Andrew S. McWilliam

Activation of Protease-Activated Receptor (PAR)-1, PAR-2, and PAR-4 Stimulates IL-6, IL-8, and Prostaglandin E2 Release from Human Respiratory Epithelial Cells

Epithelia from many tissues express protease-activated receptors (PARs) that play a major role in several different physiological processes. In this study, we examined their capacity to modulate IL-6, IL-8, and PGE2 production in both the A459 and BEAS-2B cell lines and primary human bronchial epithelial cells (HBECs). All three cell types expressed PAR-1, PAR-2, PAR-3, and PAR-4, as judged by RT-PCR and immunocytochemistry. Agonist peptides corresponding to the nascent N termini of PAR-1, PAR-2, and PAR-4 induced the release of cytokines from A549, BEAS-2B, and HBECs with a rank order of potency of PAR-2 > PAR-4 > PAR-1 at 400 μM. PAR-1, PAR-2, and PAR-4 also caused the release of PGE2 from A549 and HBECs. The PAR-3 agonist peptide was inactive in all systems tested. PAR-1, PAR-2, or PAR-4, in combination, caused additive IL-6 release, but only the PAR-1 and PAR-2 combination resulted in an additive IL-8 response. PAR peptide-induced responses were accompanied by changes in intracellular calcium ion concentrations. However, Ca2+ ion shutoff was ∼2-fold slower with PAR-4 than with PAR-1 or PAR-2, suggesting differential G protein coupling. Combined, these data suggest an important role for PAR in the modulation of inflammation in the lung.

Regulation of Dendritic Cell Recruitment into Resting and Inflamed Airway Epithelium: Use of Alternative Chemokine Receptors as a Function of Inducing Stimulus

Dendritic cells (DC) were purified by flow cytometry from rat tracheal mucosa; they exhibited the phenotypic characteristics of immature DC including high endocytic activity, low CD80/86 expression, and in vitro responsiveness to a broad range of CC chemokines. Daily treatment of adult rats with the selective CCR1 and CCR5 antagonist Met-RANTES reduced baseline numbers of tracheal intraepithelial DC by 50–60%, and pretreatment of animals with Met-RANTES before inhalation of aerosol containing heat-killed bacteria abolished the rapid DC influx into the epithelium that occurred in untreated controls, implicating CCR1 and CCR5 and their ligands in recruitment of immature DC precursors into resting airway tissues and during acute bacterial-induced inflammation. Comparable levels of DC recruitment were observed during airway mucosal Sendai virus infection and after aerosol challenge of sensitized animals with the soluble recall Ag OVA. However, Met-RANTES did not affect these latter responses, indicating the use of alternative chemokine receptors/ligands for DC recruitment, or possibly attraction of different DC subsets, depending on the nature of the eliciting stimulus.

The biology of airway dendritic cells

Recent work from our laboratory has identified a network of constitutively class II MHC (Ia) bearing dendritic cells (DC) within the epithelium of the conducting airways of laboratory animal species and in humans. The density of DC within the respiratory tract is highest in those areas exposed to greater amounts of inhaled antigen and further work has identified these DC as being critically important in controlling the induction of immune responses within the airways. The DC population in the airway epithelium is renewed every 48–72 h; this represents a more rapid turnover than DC in other tissues which are exposed to a smaller antigenic load. In addition to these results we will discuss other work which shows that airway DC are a very reactive population, comparable with neutrophils in their response to acute inflammatory stimuli and that their numbers and Ia content can be modulated following exposure to topical and systemic steroids. Finally we will discuss the development of these cells after birth and how this may influence the pathogenesis of immune regulated diseases such as asthma and allergic rhinitis.

Functional studies on dendritic cells in the respiratory tract and related mucosal tissues.

Recent reports indicate that mucosal tissues contain dendritic cell (DC) populations that exhibit phenotypic features distinct from those at more studied sites such as skin. In particular, mucosal DC populations display very rapid baseline turnover rates, which increase in response to local inflammatory stimuli. This property is likely to be central to the role of mucosal DC in surveillance of these front‐line tissues for incoming microbial pathogens. As we discuss, it may also indirectly account for the “Th2 default,” which is the recognized hallmark of mucosal immune system in the steady state. J. Leukoc. Biol. 66: 272–275; 1999.

Immunobiology of dendritic cells in the respiratory tract: steady-state and inflammatory sentinels?

A network of dendritic cells present in the epithelial lining of the respiratory tract function as sentinel cells which are able to detect the presence of foreign antigenic material and to process these antigens in such a way that they can be transported to local lymph nodes and presented to naive T cells. We will discuss the immunobiology of this network of cells as found in the rat.

Acute laryngitis in the rat induced by Moraxella catarrhalis and Bordetella pertussis : Number of neutrophils, dendritic cells, and T and B lymphocytes accumulating during infection in the laryngeal mucosa strongly differs in adjacent locations

Infectious laryngotracheitis results in fulminant respiratory distress. During the disease, the subglottic mucosa is selectively infected and swollen, the reason for this preference being unknown. Therefore, in the present study the immunoreaction of the laryngeal mucosa was studied in the rat after inhalation of either heat-killed Moraxella catarrhalis (PVG rats) or application of viable Bordetella pertussis (BN rats). The number of neutrophils, macrophages, dendritic cells, and T and B lymphocytes was determined in the mucosa of the supraglottic, glottic, and subglottic area of the larynx as well as in the trachea. After application of the pathogens, the mucosa of the subglottic area was significantly more affected than the glottic mucosa. Already 1 h after application of M. catarrhalis, not only neutrophils but also dendritic cells and T and B lymphocytes were found both subepithelially and within the epithelium. They showed a similar kinetic progression, although at a different level. Two hours after application of M. catarrhalis, at the peak of inflammation, dendritic cells (173 ± 10 cells/0.1 mm2) outnumbered neutrophils (54 ± 9 cells/0.1 mm2), T lymphocytes (25 ± 2 cells/0.1 mm2), and B lymphocytes (4.3 cells/0.1 mm2). The subglottic area (and the trachea) contained about three to five times more cells than the glottic area. In contrast, the number of local macrophages was lower in the subglottic area (24 ± 5 cells/0.1 mm2) compared with that of the glottic area (38 ± 6 cells/0.1 mm2), and did not change after application of both M. catarrhalis and B. pertussis. Thus, infectious laryngotracheitis in the rat closely resembles the clinical picture in children. In addition, the present results show a major difference in cellular influx in the mucosa of the glottic and subglottic area. This demonstrates that even in two closely adjacent locations, inflammatory responses of different magnitudes can occur, and it underlines the importance of regulatory mechanisms specific for the respective microenvironment.

Th-1/Th-2 Switch Regulation in Immune Responses to Inhaled Antigens

Until comparatively recently, allergic (atopic) disease was viewed as a manifestation of hyperreactivity to essentially non-pathogenic soluble protein antigens which are ubiquitous in the natural environment. In this context, normality would equate to non-responsiveness, resulting from either tolerance or ignorance of the antigens. However, it is now clear (reviewed inc[1]) that active T cell immunity to at least one class of these antigens (airborne ”inhalant“ allergens) is essentially universal amongst adults, clinical reactivity being a function of the cytokine profiles of CD4+ Th-cells which dominate relevant Th-memory populations. Thus, atopics who respond to allergen exposure via IgE production, eosinophilia etc., manifest Th-2-skewed memory, whereas T cell memory in non-responsive normal adults is dominated by Th- I -cytokines such as IFNγ. The situation with respect to ingested environmental allergens (ubiquitous in the diet) appears both qualitatively and quantitatively different, as T cell reactivity to this class of antigens is considerably less frequent in the adult population1.2.

Population dynamics and functions of respiratory tract dendritic cells in the rat

The epithelial surfaces of the respiratory tract are continuously exposed to environmental antigens, and the maintenance of immunological homeostasis in this vital organ system requires efficient local mechanism for antigen surveillance, both in the steady state and during episodes of inflammatory stimulation.

Failure of MHC class II expression in neonatal alveolar macrophages: potential role of class II transactivator

Neonatal peritoneal and blood macrophages are known to be ineffective in antigen‐presentation functions, and this manifests as inefficient MHC class II expression in response to IFN‐γ. Theunderlying mechanisms responsible for this maturational deficiency have not been elucidated. We show here that MHC class II expression in alveolar macrophages (AM) from neonates is also refractory to IFN‐γ stimulation. Furthermore, by examining the intracellular pathway leading to MHC class II expression, we demonstrate that the site of the impairment is at the level of transcription. Thus, expression of mRNA encoding the class II transactivator (CIITA), MHC class II (RT1.B) and invariant chain (Ii) was low or undetectable in neonatal AM stimulated with concentrations of IFN‐γ that induced adult AM to up‐regulate MHC class II expression. The failure of AM from young animals to express MHC class II was not due simply to deficient IFN‐γ receptor function since IFN‐γ‐responsive genes such as IRF‐1, IRF‐2 and IP‐10 were up‐regulated in a dose‐dependent manner from animals of all ages investigated. Importantly, the responsiveness of neonatal AM to IFN‐γ, as determined by MHC class II expression, could be modulated to adult levels when pre‐cultured in vitro. This suggests that microenvironmental factors operative in vivo may play a role in suppressing the expression of MHC class II in AM from young animals. We have investigated the role of type I interferons but did not find them to be responsible for the inability of AM from young animals to induce MHC class II in response to IFN‐γ.

Dendritic cell influx differs between the subglottic and glottic mucosae during acute laryngotracheitis induced by a broad spectrum of stimuli

Clinically, the subglottic and glottic mucosae may react differently, eg, during acute laryngotracheitis. In healthy rats, we showed previously that the composition of the mucosal immune system of the larynx also differs between these areas. Neutrophils, lymphocytes, and dendritic cells (DCs) are part of this mucosal immune system. In particular, DCs occupy a key function. They migrate into inflamed mucosae during the early phase of the immune response, which is normally characterized by an influx of neutrophils. Thus, they help to overcome the time lag between the innate and the adaptive immune responses. In the present study, the influx of DCs, neutrophils, and T lymphocytes into the subglottic and glottic mucosae of rats was examined at different time points after challenge with a broad spectrum of stimuli such as dead Moraxella catarrhalis, viable Bordetella pertussis, viable Sendai virus, and the soluble protein ovalbumin. The number of DCs increased rapidly after the application of the antigens. This increase was as rapid as the increase in neutrophils. Depending on the kind of antigen, their number in the mucosa increased up to 1,000 cells per 0.1 mm2 (Sendai virus). The comparison of different mucosal areas shows that an overwhelming number of immunocompetent cells entered the subglottic mucosa, whereas only a few cells migrated into the adjacent glottic mucosa. In conclusion, after inhalation of different kinds of antigens, the subset of immunocompetent cells investigated in this study entered the laryngeal mucosa in high numbers. The number of DCs entering the laryngeal mucosa was higher than the numbers of the other immune cells investigated. This finding underlines their function as first-line sentinels of the mucosal immune system of the larynx. The observation that the number of cells entering the laryngeal mucosa is location-dependent indicates the ability of adjacent laryngeal regions to react differently. This is similar to the clinical observation of a selective subglottic reaction during acute laryngotracheitis.