Non-eosinophilic Airway Hyper-reactivity in Mice, Induced by IFN-γProducing CD4+and CD8+Lung T cells, is Responsive to Steroid Treatment

Non-eosinophilic asthma is characterized by infiltration of neutrophils into the lung and variable responsiveness to glucocorticoids. The pathophysiological mechanisms have not been characterized in detail. Here, we present an experimental asthma model in mice associated with non-eosinophilic airway inflammation and airway hyper-responsiveness (AHR). For this, BALB/c mice were sensitized by biolistic DNA immunization with a plasmid encoding the model antigen β-galactosidase (pFascin-βGal mice). For comparison, eosinophilic airway inflammation was induced by subcutaneous injection of βGal protein (βGal mice). Intranasal challenge of mice in both groups induced AHR to a comparable extent as w…

Transcriptional targeting of dendritic cells for gene therapy using the promoter of the cytoskeletal protein fascin.

Strong cell-type-specific promoters are basic tools in gene therapy allowing for novel applications and focused strategies by transcriptionally targeting gene expression to selected cells. In immunotherapy, dendritic cells (DC) are of central importance, since they represent the principal inducers of immune responses. Here we describe isolation and use of the promoter of the murine actin-bundling protein fascin to target transcriptionally gene expression to cutaneous DC. Using the reporter gene enhanced green fluorescent protein (EGFP), we demonstrate that the fascin promoter mediates a strong antigen expression that is restricted to mature DC. DNA vaccination with antigen-encoding expressi…

Efficacy of recombinant adenovirus as vector for allergen gene therapy in a mouse model of type I allergy

DNA-based immunization represents an attractive alternative approach to the current treatment of allergic diseases by specific immunotherapy with allergen extracts. In this study, we used a replication-deficient adenovirus vector (AdCMV), to examine the in vivo efficacy of preventive and therapeutic genetic immunization in a mouse model of type I allergy. Primary immunization with a recombinant adenovirus expressing the model antigen beta-galactosidase (AdCMV-(beta)gal) induced a Th1 immune response (predominance of IgG2a antibodies, high frequency of IFN-gamma producing T cells) and large numbers of cytotoxic T lymphocytes. Prophylactic vaccination with AdCMV-(beta)gal abolished the produc…

Antigen dose-dependent suppression of murine IgE responses is mediated by CD4−CD8− double-negative T cells

Background The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. Objective The aim of the study was to identify immune cells that are involved in antigen dose-dependent regulation of IgE formation. Methods Wild-type mice as well as T helper (Th)1-deficient IL-12p40(-/-) and IFN-gamma(-/-) mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen-dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets …

Induction of regulatory T cells by leflunomide in a murine model of contact allergen sensitivity.

Allergic contact dermatitis and contact hypersensitivity (CHS) are characterized by allergen-specific activation of CD8 + and CD4 + T cells and the production of cytokines resulting in an inflammatory response and tissue damage. We show here that the immunosuppressive compound leflunomide ( N -[4-trifluoro-methylphenyl]-5-methylisoxazol-4 carboxamide, HWA 486) (LF) inhibited the contact allergic response induced in mice by epicutaneous application of the haptens dinitrofluorobenzene (DNFB) and oxazolone. The extent of ear swelling remained significantly reduced following repeated challenge with DNFB for up to 18 weeks. LF and DNFB had to be applied simultaneously for inhibition to occur. Th…

A novel plasmid DNA electroporation method allows transfection of murine DC.

Under steady state conditions dendritic cells (DC) exert tolerogenic function, but acquire potent immunogenic function due to strong upregulation of costimulatory molecules and proinflammatory cytokines. In numerous studies the potential of modified DC to induce tolerance or immune reactions towards a distinct antigen has been demonstrated. However, DC are refractory to transfection with plasmid DNA by non-viral methods. In this study we have tested the suitability of a newly developed electroporation device to transfect immature murine bone-marrow derived DC (BM-DC). Transfected BM-DC expressed reporter molecules at considerable extent which renders this method suitable to perform all kind…

21. Mainzer Allergie-Workshop

Efficiency of Biolistic DNA Vaccination in Experimental Type I Allergy

Gene gun-mediated delivery of allergen-encoding plasmid DNA has been in focus for many years now as being a needle-free alternative to the protein-based desensitization regimen used in specific immunotherapy. Biolistic immunization with the Helios gene gun has proven to be potent in the induction of antigen-specific CD4(+) and CD8(+) T cells. Here we describe biolistic vaccination in experimental mouse models of IgE-mediated type I allergy as well as allergen-induced airway inflammation.

Mast Cells Induce Migration of Dendritic Cells in a Murine Model of Acute Allergic Airway Disease

<i>Background: </i>The migration of dendritic cells (DCs) from the lungs to the regional lymph nodes is necessary for the development of allergic airway disease. Following activation, mast cells release a variety of stored or de novo-produced inflammatory mediators, several of them being capable of activating DCs. In this study, the role of mast cells on DC migration from the lungs to the thoracic lymph nodes was investigated in sensitized mice. <i>Methods:</i> Mast cell-deficient mice (Kit<sup>W-sh/W-sh</sup>) and their wild-type counterparts were sensitized intraperitoneally with ovalbumine (OVA) in saline and challenged by a single intranasal administr…

Prophylactic and therapeutic intervention in IgE responses by biolistic DNA vaccination primarily targeting dendritic cells.

Background Allergen gene transfer represents an alternative approach to specific immunotherapy with allergen extracts. Gene gun–mediated DNA immunization with plasmid vectors expressing a transgene under control of the promoter of the fascin gene (pFascin) allows for antigen production predominantly by dendritic cells and resulted in the generation of CD8 + cytotoxic T lymphocytes as well as in the development of a type 1 immune response. Objective We compared the in vivo efficiency of biolistic transfection with pFascin and plasmids containing the cytomegalovirus promoter (pCMV) in a mouse model of type I allergy. Methods BALB/c mice were sensitized with the model allergen β-galactosidase …

15. Mainzer Allergie-Workshop 2003

Comparison of direct and indirect antioxidant effects of linagliptin with other gliptins — Evidence for antioxidant and antiinflammatory properties of linagliptin

Regulation of IgE production and airway reactivity by CD4(-)CD8(-) regulatory T cells

The mechanisms of tolerance induction occurring in the course of allergen-specific immunotherapy have not been elucidated in full detail. Our study aimed to characterize high zone tolerance in mouse models of type I allergy and of allergic airway inflammation induced by subcutaneous sensitization of mice with high doses of the model allergen ovalbumin (OVA) without the use of adjuvant. Mice were immunized by subcutaneous injection of high doses (HD) of OVA or, for comparison, low doses (LD) of OVA in saline. HD-mice showed lower specific IgE, but augmented IgG in sera than LD-mice. Pre-treatment of mice with HD-OVA antigen-specifically inhibited IgE production subsequently induced by LD-OVA…

Transcriptional targeting of dendritic cells in gene gun-mediated DNA immunization favors the induction of type 1 immune responses

Cutaneous dendritic cells (DC) are pivotal for the elicitation of antigen-specific immune responses following gene gun-mediated biolistic transfection of the skin. We transcriptionally targeted transgene expression to DC using vectors containing the murine fascin promoter (pFascin) to control antigen production and compared the immune response elicited with conventional DNA immunization using plasmid constructs with the ubiquitously active CMV promoter (pCMV). Biolistic transfection with pFascin initiated a marked type 1 immune response characterized by the occurrence of a large population of IFN-gamma-producing T helper (Th) cells in spleen and draining lymph nodes. Consistently, immunoglo…

Nutritional control of IL-23/Th17-mediated autoimmune disease through HO-1/STAT3 activation

AbstractThe nutritional curcumin (CUR) is beneficial in cell-mediated autoimmune diseases. The molecular mechanisms underlying this food-mediated silencing of inflammatory immune responses are poorly understood. By investigating antigen-specific immune responses we found that dietary CUR impairs the differentiation of Th1/Th17 cells in vivo during encephalomyelitis and instead promoted Th2 cells. In contrast, feeding CUR had no inhibitory effect on ovalbumin-induced airway inflammation. Mechanistically, we found that CUR induces an anti-inflammatory phenotype in dendritic cells (DC) with enhanced STAT3 phosphorylation and suppressed expression of Il12b and Il23a. On the molecular level CUR …

Experimentelle Allergologie/Immunologie

Formalin-Fixed <i>Staphylococcus aureus</i> Particles Prevent Allergic Sensitization in a Murine Model of Type I Allergy

<i>Background:</i> Bacterial infections are supposed to act counterregulatory to the development of allergen-specific Th2 immune responses. We analyzed whether administration of extracellular <i>Staphylococcus aureus</i> inhibited experimental sensitization against allergens. <i>Methods:</i> BALB/c mice were immunized with alum-adsorbed ovalbumin (OVA) together with formalin-fixed <i>Staphylococcus</i> particles. OVA-specific antibody production and cytokine synthesis by spleen cells was analyzed. Airway reactivity and cellular infiltration into the airways was assessed after intranasal challenge of mice with OVA. In addition, the capacity of …

Allergen-induced IgE-dependent gut inflammation in a human PBMC-engrafted murine model of allergy.

Background Humanized murine models comprise a new tool to analyze novel therapeutic strategies for allergic diseases of the intestine. Objective In this study we developed a human PBMC–engrafted murine model of allergen-driven gut inflammation and analyzed the underlying immunologic mechanisms. Methods Nonobese diabetic (NOD)– scid -γc −/− mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or not. Three weeks later, mice were challenged with the allergen orally or rectally, and gut inflammation was monitored with a high-resolution video miniendoscopic system, as well as histologically. Results Using the aeroallergens birch or gra…

A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid

AbstractThe phenotype and function of murine dendritic cells (DCs) are primarily studied using bone-marrow–derived DCs (BM-DCs), but may be hampered by the heterogenous phenotype of BM-DCs due to their differential state of maturation. Here we characterize a newly established murine DC line (SP37A3) of myeloid origin. During maintainance in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and M-CSF, SP37A3 cells resemble immature DCs characterized by low expression of major histocompatibility complex (MHC) II and costimulatory molecules and low T-cell stimulatory capacity. Upon stimulation, SP37A3 cells acquire a mature phenotype and activate naive T cells as potent…

Prevention of long-term IgE antibody production by gene gun-mediated DNA vaccination.

Background Vaccination with allergen-encoding DNA represents a promising approach for the treatment of allergic diseases. Objective In a mouse model of type I allergy, we analyzed the ability of biolistic transfection to inhibit antigen-specific IgE production and to modulate T H 2 responses. Methods BALB/c mice were vaccinated by means of gene gun–mediated DNA immunization with plasmid vector pCMV-βGal, encoding β-galactosidase as a model allergen. Subsequently, mice were immunized by means of repeated intraperitoneal injection of β-galactosidase adsorbed to the adjuvant aluminum hydroxide. Development of IgE, IgG1, and IgG2a antibody titers during the course of immunization was followed, …

Glucose-independent improvement of vascular dysfunction in experimental sepsis by dipeptidyl-peptidase 4 inhibition.

Aims Dipeptidyl peptidase-4 (DPP-4) inhibitors are a novel class of drugs for the treatment of hyperglycaemia. Preliminary evidence suggests that their antioxidant and anti-inflammatory effects may have beneficial effects on the cardiovascular complications of diabetes. In the present study, we investigate in an experimental sepsis model whether linagliptin exerts pleiotropic vascular effects independent of its glucose-lowering properties. Methods and results Linagliptin (83 mg/kg chow for 7days) was administered in a rat model of lipopolysaccharide (LPS) (10 mg/kg, single i.p. dose/24 h)-induced sepsis. Vascular relaxation, reactive oxygen species (ROS) formation, expression of NADPH oxida…

Divergent effects of biolistic gene transfer in a mouse model of allergic airway inflammation.

Particle-mediated epidermal delivery (PMED) of allergen genes efficiently prevents systemic sensitization and suppresses specific immunoglobulin E synthesis. We investigated in a mouse model of allergic airway disease the effect of PMED on the elicitation of local inflammatory reactions in the lung. BALB/c mice were biolistically transfected with plasmids encoding beta-galactosidase (betaGal) as model allergen under control of the DC-targeting fascin promoter and the ubiquitously active cytomegalovirus promoter, respectively. Mice were challenged intranasally with betaGal-protein with or without intermediate sensitization with betaGal adsorbed to aluminiumhydroxide. Subsequently, local cyto…

Dendritic Cell-Specific Biolistic Transfection Using the Fascin Gene Promoter

The transcriptional targeting of gene expression to selected cells by cell type-specific promoters displays a fundamental tool in gene therapy. In immunotherapy, dendritic cells (DCs) are pivotal for the elicitation of antigen-specific immune responses following gene gun-mediated biolistic transfection. Here we report on transcriptional targeting of murine skin DCs using plasmids which include the promoter of the gene of the cytoskeletal protein fascin to control antigen production. Fascin, which is mandatory for the formation of dendrites, is synthesized among the hematopoietic cells exclusively by activated DCs. The activity of the promoter of the fascin gene reflects the endogenous produ…

Uptake and presentation of exogenous antigen and presentation of endogenously produced antigen by skin dendritic cells represent equivalent pathways for the priming of cellular immune responses following biolistic DNA immunization

Gene gun-mediated biolistic DNA vaccination with beta-galactosidase (betaGal)-encoding plasmid vectors efficiently modulated antigen-induced immune responses in an animal model of type I allergy, including the inhibition of immunoglobulin E (IgE) production. Here we show that CD4(+) as well as CD8(+) T cells from mice biolistically transfected with a plasmid encoding betaGal under the control of the fascin promoter (pFascin-betaGal) are capable of inhibiting betaGal-specific IgE production after adoptive transfer into naïve recipients. Moreover, suppression of IgE production was dependent on interferon (IFN)-gamma. To analyse the modalities of activation of CD4(+) and CD8(+) T cells regardi…

Interleukin-10-treated dendritic cells do not inhibit Th2 immune responses in ovalbumin/alum-sensitized mice.

<i>Background:</i> It is well known that the immunoregulatory cytokine interleukin (IL)-10 inhibits the accessory function of human dendritic cells (DC) in vitro. Recently, we have shown that these IL-10 DC inhibit the production of T helper cell 1 (Th1) and T helper cell 2 (Th2) cytokines by T cells from atopic individuals in vitro. The current study was set out to analyze whether IL-10 DC also exert inhibitory effects in vivo in a murine model of allergy to ovalbumin adsorbed to the adjuvant aluminium hydroxide (OVA/alum). <i>Methods:</i> OVA-pulsed or unpulsed bone marrow-derived DC, treated with IL-10 or left untreated during generation, were injected intravenous…

17. Mainzer Allergie-Workshop