of AHR (Table 1). The highest levels of IgE and eosinophil infiltration (approximately 20-fold increases) were achieved after systemic sensitization with allergen (plus adjuvant) followed by repeated airway challenge. Passive sensitization with allergen-specific IgE followed by limited airway challenge induced a modest eosinophilic inflammatory response in the airways despite high levels of serum IgE. Exposure to allergen exclusively via the airways also resulted in a modest serum IgE response and a limited eosinophilic inflammatory response (approximately fourfold increases). Under all of these conditions, inhibition of IL-5-mediated eosinophilic airway inflammation was associated with attenuation of AHR. In contrast, the differences in the responses to the different modes of allergen exposure were associated with differences in the requirements for IgE in the development of AHR (Table 1). In the two models associated with mild eosinophil infiltration (passive sensitization and exclusive airway exposure), IgE was required for the development of AHR but did not substantially enhance airway inflammation on its own. However, IgE-allergen interaction was able to enhance T-cell function in vitro and induce T-cell expansion in vivo. In mice systemically sensitized and challenged via the airways, IgE (or IgE-mediated mast-cell activation) was not required for T-cell activation, eosinophilic inflammation and activation in the airways, or development of AHR. This was most clearly seen in B-cell-deficient and mast-cell-deficient, low-IgE-responder mouse strains (B6, B10) and in anti-IgE-treated high-IgEresponder mice (BALB/c). At the same time, we confirmed the importance of IgE in the induction of immediate-type hypersensitivity (mast-cell activation, immediate cutaneous hypersensitivity, passive cutaneous and systemic anaphylaxis). These differences were also highlighted by the means used to detect altered airway function. Passive sensitization and limited airway challenge or exclusive airway exposure to allergen over 10 days elicited changes in airway function that could be detected only in tracheal smooth-muscle preparations exposed to EFS. In contrast, systemic sensitization followed by repeated airway challenge resulted not only in changes in the contractile response to EFS but also in increased responsiveness to inhaled MCh. Thus, these results distinguish not only the differential involvement of IgE and eosinophil numbers but also their contribution to the readouts used to monitor airway function. Based on these studies, we conclude that IgE plays an important role in the development of airway inflammation and AHR under conditions in which limited IL-5-mediated eosinophilic airway infiltration is induced. In conditions where a robust eosinophilic inflammation of the airways is elicited, IgE (and IgE-mediated mast-cell activation) does not appear to be essential for airway inflammation and the development of AHR, detected as increased responsiveness to inhaled MCh. These findings reveal the potential importance of differential targeting in the treatment of allergic diseases with a predominance of IgE-mediated symptoms, e.g., allergic rhinitis and conjunctivitis, where anti-IgE may be an effective therapy, compared to those diseases with a predominant inflammatory component, e.g., AHR in atopic bronchial asthma, where anti-inflammatory or anti-IL-5 therapy may be more beneficial.

Development of eosinophilic airway inflammation and airway hyperresponsiveness in mast cell-deficient mice.

Mast cells are the main effector cells of immediate hypersensitivity and anaphylaxis. Their role in the development of allergen-induced airway hyperresponsiveness (AHR) is controversial and based on indirect evidence. To address these issues, mast cell-deficient mice (W/W v) and their congenic littermates were sensitized to ovalbumin (OVA) by intraperitoneal injection and subsequently challenged with OVA via the airways. Comparison of OVA-specific immunoglobulin E (IgE) levels in the serum and numbers of eosinophils in bronchoalveolar lavage fluid or lung digests showed no differences between the two groups of mice. Further, measurements of airway resistance and dynamic compliance at baseline and after inhalation of methacholine were similar. These data indicate that mast cells or IgE-mast cell activation is not required for the development of eosinophilic inflammation and AHR in mice sensitized to allergen via the intraperitoneal route and challenged via the airways.

Mast cells do not contribute to nonsteroidal anti-inflammatory drug-induced gastric mucosal injury in rodents.

By releasing pro-ulcerogenic mediators, mast cells may contribute to the mucosal injury associated with the use of nonsteroidal anti-inflammatory drugs (NSAIDs). To study this, rat and mouse models of NSAID-induced gastric damage were used in which administration of indomethacin causes haemorrhagic injury in the corpus region of the stomach, and the "re-feeding" model in which penetrating antral ulcers are induced in the rat by naproxen. Mast cell degranulation was determined histologically and by measurement of tissue and serum levels of rat mast cell protease-II, a mediator specific to mucosal mast cells. The effects of either increasing or decreasing the number of gastric mucosal mast cells on the susceptibility of the stomach to injury induced by indomethacin were also studied. Gastric injury induced by indomethacin was not accompanied by significant mast cell degranulation. Moreover, neither increasing nor decreasing the number of gastric mucosal mast cells had a significant effect on the susceptibility of the gastric mucosa to damage induced by indomethacin. In the re-feeding model, prior depletion of gastric mucosal mast cells did not significantly affect the severity of antral ulceration induced by naproxen, nor the ability of prostaglandin E2 to prevent this damage. Finally, indomethacin-induced damage was similar in severity in mice with a genetic defect resulting in the complete absence of mast cells as it was in normal, congenic littermates. Mast cells do not play a significant role in the development of gastric injury induced by acute NSAID administration in the rat or mouse.