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Asthma is a type-I allergic airway disease characterized by Th(2) cells and IgE. Episodes of bronchial inflammation, eosinophilic in nature and promoting bronchoconstriction, may become chronic and lead to persistent respiratory symptoms and irreversible structural airway changes. Representative mostly of mild to moderate asthma, this clinical definition fails to account for the atypical and often more severe phenotype found in a considerable proportion of asthmatics who have increased neutrophil cell counts in the airways as a distinguishing trait. Neutrophilic inflammation is a hallmark of another type of allergic airway pathology, hypersensitivity pneumonitis. Considered as an immune counterpart of asthma, hypersensitivity pneumonitis is a prototypical type-III allergic inflammatory reaction involving the alveoli and lung interstitium, steered by Th(1) cells and IgG and, in its chronic form, accompanied by fibrosis. Although pathologically very different and commonly approached as separate disorders, as discussed in this review, clinical studies as well as data from animal models reveal undeniable parallels between both airway diseases. Danger signaling elicited by the allergenic agent or by accompanying microbial patterns emerges as critical in enabling immune sensitization and in determining the type of sensitization and ensuing allergic disease. On this basis, we propose that asthma allergens cause severe noneosinophilic asthma because of sensitization in the presence of hypersensitivity pneumonitis-promoting danger signaling.

Rationale: Mycolic acid (MA) constitutes a major and distinguishing cell wall biolipid from Mycobacterium tuberculosis. MA interferes with the lipid homeostasis of alveolar macrophages, inducing differentiation into foamy macrophages exhibiting increased proinflammatory function., Objectives: We verified the interference of this altered macrophage function with inhaled antigen-triggered allergic airway inflammation and underlying Th2 lymphocyte reactivity., Methods: Using ovalbumin (OVA) as model allergen, C57BL/6 or BALB/C mice were sensitized by OVA-alum immunization. Experimental asthma, triggered subsequently by repetitive nebulized OVA inhalation, was assessed, using as readout parameters eosinophilia, peribronchial inflammation, and Th2 cytokine function., Measurements and Main Results: A single intratracheal treatment of sensitized mice with MA, inserted into liposomes as carriers, prevented the onset of OVA-triggered allergic airway inflammation and promoted unresponsiveness to a secondary set of allergen exposures. The development of this tolerant condition required an 8-d lapse after MA instillation, coinciding with the appearance of foamy alveolar macrophages. MA-conditioned CD11b(+)F4/80(+) macrophages, transferred to the airways, mimicked the tolerogenic function of instilled MA; however, without the 8-d lapse requirement. Indicative of a macrophage-mediated tolerogenic antigen-presenting function, major histocompatibility complex (MHC)-mismatched donor macrophages failed to promote tolerance. Furthermore, Treg markers were strongly increased and established tolerance was lost after in situ depletion of CD25(+) Treg cells. Contrary to the interleukin-10 dependence of tolerogenic dendritic cells, IFN-gamma deficiency but not interleukin-10 deficiency abrogated the tolerogenic capacity of MA-conditioned macrophages., Conclusions: These results document an innate-driven Mycobacterium tuberculosis MA-triggered immune regulatory mechanism in control of pulmonary allergic responses by converting macrophages into IFN-gamma-dependent tolerogenic antigen-presenting cells.

Polymethacrylates are vinyl-based polymers that are used for DNA transfection. Cationic polymethacrylates efficiently condense DNA by forming inter-polyelectrolyte complexes. Their use for DNA transfection is, however, limited due to their low ability to interact with membranes. In order to increase their transfection efficiency, we combined polymethacrylates with Penetratin, a 16-residue water-soluble peptide that internalises into cells through membrane translocation. DNA condensation was assessed using physicochemical methods, while transfection efficiency and cellular internalisation were studied using Cos-1 cells. Agarose gel electrophoresis retardation, ethidium bromide exclusion tests and dynamic light scattering measurements showed that the stability of the polymethacrylate-DNA complexes is not affected by addition of Penetratin. Transfection efficiency of polymethacrylate-DNA complexes into Cos-1 cells increased by addition of Penetratin and was higher than that of polyethylenimine (PEI)-DNA complexes and comparable to Lipofectamine. Confocal microscopy and flow cytometry indicated that Penetratin mainly enhances endolysosomal escape polymethacrylate-DNA complexes and increases their cellular uptake. Since the cellular toxicity of polymethacrylate-DNA-Penetratin complexes remains low, especially compared to PEI, this transfection system opens new perspectives for gene therapy.

The cytokine IL-15 performs numerous functions, such as promotion of growth and survival, on a plethora of cell types from both the lymphoid and non-lymphoid compartments. Therefore, mice genetically engineered to either lack or overexpress functional IL-15 display reduced immunological responses and leukemia, respectively. Surprisingly, IL-15 protein is hardly found in serum or body fluids. Due to the lack of a clear demonstration of its presence as protein,IL-15 was often referred to as a "ghost cytokine ". Recently, however, membrane-bound IL-15 was detected in both a membrane-anchored form and an IL-15Ralpha -bound form on monocytes. Interestingly, the latter complex can ben transpresented to cells expressing the intermediate-affinity IL-2/15Rbeta-gamma C receptor and thereby support the survival and proliferation of T cells. Moreover, overlapping promoter elements indicate a model of co-regulation of IL-15 and IL-15Ralpha by which IL-15 activities are controlled in a cell-contact-dependent manner. In this review, recent reports on IL-15 are combined with previous observations and discussed in terms of their functional consequences for CD4+ T cell responses.

Penetratin is a 16-residue peptide [RQIKIWFQNRRMKWKK(43-58)] derived from the Antennapedia homeodomain, which is used as a vector for cellular internalization of hydrophilic molecules. In order to unravel the membrane translocation mechanism, we synthesized new penetratin variants. The contribution of the positively charged residues was studied by double substitutions of Lys and/or Arg residues to Ala, while the specific contribution of Trp48 and Trp56 was studied by individual substitution of these residues to Phe. Trp fluorescence titrations demonstrated the importance of the positively charged residues for the initial electrostatic interaction of the peptide with negatively charged vesicles. In contrast, none of the Trp residues seemed critical for this initial interaction. Trp fluorescence quenching experiments showed that penetratin lies close to the water-lipid interface in a tilted orientation, while circular dichroism indicated that lipid binding increased the alpha-helical structure of the peptides. The R53A/K57A and R52A/K55A substitutions increased calcein leakage and decreased vesicle aggregation compared to wild-type penetratin. These variants insert deeper into the lipid bilayer, due to an increased hydrophobic environment of Trp56. The W48F and W56F substitutions had a minor effect on membrane insertion and destabilization. Cellular internalization of the R53A/K57A, R52A/K55A and K46A/K57A variants by MDCK cells was similar to wild-type penetratin, as shown by flow cytometry. Moreover, residue Trp48 specifically contributed to endocytosis-independent internalization by MDCK cells, as demonstrated by the lower uptake of the W48F variant compared to wild-type penetratin and to the W56F variant. None of the penetratin variants was haemolytic or cytotoxic.

Local inflammatory responses involve relocating immune functions generated by previous immunization to confined parts of the body, and hence are presumed to reflect the prevailing systemic immune bias. To verify to what extent local antigen-presenting cells (APCs) may modulate immune inflammation, we analyzed the consequences of antigen presentation by macrophages on Th2-dependent airway inflammation in ovalbumin (OVA)-sensitized mice. In contrast to challenge with free OVA, which triggers airway eosinophilia and Th2 cell recruitment, intratracheal instillation of immortalized spleen macrophages (Mf4/4 cells), pulsed with OVA, promoted a nonallergic airway response featuring recruitment of interferon-gamma-producing Th1 cells. Combining OVA-Mf4/4 instillation with OVA inhalation strongly reduced airway eosinophilia. Inflammation repression persisted after secondary OVA challenge and depended on the antigen-presenting ability of the macrophages. Arguing against Th1-mediated counter-regulation, Th1/Th2 ratios remained unaltered in macrophage-treated/OVA-challenged mice. In contrast, levels of interleukin-4 and interleukin-13 mRNA in lung tissue CD4+ T cells were strongly downregulated, indicating a suppression of Th2 cell activation. These results document a role for local macrophages/APCs in controlling the nature and intensity of local immune inflammatory responses. The resulting segregation of systemic and local levels of immune reactivity may enable local inflammation tolerance; it is a nonallergic airway response despite systemic sensitization.

Besides IgE, the Ab isotype that gives rise to sensitization and allergic asthma, the immune response to common inhalant allergens also includes IgG. Increased serum titers of allergen-specific IgG, induced spontaneously or by allergen vaccination, have been implicated in protection against asthma. To verify the interference of topical IgG with the allergen-triggered eosinophilic airway inflammation that underlies asthma, sensitized mice were treated by intranasal instillation of specific IgG, followed by allergen challenge. This treatment strongly reduced eosinophilic inflammation and goblet cell metaplasia, and increased Th1 reactivity and IFN-gamma levels in bronchoalveolar lavage fluid. In contrast, inflammatory responses were unaffected in IFN-gamma-deficient mice or when applying F(ab')(2). Although dependent on specific allergen-IgG interaction, inflammation triggered by bystander allergens was similarly repressed. Perseverance of inflammation repression, apparent after secondary allergen challenge, and increased allergen capture by alveolar macrophages further characterized the consequences of topical IgG application. These results assign a novel protective function to anti-allergen IgG namely at the local level interference with the inflammatory cascade, resulting in repression of allergic inflammation through an FcgammaR- and IFN-gamma-dependent mechanism. Furthermore, these results provide a basis for topical immunotherapy of asthma by direct delivery of anti-allergen IgG to the airways.