Anke Lührmann

In Vivo Effects of a Synthetic 2-Kilodalton Macrophage-Activating Lipopeptide of Mycoplasma fermentans after Pulmonary Application

ABSTRACT 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 μg 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.

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.

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.

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.

Stimulation of Bronchus-Associated Lymphoid Tissue in Rats by Repeated Inhalation of Aerosolized Lipopeptide MALP-2

Objective: Bronchus-associated lymphoid tissue (BALT) is a part of the integrated mucosal immune system. It may play an important functional role for antigen uptake and induction of specific immune reactions. The aim of this study was to investigate whether it is possible to induce or modulate BALT by the repetitive inhalation of the synthetic lipopeptide MALP-2. Methods: Female Lewis rats (245 ± 19 g) inhaled 25 µg of MALP-2 six times at intervals of 1 week. One week after the last inhalation, they were sacrificed. Cells of the bronchoalveolar lavage and the left lung were investigated by flow cytometry. The middle lobe of the right lung was embedded in paraffin. BALT was semiquantitatively measured in 15 serial cross sections per animal. Results: After repetitive inhalation of the diluent as well as MALP-2, BALT was found. The total area was increased after repetitive treatment with MALP-2. In addition, the preferential incidence of BALT was higher after MALP-2 application, in association with a bronchial diameter of 0.6–1 mm. The cellular analysis revealed no differences in the number of leukocyte subsets between the control and MALP-2 group. Conclusion: MALP-2 is a potent local stimulator and can be used to modulate BALT by repetitive inhalant treatment. The functional significance of enlarged or activated BALT has to be elucidated in future studies.

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

ABSTRACT 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.

Increased Surfactant Protein A and D Expression in Acute Ovalbumin-Induced Allergic Airway Inflammation in Brown Norway Rats

Background: The surfactant proteins SP-A and SP-D, components of the innate immune system, are involved in host defence. Objective: We tested the hypothesis that ovalbumin (OVA) challenge leads to an upregulation of both proteins in alveolar epithelial type II cells (AEII) and Clara cells and to an enhanced uptake by macrophages. Methods: After sensitization with OVA and heat-killed Bordetella pertussis challenge followed intratracheally with 0.5% OVA on day 13. One day after challenge lung tissue and bronchoalveolar lavage fluid (BALF) of sensitized NaCl- and OVA-challenged Brown Norway rats were compared with home cage controls using qRt-PCR, Western blot and immunohistochemistry. Results: After OVA challenge (1) eosinophils increased significantly in the BALF, (2) the total amount of SP-A and SP-D was significantly increased in lung tissue, (3) the amount of SP-A was significantly and the amount of SP-D was remarkably elevated in BALF, and (4) the levels of SP-A and SP-D mRNA in lung tissue were significantly elevated. Using quantitative immunohistochemistry, we found (5) significantly higher surface fractions of SP-A- and SP-D-labelled AEII, (6) no differences in the surface fractions of SP-A- and SP-D-labelled bronchial Clara cells, and (7) a significantly increased cell density of unlabelled and SP-A-labelled macrophages. Conclusions: Thus, combining molecular biological and histological methods we suggest that after OVA challenge (1) AEII but not Clara cells show a significantly higher expression of SP-A and SP-D leading also to higher amounts of both SPs in BALF and (2) macrophages gather predominantly SP-A.

Recruitment of Lymphocytes and Dendritic Cells from the Blood to the Bronchoalveolar Space and the Draining Lymph Nodes after a Single Intrabronchial Application of the Lipopeptide MALP-2

Objective: It has been shown previously that the synthetic macrophage-activating lipopeptide, MALP-2, is a potent stimulator of the respiratory immune system and an effective adjuvant in the induction of mucosal immune responses. In this study, the migration route of leukocytes from the blood to the bronchoalveolar space and then to the draining lymph nodes was investigated. Methods: MALP-2 was intratracheally instilled into lungs of Lewis rats. Bronchoalveolar lavage cells as well as cell preparations of other lung compartments such as the marginal vascular pool, the interstitial pool and also the draining lymph nodes were examined 3 days later. Results: The application of MALP-2 induced a pronounced leukocyte accumulation in the bronchoalveolar space and the lung interstitium but not in the marginal vascular pool. A tendency to increased lymphocyte and dendritic cell numbers was observed in the draining lymph nodes. Conclusion: Our data indicate the migration of blood cells into the lung interstitium and the bronchoalveolar space in response to MALP-2. Thus, the immune reaction induced by MALP-2 might be of relevance as an adjuvant treatment in inhalant vaccination strategies 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.

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

Background: The kinetics of regulatory T cells (TReg) 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 TReg 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 TReg. The TReg 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+ TReg and dendritic cells showed different kinetics after two challenges. The leukocyte numbers in the lung did not correlate with lung function. Conclusion: TReg 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.