A mouse GM-CSF receptor antibody attenuates neutrophilia in mice exposed to cigarette smoke.

We investigated the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a subchronic exposure model of cigarette smoke (CS)-induced inflammation using antibodies directed against GM-CSF or the GM-CSF receptor (GM-CSFR) α-chain. CS-induced mononuclear and neutrophilic inflammation following 4 days of CS exposure in BALB/c mice was assessed in bronchoalveolar lavage (BAL) fluid. An increase in mature dendritic cells (DCs) (CD11c+ and major histocompatibility complex II+) and Gr-1-high neutrophils was also observed by flow cytometric analysis of whole-lung tissue. Daily i.p. injection of 400 µg GM-CSF or GM-CSFR antibody prior to daily smoke exposure attenuated the accumulation of neutrophils within the BAL by 60%. A reduction in mature DCs was also observed. Anti-GM-CSFR antibody administration did not have an effect on the percentage of lung T-cells; however, a significant decrease in activated CD69+ CD8+ T-cells was observed. Anti-GM-CSFR antibody administration decreased the mRNA and protein expression of interleukin-12 p40 and matrix metalloproteinase 12. Taken together, intervention with this receptor antibody implicates the GM-CSF pathway as an important mediator of smoke-induced inflammation.

Mechanisms of clearance of nontypeable Haemophilus influenzae from cigarette smoke-exposed mouse lungs.

Inflammation is prevalent in all stages of chronic obstructive pulmonary disease, and, furthermore, individuals undergo periods of exacerbation, during which pulmonary inflammation increases, often a result of bacterial infection. The present study investigates the in vivo consequences of cigarette smoke exposure on bacterial challenge with nontypeable Haemophilus influenzae (NTHi). BALB/c and C57 black 6 (C57BL/6) mice were exposed to cigarette smoke once or twice daily for a total period of 8 weeks. Exacerbated inflammation was observed in cigarette smoke-exposed compared to room-air-exposed mice following challenge with live or heat-inactivated NTHi. Accelerated clearance of live NTHi from cigarette smoke-exposed mice was independent of the establishment of chronic inflammation or direct toxic effects of cigarette smoke components on bacteria. Mechanistically, a cell-free factor in the bronchoalveolar lavage fluid contributed to accelerated clearance following passive transfer to naive mice. Further investigation demonstrated increased titres of immunoglobulin A in the bronchoalveolar lavage fluid, but not the blood, of cigarette smoke-exposed mice, including increased titres of NTHi-specific immunoglobulin A, whereas heavy chain joining element (J(H))(-/-) B-cell-deficient cigarette smoke-exposed mice did not demonstrate decreased bacterial burden following challenge. The present results demonstrate that cigarette smoke exposure results in exacerbated inflammation following challenge with NTHi, as well as increased titres of antibodies that contribute to bacterial clearance.

Disruption of antigen-induced inflammatory responses in CD40 ligand knockout mice.

The objective of this study was to investigate the contribution of the interaction between CD40 and its ligand (CD40L) to antigen-induced airways inflammatory responses. To this end, we used a model involving ovalbumin (OVA) sensitization followed by OVA aerosol challenge in CD40L knockout (KO) mice. OVA-specific IgE and IgG1 were detected in the serum of the sensitized control, but not in CD40L-KO mice. After antigen challenge, sensitized control mice developed airway inflammation that was primarily eosinophilic. This inflammatory response was dramatically reduced in CD40L-KO mice. In contrast, similar numbers of eosinophils were observed in both the bone marrow and the peripheral blood in the sensitized controls and mutant strains after antigen challenge. To investigate the mechanisms underlying these findings, we examined levels of the cytokines IL-5, IL-4, and TNFalpha in both bronchoalveolar lavage (BAL) and serum. Similar levels of IL-5 were detected in BAL and serum of control and CD40L-KO mice; however, negligible levels of IL-4 in BAL and serum and of TNFalpha in BAL were detected in CD40L-KO mice when compared with control mice. Furthermore, we demonstrated that endothelial cell expression of vascular cell adhesion molecule 1 in OVA-sensitized and -challenged CD40L-KO mice was, as detected by immunohistochemistry, markedly decreased compared with that observed in similarly treated control mice. In addition, we locally overexpressed IL-4 and TNFalpha by using an adenoviral (Ad)-mediated gene transfer approach. Intranasal administration of either Ad/TNFalpha or Ad/IL-4 into OVA-sensitized and -challenged CD40L-KO mice did not reconstitute airway eosinophilia. However, concurrent administration of Ad/TNFalpha and Ad/IL-4 upregulated endothelial expression of vascular cell adhesion molecule 1, and resulted in full reconstitution of the inflammatory response in the airways. Together, these findings demonstrate the importance of the CD40-CD40L costimulatory pathway in the full expression of the inflammatory response in the airways.

GM-CSF transgene expression in the airway allows aerosolized ovalbumin to induce allergic sensitization in mice.

The purpose of this study was to explore whether repeated exposure to aerosolized ovalbumin (OVA) in the context of local expression of GM-CSF can initiate a Th2-driven, eosinophilic inflammation in the airways. On day -1, Balb/c mice were infected intranasally with an adenovirus construct expressing GM-CSF (Ad/GM-CSF). From day 0 to day 9 mice were exposed daily to an OVA aerosol. Mice exposed to OVA alone did not show any evidence of airway inflammation. Mice receiving both Ad/GM-CSF and aerosolized OVA exhibited marked airway inflammation characterized by eosinophilia and goblet cell hyperplasia. Migration of eosinophils into the airway was preceded by a rise in IL-5 and IL-4. Both IL-5 and class II MHC were critically required to generate airway eosinophilia. After resolution, airway eosinophilia was reconstituted after a single OVA exposure. Flow cytometric analysis of dispersed lung cells revealed an increase in macrophages and dendritic cells expressing B7.1 and B7.2, and expansion of activated (CD69-expressing) CD4 and CD8 T cells in mice exposed to OVA and Ad/GM-CSF. Our data indicate that expression of GM-CSF in the airway compartment increases local antigen presentation capacity, and concomitantly facilitates the development of an antigen-specific, eosinophilic inflammatory response to an otherwise innocuous antigen.

Generation of experimental allergic airways inflammation in the absence of draining lymph nodes.

The objective of this study was to investigate the contribution of secondary lymphoid organs in the generation and maintenance of experimental allergic airway inflammation. We employed a previously reported murine model of respiratory mucosal allergic sensitization, induced by repeated aerosolizations of ovalbumin in the context of a GM-CSF airway environment. We executed this protocol in wild-type (WT) and lymphotoxin-alpha-deficient mice (LTalpha-KO) mice, which are devoid of lymph nodes (LNs) and possess rudimentary spleen structures. Despite the lack of pulmonary LNs draining the airway compartment, LTalpha-KO mice were fully capable of mounting a robust inflammatory response in the airways, consisting of Th2 polarized CD4+ T cells and eosinophils. This was accompanied by IL-5, IL-13, and IFN-gamma production by splenocytes and generation of ovalbumin-specific serum IgE. Exposure to the same antigen 7 weeks after complete resolution of airway inflammation once again induced a Th2 polarized infiltrate, demonstrating intact immunological memory. To investigate inherent plasticity in establishing antigen-specific immunity, mice were splenectomized before sensitization. Allergic sensitization was completely abrogated in splenectomized LTalpha-KO mice, compared with eusplenic LTalpha-KO controls. These data demonstrate that secondary lymphoid organs, either LN or spleen, are essential for the generation of allergic airway responses.

Hydroxychloroquine attenuates cigarette smoke induced autophagic signaling in the mouse ovary.

We previously demonstrated that Cigarette Smoke (CS) induces autophagy in the ovary. Therefore we aimed to determine if chloroquine (CQ) could inhibit CS-induced autophagy in the ovary. Eight week old mice were implanted with CQ pellets; 0, 25, and 50mg CQ/kg. Half of the animals in each group were exposed to room air and the other half were exposed to CS twice daily for 8 weeks. Ovaries were harvested for electron microscopy, gene and protein expression analysis. There was a significant increase in the production of autophagosomes in granulosa cells of mice exposed to CS (p=0.0297). However 25 and 50mg/kg CQ treatment significantly decreased the CS-induced autophagosomes (p=0.0505; p=0.0065) and attenuated the effects of CS on LC3B and BECN1 expression. In summary, this suggests that CQ attenuates CS-induced autophagy in the ovary and that ovarian protection from toxic insult is potentially feasible.

Transient Transgenic Approaches for Investigating the Role of GM-CSF in Pulmonary Inflammation and Immune Diseases.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), a 23-kDa polypeptide, was originally identified as a hematopoietic growth factor, but has recently been found to be a multifunctional cytokine with many proinflammatory activities (1,2). GM-CSF can be produced by, and act upon, a broad range of cell types, including both immature and mature granulocyte and monocyte lineage cells, dendritic cells, and tissue structural cells. Abundant in vitro observations have suggested that GM-CSF is able to induce both differentiation and activation of these cells (1).

Eosinophilia in antigen-induced airways inflammation.

Airways eosinophilia is a hallmark of asthmatic inflammation. Accumulation of eosinophils in the airways of asthmatic patients is a terminal event of a process that involves complex cellular and molecular interactions. In the past five years, murine models of experimental asthmatic inflammation have provided insight into its complexity and regulation. The three main steps involved in the development of airways eosinophilia are discussed. The first step is the elicitation of an allergen-specific immune response whereby the allergen, captured and processed by antigen-presenting cells, is presented to T lymphocytes to initiate a specific immunological response. In addition to the class II major histocompatibility T cell receptor interaction, this process critically requires costimulation through two independent pathways. The second step is eosinopoiesis in the bone marrow. Under normal conditions, eosinophils represent only 1% to 3% of the white blood cell pool. Therefore, an eosinopoietic event must precede peripheral blood and tissue eosinophilia. The third step is the recruitment of eosinophils from the vascular compartment into the airways. Migration through the endothelium is an active process that involves a number of molecules such as integrins and adhesion molecules. Understanding these three key steps in the development of airways eosinophilia will help to identify new targets and unveil novel strategies for an even more effective treatment for asthma.

The effect of needle biopsy procedure on prepubertal rat testis.

Testicular needle biopsies were performed in 20 prepubertal rats. Ten rats were used as a control group. Spermatohistogenesis was observed in the tubuli (mean number: 17) of each biopsy. The biopsy procedure caused tubular damage which extended to 0.2-4% of the entire testicular volume. Twenty percent of the rats had severe unilateral testicular atrophy at the site of the procedure. A significant compensatory tubular hypertrophy occurred in the contralateral testis that was observed 50 days after the procedure. Eighty percent of the rats produced the normal number of offspring, independent of the occurrence of unilateral testicular atrophy. The histology of the contralateral testis was normal in all animals; thus, no adverse effect from the biopsied testis occurred.

[Current value of mediastinoscopy]. / Intérêt actuel de la médiastinoscopie.

Between 1976 and 1985 a total of 367 mediastinoscopies were performed in the ENT Clinic, Kantonsspital of Lucerne, Switzerland. Between 1976 and 1981 and average of 50 cases were carried out annually, but the introduction of CT-scanning has provoked a considerable fall in their number. Although mediastinoscopy involves few risks, with a morbidity of 0.8% and a mortality of 0% it has increasingly been replaced by CT-scanning as the sensitivity of both procedures is comparable. The only remaining indication for mediastinoscopy is when lymph node biopsy is necessary in systemic diseases with primary mediastinal lesions.

Influenza A facilitates sensitization to house dust mite in infant mice leading to an asthma phenotype in adulthood.

The origins of allergic asthma, particularly in infancy, remain obscure. Respiratory viral infections and allergen sensitization in early life have been associated with asthma in young children. However, a causal link has not been established. We investigated whether an influenza A infection in early life alters immune responses to house dust mite (HDM) and promotes an asthmatic phenotype later in life. Neonatal (8-day-old) mice were infected with influenza virus and 7 days later, exposed to HDM for 3 weeks. Unlike adults, neonatal mice exposed to HDM exhibited negligible immune responsiveness to HDM, but not to influenza A. HDM responsiveness in adults was associated with distinct Ly6c+ CD11b+ inflammatory dendritic cell and CD8α+ plasmacytoid (pDC) populations that were absent in HDM-exposed infant mice, suggesting an important role in HDM-mediated inflammation. Remarkably, HDM hyporesponsiveness was overcome when exposure occurred concurrently with an acute influenza infection; young mice now displayed robust allergen-specific immunity, allergic inflammation, and lung remodeling. Remodeling persisted into early adulthood, even after prolonged discontinuation of allergen exposure and was associated with marked impairment of lung function. Our data demonstrate that allergen exposure coincident with acute viral infection in early life subverts constitutive allergen hyporesponsiveness and imprints an asthmatic phenotype in adulthood.

Kinetics of in vitro bronchoconstriction in an elastolytic mouse model of emphysema.

Thin-slice videomicroscopy was used to examine the kinetics of constriction in small airways in situ. Balb/C mice inhaled elastase (0-20 IU), and were then left to recover for 14 days before euthanisation and lung removal. Cholinergic responsiveness was assessed in thin lung slices. Magnitude and velocity of narrowing in response to 10(-5) M acetylcholine (ACh), as well as the full concentration-response relationship for ACh (10(-8)-10(-5) M) were assessed. In vivo exposure to elastase was accompanied by statistically significantly decreased magnitudes and velocities of contraction, but no change in the ACh concentration-response relationship. Conversely, overnight, in vitro exposure of slices from control animals to elastase (2.5 microg.mL(-1)) resulted in increased magnitudes and velocities of airway narrowing, with impaired relaxation, as well as marked tearing of the airways from the surrounding parenchyma. These changes are characteristic of decreased tethering forces on the airway wall. Thus, the lung slice technique coupled with videomicroscopic analysis of airway contraction velocities provides a powerful tool to study airway-parenchymal interactions. The elastolytic model of emphysema, which manifests with airspace enlargement and loss of parenchymal attachments, is accompanied by decreased airway contraction kinetics. The mechanism(s) underlying this loss of function remain to be elucidated.

Biological activities of anti-IgE antibodies.

Naturally occurring anti-IgE autoantibodies represent a heterogeneous mixture of antibodies with diverse specificities and biological functions. By using murine monoclonal anti-IgE autoantibodies directed against different epitopes on the IgE molecule as a model for autoantibodies, we could show that only a minority of antibodies combine all beneficial biological activities, such as the activity of inhibiting in vitro IgE synthesis, removing IgE from the surface of CD23+ cells and not being anaphylactogenic. While it is difficult to isolate and measure anti-IgE antibodies in human serum, it is now possible to generate such human anti-IgE antibodies by the method of repertoire cloning. Thus, human recombinant antibodies against IgE may become available for the treatment of atopic disease.

Cloning of human anti-IgE autoantibodies and their role in the regulation of IgE synthesis.

In vivo experiments using anti-IgE antibodies have clearly documented that they inhibit IgE production. In vitro experiments showed that not only IgE synthesis but also the effector phase of the allergic response may be influenced, because anti-IgE antibodies can prevent basophil sensitization with IgE or even remove IgE-receptor-bound IgE molecules. However, the question remains whether naturally occurring anti-IgE autoantibodies possess similar biological activity. To generate such antibodies for the necessary in vitro studies, we have cloned human Ig variable genes and selected anti-IgE antibodies using phage display libraries. Most of the human anti-IgE antibodies were anti-idiotypes, but anti-isotypes were also isolated.

Antigen-specific inhibition of IgE binding to the high-affinity receptor.

Since the beginning of this century, allergen immunotherapy has been widely used to treat allergic disorders. In addition to the long-term clinical efficacy of this therapy, there are immediate beneficial effects as observed in rush immunotherapy for which there is no clear mechanism. We investigated the direct impact of an Ag on its specific IgE in terms of IgE measurability in immunoassays and subsequent binding of IgE to the high-affinity receptor for IgE. As a model we used a chimeric IgE specific for NIP that exhibits similar biologic properties as serum or myeloma IgE. To mimic particulate and soluble allergens we coupled 15 NIP molecules to BSA. Using this "artificial allergen" we could show that the presence of the Ag reduced the IgE measurability in immune assays. Furthermore, IgE binding to the Fc epsilon RI was 60% inhibited in the presence of the Ag shown with an optical biosensor that monitors molecular interactions. In normal basophils passively sensitized with IgE that was preincubated with the Ag, no sulfidoleukotriene release could be induced. Rush immunotherapy may invoke a similar phenomenon, resulting in the short-term alteration of symptoms by blocking or substantially reducing binding of IgE to its high-affinity receptor. Thus, our result may explain some of the short-term beneficial effects observed in rush immunotherapy.

Inhibition of human IgE synthesis by anti-IgE antibodies requires divalent recognition.

We used a selection of well-characterized murine monoclonal anti-IgE antibodies to investigate their effect on human in vitro IgE synthesis. We found anti-IgE antibodies that either inhibited or enhanced interleukin-4 plus anti-CD40-induced in vitro IgE synthesis in peripheral blood mononuclear cells (PBMC). This differential activity was isotype specific as neither IgM nor IgG synthesis were affected. Interestingly, only coding IgE mRNA was down-regulated, whereas germ-line epsilon RNA expression was not influenced by anti-IgE monoclonal antibody (mAb). On purified B cells all anti-IgE mAb inhibited interleukin-4 plus anti-CD40-induced IgE synthesis, implying a role of non-B cells for the enhancing activity observed in PBMC. Using Fab and F(ab')2 of an inhibitory anti-IgE mAb we could show that divalent recognition was required for inhibition of IgE synthesis.

Determinants of the immune-inflammatory response in allergic airway inflammation: overview of antigen presentation and cellular activation.

Under normal circumstances the lung is in a state of immunologic homeostasis, a condition in which exposure to innocuous antigens does not lead to immune-inflammatory responses. This is the only reasonable solution to the dilemma faced by the lung: The need to interact with the external environment and the need to avoid responding to most of the environmental antigens to which it is exposed. In allergic diseases, such as asthma, this homeostasis is undermined, and immuneinflammatory responses to harmless aeroallergens are activated. We describe the changes in antigen presentation and cellular activation observed in a model of allergic airway inflammation. Further, we present a summary of our work that investigated the impact of the airway cytokine microenvironment on the development of immune responses in the respiratory tract.

Neuropeptides accentuate interleukin-4 induced human immunoglobuline E synthesis in vitro.

Corticotropin releasing factor, adrenocorticotropic hormone (ACTH) and alpha-melanocyte stimulating hormone either inhibit or enhance in a dose-dependent fashion an interleukin-4 (IL-4) driven human IgE synthesis in vitro. Here, we show that culture conditions strongly influence the earlier observed dose- and donor-dependent effects of adrenocorticotropic hormone. The effect of ACTH on IgE synthesis became only apparent late during culture periods, suggesting an indirect effect via the cellular microenvironment rather than by acting directly at the level of B-cell isotype switching. Thus, we studied other proopiomelanocortin (POMC) derived peptides and neuropeptides known to influence the cellular microenvironment. Indeed, similar modulatory effects on IgE synthesis were also observed by the addition of other proopiomelanocortin-derived peptides such as alpha-, beta-, and gamma-endorphins as well as by the opioid binding pentapeptide Leu-enkephalin. Furthermore the neuropeptide substance P accentuated an IL-4 or an IL-4 and anti-CD40 antibody driven class switch to IgE. In contrast to ACTH, substance P interfered not only with IgE synthesis but also with the synthesis of the other immunoglobulin isotypes. Thus, systemically acting neuroendocrine peptides such as ACTH and locally acting neuropeptides such as the enkephalins and substance P can modulate the magnitude of an IL-4 induced IgE response.

Domain-specific anti-IgE antibodies interfere with IgE binding to Fc epsilon RII.

Human anti-IgE autoantibodies have been identified and implicated in the regulation of IgE-mediated reactions and IgE synthesis. In order to study the potential regulatory role of anti-IgE antibodies on IgE binding to the Fc epsilon RII we used a panel of IgE-specific monoclonal antibodies that were mapped by Western blotting against a series of recombinant epsilon domain peptides. Antibodies specific for all epsilon domains were detected except those against C epsilon H1. Using a competitive inhibition cell-binding assay on Fc epsilon RII + 8866 cells, we identified two major patterns of anti-IgE activity. Antibodies specific for the C epsilon H3 domain removed IgE whereas those specific for the C epsilon H2 domain enhanced IgE binding to the Fc epsilon RII. The anti-C epsilon H2 antibodies, in contrast to the anti-C epsilon H3 antibodies, could not dissociate cell-bound IgE from the Fc epsilon RII. Using preformed immune complexes of IgE and anti-IgE antibodies, it was clear that the anti-C epsilon H2 antibodies bound more IgE to the Fc epsilon RII by addition of immune complexes to the cell surface. Our results suggest that the opposing actions of either inhibition or enhancement of IgE binding by anti-IgE antibodies are related to their epsilon domain specificity.