Decoy oligodeoxynucleotide against STAT transcription factors decreases allergic inflammation in a rat asthma model.

Leukocyte infiltration and activation of the CD40-CD40 ligand costimulatory pathway may promote inflammatory processes such as asthma. The aim of this study was to investigate whether a single intratracheal application of a decoy oligodeoxynucleotide (ODN) specific for signal transducer and activator of transcription (STAT) family members 1 and 3 influences leukocyte influx and pulmonary CD40 expression in a rat model of allergic airway inflammation. In comparison with the corticosteroid budesonide, the authors investigated the efficacy of the STAT decoy ODN in ovalbumin-induced allergic asthma in a Brown Norway rat asthma model. Leukocytes of the bronchoalveolar lavage (BAL) and lung tissue were analyzed and expression of CD40 was assessed by Western blotting. Single administration of the STAT decoy ODN but not of a mutated control ODN or budesonide resulted in a significant decrease of eosinophils and T lymphocytes in the BAL fluid. Cell numbers of CD4+ and CD8+ lymphocytes were significantly decreased in the lung tissue after decoy ODN application. CD40 expression in protein extracts from lung tissue was also reduced significantly following STAT decoy ODN treatment. These findings indicate that a single, local application of a transcription factor decoy ODN specific for STAT1 and STAT3 caused an attenuation of the allergen-induced cellular inflammatory reaction and is at least as effective as a topical steroid.

Immunostimulation with macrophage-activating lipopeptide-2 increased survival in murine pneumonia.

Community-acquired pneumonia (CAP) is associated with high morbidity and mortality, and Streptococcus pneumoniae is the most prevalent causal pathogen identified in CAP. Impaired pulmonary host defense increases susceptibility to pneumococcal pneumonia. S. pneumoniae may up-regulate Toll-like receptor (TLR)-2 expression and activate TLR-2, contributing to pneumococcus-induced immune responses. In the current study, the course of severe murine pneumococcal pneumonia after pulmonary TLR-2-mediated immunostimulation with synthetic macrophage-activating lipopeptide-2 (MALP-2) was examined. Intratracheal MALP-2 application evoked enhanced proinflammatory cytokine and chemokine release, resulting in recruitment of polymorphonuclear neutrophils (PMN), macrophages, and lymphocytes into the alveolar space in WT, but not in TLR-2-deficient mice. In murine lungs as well as in human alveolar epithelial cells (A549), MALP-2 increased TLR-2 expression at both mRNA and protein level. Blood leukocyte numbers and populations remained unchanged. MALP-2 application 24 hours before intranasal pneumococcal infection resulted in increased levels of CCL5 associated with augmented leukocyte recruitment, and decreased levels of anti-inflammatory IL-10 in bronchoalveolar lavage fluid. Clinically, MALP-2-treated as compared with untreated mice showed increased survival, reduced hypothermia, and increased body weight. MALP-2 also reduced bacteremia and improved bacterial clearance in lung parenchyma, as examined by immunohistochemistry. In conclusion, pulmonary immunostimulation with MALP-2 before infection with S. pneumoniae improved local host defense and increased survival in murine pneumococcal pneumonia.

TLR2/6 stimulation of the rat lung: effects on lymphocyte subsets, natural killer cells and dendritic cells in different parts of the air-conducting compartments and at different ages.

The composition of lymphocyte subsets in the lung has been found to be compartment-specific. To characterize the effect of age, weanling, young adult and adult rats were studied in control conditions and after a single intratracheal dose of the Toll-like receptor 2/6 (TLR2/6) agonist macrophage activating lipopeptide-2 (MALP-2). In all age groups, T, B and natural killer (NK) cells increased dramatically in the epithelium and lamina propria of the bronchi. Male adult rats were found to have responded to MALP-2 to a much greater extent than females when lymphocyte subsets were counted in the epithelium and the lamina propria. In a second series of experiments the time kinetics of regulatory T-cell (Treg) subsets and dendritic cells (DCs) in the lung was studied after local stimulation with MALP-2. Different time-dependent patterns were found in the Treg subsets CD4(+) CD25(+), CD4(+) CD25(+) neuropilin 1(+) and CD4(+) CD25(+) Foxp3(+) cells. Neutrophils and DCs also showed different patterns. Thus, the local application of a TLR agonist increased the number of lymphocyte subsets in a compartment-specific pattern. However, data should not be generalized or extrapolated from one age group, sex or lymphocyte subpopulation to another.

Kinetics of regulatory T cells in the ovalbumin asthma model in the rat.

BACKGROUND: The kinetics of regulatory T cells (T(Reg)) in allergic diseases such as asthma are only partly known. METHODS: The asthma model in the Fischer rat with ovalbumin (OVA) sensitization and aerosol challenge was used. The relative and absolute numbers of leukocytes, lymphocytes and T(Reg) subsets were determined by flow cytometry in the lung interstitium and draining bronchial lymph nodes at different time points after two challenges, and lung function was tested in parallel. RESULTS: The progressive number of challenges resulted in increased relative and absolute numbers of lymphocytes and in particular of T(Reg). The T(Reg) number was augmented with each aerosol challenge and was already significantly increased 6 h after the second challenge. The relative (%) and absolute numbers of CD4+ and CD8+ T(Reg) and dendritic cells showed different kinetics after two challenges. The leukocyte numbers in the lung did not correlate with lung function. CONCLUSION: T(Reg) increased surprisingly early after challenge in the lung tissue. Relative and absolute numbers of leukocyte subsets should always be calculated. The kinetics of different leukocyte subsets can only be determined when several time points are studied.

Local pulmonary immune stimulation by the Toll-like receptor 2 and 6 ligand MALP-2 in rats is age dependent.

Recent studies indicate that the pulmonary immune response in humans and experimental animals is different in newborn, adult and elderly age groups. The aim of this study was to investigate the influence of age on the leukocyte composition in different lung compartments and peripheral blood of weaned and adult rats. A mycoplasma-like inflammatory response was mimicked by intratracheal application of the synthetic macrophage-activating lipopeptide-2 (MALP-2) which activates macrophages and other cells via the Toll-like receptor (TLR) 2 and 6. TLR 2 and 6 mRNA expressions were investigated by semiquantitative RT-PCR in cells of the bronchoalveolar lavage (BAL) and lung interstitium. Weaned Lewis rats (3-4 weeks old) and adults (12-14 months old) were treated with vehicle control or MALP-2. Cytokines and cell infiltration were measured in the BAL and lung interstitium. In control rats, no differences in TLR 2 and 6 mRNA expression level were found between the age groups. After MALP-2 treatment, the maximum of MCP-1 concentration in the BAL fluid was reached in weaned rats after 4h and in adults after 2h. The TNF-alpha maximum was measured after 2h in both age groups. Three days after MALP-2 the numbers of different leukocyte subsets were significantly increased in the BAL of both groups. In contrast, in the lung interstitium MALP-2 induced a leukocyte increase in adult rats but not in weaned rats. In conclusion, data on pulmonary immune responses from one age group and one lung compartment should not be generalized or extrapolated to other groups.

Down-regulation of the non-neuronal acetylcholine synthesis and release machinery in acute allergic airway inflammation of rat and mouse.

Acetylcholine (ACh), derived both from nerve fibres and from non-neuronal sources such as epithelial cells, is a major regulator of airway function. There is evidence that dysfunction of the neuronal cholinergic system is involved in the pathogenesis of asthma. Here, we asked whether the pulmonary non-neuronal ACh-synthesis and release machinery is altered in a rat and a mouse model of allergic airway disease. Animals were sensitized against ovalbumin, challenged by allergen inhalation, and sacrificed 24 or 48 h later. Targets of investigation were the high-affinity choline transporter-1 (CHT1), that mediates cellular uptake of choline, the ACh-synthesizing enzyme choline acetyltransferase (ChAT), the vesicular ACh transporter (VAChT), and the polyspecific organic cation transporters (OCT1-3), which are able to translocate choline and ACh across the plasma membrane. With cell-type specific distribution patterns, immunohistochemistry identified these proteins in airway epithelial cells and alveolar macrophages. Real-time RT-PCR revealed significant decreases in ChAT-, CHT1-, VAChT-, OCT-mRNA in the lung of sensitized and allergen challenged animals. These data were supported by immunohistochemistry, demonstrating reduced labeling intensity of airway epithelial cells. ChAT-, CHT1-, VAChT-, and OCT1-mRNA were also significantly reduced in cells recovered by bronchoalveolar lavage from sensitized and challenged rats. In conclusion, the pulmonary non-neuronal cholinergic system is down-regulated in acute allergic airway inflammation. In view of the role of ACh in maintenance of cell-cell-contacts, stimulation of fluid-secretion and of ciliary beat frequency, this down-regulation may contribute to epithelial shedding and ciliated cell dysfunction that occur in this pathological condition.

The alveolar epithelial type I-like cell line as an adequate model for leukocyte migration studies in vitro.

The lung is unique as leukocytes not only migrate into the bronchoalveolar space but also return to the parenchyma and then via the lymphatics to the draining lymph node. The aim of this study was to investigate the migration of leukocytes via an epithelial monolayer in a Transwell system against a chemokine gradient. Rat type I-like R3/1 alveolar epithelial cells were cultivated on a Transwell polyester membrane (pore diameter 3 microm) for 3 days until a monolayer was formed. The tightness of the monolayer was determined by transepithelial transport of horseradish peroxidase. Isolated human and rat peripheral blood mononuclear cells (PBMC) were placed in the upper chamber, and different concentrations of monocyte chemotactic protein-1 (MCP-1) in the lower chamber. The transmigration of PBMC was quantified and investigated by light and transmission electron microscopy. PBMC migrated through the epithelial cell barrier intercellularly as well as transcellularly. The migration of PBMC against the MCP-1 gradient was dose dependent. The results indicate that this model could help in the study of key events involved in chemokine-induced cell migration from the airways into tissue.

Effects of cilomilast on dendritic cell function in contact sensitivity and dendritic cell migration through skin.

The phosphodiesterase 4 inhibitor cilomilast demonstrated strong inhibitory effects in a model of allergic contact dermatitis. In this study, we examined whether this inhibitory effect is at least partly due to modulation of dendritic cell function. Bone marrow-derived dendritic cells were pulsed with the sensitizer toluene-2,4-diisocyanate and administered subcutaneously to nonsensitized mice. Five days later, the mice were challenged with a low dose of toluene-2,4-diisocyanate onto the ears. In contrast to sham-treated mice, mice obtaining toluene-2,4-diisocyanate pulsed dendritic cells showed a significant increase in ear swelling. This swelling was not influenced when the dendritic cells were pre-incubated with cilomilast. When cilomilast was administered systemically simultaneously to the application of toluene-2,4-diisocyanate pulsed cells, there was an impaired allergic reaction provoked 5 days later. Additionally, a topical treatment with cilomilast resulted in a significant inhibition of skin dendritic cell migration. These results indicate that the antigen-presenting function of dendritic cells is not influenced by cilomilast but the dendritic cell T cell interaction and dendritic cell migration is modulated.

Beneficial effects of TLR-2/6 ligation in pulmonary bacterial infection and immunization with Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is the major pathogen in nosocomial and life-threatening infections of immunocompromised or critically ill patients. The macrophage-activating lipopeptide-2 (MALP-2) activates the immune system via Toll-like receptors (TLR) 2 and 6 and leads to an accumulation of immune cells in lungs of young adult (8-10 week old) rats after intratracheal application. This is characterized by a high increase of granulocyte numbers in the BAL 24 h after MALP-2 treatment. It was hypothesized that MALP-2 may have a positive effect on the clinical course of an experimental infection. Therefore, rats were treated with MALP-2 at different time points following an infection with P. aeruginosa. The effect of MALP-2 in combination with immunization with inactivated P. aeruginosa was also investigated. Rats (n = 10) were infected intratracheally (i.t.) with 1 x 10(8) CFU P. aeruginosa on day 0. They were treated on day -3, -1, 0 and +1 with 2.5 microg MALP-2 or the vehicle i.t. In additional experiments, rats were immunized on day -21 and -14 with 1 x 10(8) CFU of inactivated P. aeruginosa bacteria and 2.5 microg MALP-2 or vehicle with 1 x 10(8) CFU of inactivated bacteria and isopropanol. The clinical score, rectal temperature and weight of the rats were checked in both treatment and immunization experiments twice a day. On day 2 they were sacrificed, CFU were determined in the left lung, the right lung being used for histology. In the group treated with MALP-2 1 day prior to infection significant effects were seen: The rectal temperature was about 2 degrees C higher in comparison to the controls at 6 h and also 1 day after infection. Both the symptoms of the infection and the weight loss were significantly reduced. In addition, the CFU and the inflammation in the lung tissue were significantly lower. These effects were not observed after treatment on day -3, 0 or +1. The MALP-2 enhanced immunization only resulted in a tendency to clinical improvement. In conclusion, local immunostimulation at the appropriate time can enhance the host defense against bacteria in the lung.

Leukocyte infiltration of the periarterial space of the lung after allergen provocation in a rat asthma model.

The periarterial space has recently been described and its physiological and pathophysiological role during inflammatory and allergic reactions has been reviewed. The present studies used a light-/electron-microscopic approach to characterize the periarterial space in an asthma model in Brown Norway rats. After repeated sensitization with ovalbumin and heat-killed Bordetella pertussis bacilli, airway challenge was carried out after 1 further week. Four or 24 h after challenge, rats were fixed by perfusion or instillation and processed for microscopy. Several periarterial capillaries and connective tissue characterized the tissue between small pulmonary arteries, bronchioles and alveolar septa. Additionally, a partly pronounced interstitial edema was seen independent of the kind of fixation. Not only small arteries but also arterioles and venules were partly surrounded by edematous fluid already visible by light microscopy. Within the connective tissue and within the periarterial fluid, numerous leukocytes, predominantly eosinophils, were found. However, leukocytes were detected only rarely in the vascular lumen. Only sporadically were eosinophils seen in the wall of small arteries or venules. Eosinophils transmigrating the endothelium of capillaries or arterioles were not visible 4 or 24 h after challenge. Thus, granulocytes transmigrate in the periarterial space very rapidly or even earlier than 4 h after challenge. The location of transmigration in the periarterial space needs further investigation.

Improved intranasal immunization with live-attenuated measles virus after co-inoculation of the lipopeptide MALP-2.

The macrophage-activating lipopeptide with a molecular weight of 2kDa (MALP-2) activates antigen presenting cells of human, mouse and rat origin in vitro and in vivo. Here, we demonstrate that MALP-2 induces MIP1alpha and beta, MIP-2, Gro, TNFalpha, IL1alpha and IL6 in cells of cotton rats (Sigmodon hispidus) in vitro. Intranasal inoculation into cotton rats leads to migration of neutrophils and other leucocytes into the lung lumen and lung tissue. After intranasal co-inoculation of MALP-2 with live-attenuated measles vaccine virus, higher titers of neutralizing antibodies are induced but the proliferative T cell response did not increase. Immunization leads to protective immunity in the absence, but not in the presence of passively transferred measles virus (MV) specific antibodies.

A single intratracheal dose of the growth factor Fms-like tyrosine kinase receptor-3 ligand induces a rapid differential increase of dendritic cells and lymphocyte subsets in lung tissue and bronchoalveolar lavage, resulting in an increased local antibody production.

Repetitive doses of the growth factor Fms-like tyrosine kinase receptor-3 ligand (Flt3L) have resulted in increased numbers of dendritic cells (DC) in various organs, and the effect on protective or tolerogeneic responses in the gut wall has been documented in the literature. In this study, for the first time, Flt3L was locally applied in the trachea of rats using a single dose only. A dose-dependent increase not only of DC, but also of T lymphocytes (CD4(+) and CD8(+)), was seen with a maximum on day 3. The effects on the cells in the lung interstitium and the bronchoalveolar space showed some differences. The use of tetanus toxoid as a model Ag applied intratracheally after the local Flt3L stimulation resulted in increased levels of specific IgA and IgG in the lung. Thus, this novel approach of locally stimulating APCs by topical application of a DC growth factor before applying the Ag offers a new vaccination strategy.

Intratracheal macrophage-activating lipopeptide-2 reduces metastasis in the rat lung.

Primary surgery of tumors bears the risk of metastasis to organs such as the lungs. In order to prevent such metastatic processes, in the present study, local intratracheal instillation of macrophage-activating lipopeptide-2 (MALP-2) as a bacterial-derived immunomodulator of cellular host defense responses was performed, and the effects on tumor cell clearance as well as tumor colonization were investigated in the lungs of Fischer 344 (F344) rats. Compared with vehicle controls, local administration of MALP-2 parallel to intravenous inoculation of MADB106 mammary adenocarcinoma tumor cells resulted in a significant reduction of lung colony numbers, whereas MALP-2 application 1 or 3 d afterwards was not effective. Quantification of leukocyte subsets in the lung tissue by immunohistochemistry revealed a significant increase of the number of monocytes in situ, as well as an increased co-localization of Natural Killer (NK) cells with tumor cells. Synthetic MALP-2 is easily available, with virtually no limitation to the amount of compound, and easily applicable by inhalation. Therefore, as local immunostimulative effects of the bacterial antigen MALP-2 have successfully been demonstrated, its use as an immunotherapeutic agent is worth further investigation.

In vivo effects of a synthetic 2-kilodalton macrophage-activating lipopeptide of Mycoplasma fermentans after pulmonary application.

Mycoplasmas can cause interstitial pneumonias inducing critical illness in humans and animals. Mycoplasma infections are characterized by an influx of neutrophils, followed by an accumulation of macrophages and lymphocytes. The present study deals with the question of which mycoplasmal components cause this host reaction. The mycoplasma-derived, macrophage-activating lipopeptide 2S-MALP-2 was used to mimic the sequelae of a mycoplasma infection. To this end, 2S-MALP-2 was intratracheally instilled into the lungs of Lewis rats, and the bronchoalveolar lavage cells were examined at different times after different doses of 2S-MALP-2. Application of 2.5 microg induced a pronounced leukocyte accumulation in the bronchoalveolar space. At 24 h after 2S-MALP-2 administration, the majority of leukocytes consisted of neutrophils, followed by macrophages, peaking on days 2 and 3. Lymphocyte numbers, although amounting to only a few percent of the total bronchoalveolar lavage cells, also increased significantly, with maximal lymphocyte accumulation occurring by 72 h after instillation. The leukocyte count of the lung interstitium was increased on day 3 after treatment. After 10 days all investigated cell populations returned to control levels. Transient chemotactic activity for neutrophils was detected in the bronchoalveolar lavage fluid early after 2S-MALP-2 application, followed by monocyte chemoattractant protein-1 activity (MCP-1) in lung homogenates. MCP-1 was produced by bronchoalveolar lavage cells upon stimulation with 2S-MALP-2. Our data indicate that mycoplasmal lipoproteins and lipopeptides are probably the most relevant mycoplasmal components for the early host reaction. The primary target cells are likely to be the alveolar macrophages liberating chemokines, which attract further leukocytes.