The biology of airway dendritic cells - Wiley Online Library

Immunology and Cell Biology (1995) 73, 405-413

The biology of airway dendritic cells ANDREW S McWlLLIAM, DELIA J NELSON and PATRICK G HOLT§ ' Telethon Institute For Child Health Research, West Perth. Western Australia, Australia Summary Recent work from our laboratory has identified a network of constitutively class II MHC (Ia) bearing dendritic cells (DC) within the epithelium ofthe 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. Key words: airways, dendritic, epithelium. Ia, inflammation, MHC, ontogeny, rat, steroids, tumover. Introduction

Antigen presentation

It is the function of the respiratory tract and lungs to deliver oxygen to the vascular system and. in doing so, they represent a uniquely sensitive interface between the tissues ofthe body and a potentially hostile external environment, which in an adult human may comprise up to 75 square metres of epithelial surface and may be exposed to as much as 15 000 L of air per day. It is critically important for survival that these airways maintain their function whilst dealing with a constant barrage of potentially damaging pollutants carried within this airstream; to highlight this point, it has been estimated that in humans, the airways may be exposed to more than 7 kg of pollutant per year compared to 1.4 kg in the gastrointestinal tract.' The range of differing types of material which may be carried into the lungs and which must be either contained or eliminated by the local defence mechanisms is potentially enormous. Thus, the lungs may be exposed to inorganic compounds such as soot, diesel exhaust, mineral dust or chemical fumes. Organic exposure may comprise a wide variety of non-reproducing material such as pollens and danders or live agents such as fungai spores, bacteria or viruses. The nature and amount of material which enters the airways will ultimately determine the type of response initiated and hence whether or not the encounter results in disease. In this article we will review the work carried out in our laboratory on the role of airway dendritic cells (DC) in providing a 'front-line' damage control system within the airways, and in initiating both local and systemic immune responses.

Perhaps the most important resident defence cell, the macrophage, with its versatile response to stimulation, occupies a critically important niche in the frontline defence of the respiratory tract. However, before a more prolonged and specific immune response can be initiated, other elements of the immune system such as the T lymphocyte must be activated and put into place. The problem that confronts the immune system is that the T lymphocyte cannot, by itself, initiate this process nor can it respond to antigens without the assistance of a second or accessory cell. For many years the macrophage was thought to be the cell most likely to have the necessary capabilities to fulfil this function, presumably via expression of Class II antigens. Within the environment ofthe lung and airways, it is clear that the alveolar macrophage is capable of acting as an accessory cell; however, there are now a number of studies in mice, rats and humans which have highlighted the fact that alveolar macrophages are in fact very poor accessory cells.- Indeed, perhaps the most elegant but indirect demonstration of this fact has been provided by the work of Thepen-^ in which the alveolar macrophage population was almost totally ablated using the so-called 'liposome suicide' technique. Despite the absence of alveolar macrophages, the authors were able to demonstrate that the ability of these animals to mount an immune response to intratracheally administered antigens was significantly enhanced, suggesting of course that the presence of the alveolar macrophage was unnecessary for effective antigen presentation and that the macrophage itself may be inimicable to this process.

Correspondence: Dr AS McWilliam, TVW Telethon Institute for Child Health Research, PO Box 855, West Perth. Westem Australia 6872. Australia. Received 20 June 1995; accepted 20 June 1995.

The action of these accessory cells in initiating T cell responses is commonly considered under the umbrella term of'antigen presentation' (AP) and can be dismantled and discussed as a series of separate events. First, as our current notion of AP necessitates that at some stage the antigen must be broken down or digested into small pep-

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tide size pieces which can be readily handled and which also must represent an important antigenic element ofthe antigen. This process must occur either extracellularly as a result of secreted enzymes or it must take place within dedicated phagocytes such as the macrophage or neutrophil which then releases these peptides, or the accessory cell itself must be capable of intemalization of either the digested peptide or uptake and digestion of the whole antigen. As DC have relatively poor phagocytic apparatus, an accurate picture of how this actually takes place is still slowly evolving. Second, the accessory cell must process these peptides in such a way that they become associated with MHC molecules (HLA in man) on the cell surface. Once accomplished this binding to polymorphic MHC molecules then introduces the essential element of restriction into the process. Third, the accessory cell must be able to migrate to a location where there are sufficient numbers of naive T lymphocytes to enable the final interaction to occur. Finally, the accessory cell, bearing its burden of peptide-MHC complex, must engage an appropriate T lymphocyte through its T cell antigen receptor complex and deliver an activation signal which sets the lymphocyte off on its path of antigen specificity and effector functions, namely cytotoxicity, cytokine production etc. A consideration of these events makes it clear that surface expression of HLA/Ia is a necessary prerequisite for presentation of the peptide to the T cell receptor and therefore becomes the quintessential definition of the APC and, in general terms, the concentration of Ia on the surface correlates with the potency of APC in T cell activation."* Thus, our operational definition of a potential APC distils to include only those cells on which Ia can be identified by binding or staining with monoclonal antibodies specific for Ia. Within healthy or diseased lung tissues a number of cell types have been identified as potential APC based on their expression of surface Ia. These cells can be broadly categorized as either 'professional' APC which include B cells, macrophages and dendritic cell (DC) or 'opportunistic' APC which include fibroblasts and airway epithelial cells, either ciliated or Type II pneumocytes. The potential for each population to participate in an immune induction event is ultimately determined by factors such as the host immune status and the physicochemical properties ofthe inhaled material. Thus, by their nature, larger insoluble particulate antigens are restricted in their access to cell populations residing below the epithelial surfaces and therefore, before a Tcell response can be initiated to these antigens, the participation of actively phagocytic cells at the airway lumenal surface is mandatory. In contrast, low molecular weight soluble antigens are readily able to translocate to intraepithelial or submucosal micro-environments via pinocytotic or intercellular pathways and in this way gain ready access to the potential APC populations listed earlier.* The mechanisms by which potential environmental allergens might breach the epithelial barrier have been reviewed elsewhere.^ It is becoming increasingly clear that we are able to differentiate between those cells involved in antigen pro-

cessing and presentation which operate during inflammation and in diseased tissue and those which operate in normal non-inflamed tissues.^ Thus, whereas macrophages, B cells and epithelial cells may have increased APC function during inflammatory reactions, it is the local population of DC which appear to be the principal resident APC. However, despite their constitutive expression of functional Ia antigen, the capacity of these cells to present processed antigen to T cells is a tightly regulated process in vivo and there is now convincing evidence that secreted factor(s) from resident tissue macrophages are active in suppression of DC functions during their residence in the lung.

Distribution of airway DC Analogous to the mononuclear phagocyte system, there appears to be an extensive network of DC throughout most tissues of the body linked by common pathways of movement.^ Within this network a number of terms have been used to describe those DC originating in different tissues; for example, epidermal Langerhans cells, lymph veiled cells and interdigitating cells. Although arising from common bone marrow precursors, the precise relationship of these populations to each other is not at all clear and, likewise, possible differentiation of tissue DC by micro-environmental factors is still an unexplored field. For the purposes of this article we will refer to members of this network within the respiratory tract as DC and although the distinction in the literature is not always clear, to those cells having elements in common with epidermal Langerhans cells (i.e. Birbeck granules [BG]) as Langerhans cells (LC). Within the human lung. DC and/or LC were first identified in tissues with inflammatory granulomatous and fibrotic disease.^"'^ However, since these initial observations were made, a number of studies have succeeded in identifying DC as being present in the normal human airway epithelium, alveolar parenchyma and the nasal mucosa.'"*"'^ In addition they can be found, albeit in small numbers, resident on the human alveolar surface and thus may be present in bronchoalveolar Iavage fluid.'* In laboratory animals DC have now been identified in sections of parenchymal lung tissue and airway Besides their constitutive surface expression of Class II antigen, epidermal LC possess a unique organelle known as the Birbeck granule. Although the function of this organelle is unknown, the observation that 10-50% per cent of Birbeck granules in human LC are acidic and that acidic organelles such as endosomes and Birbeck granules disappear from the LC during periods of culture when there is a concomitant loss of antigen processing capacity, suggest that the Birbeck granule is involved in processing of antigen by the L C . " This conclusion is further supported by evidence that membrane bound molecules can reach intracellular Birbeck granules following endocytosis.-^^ Birbeck granules are normally only demonstrable by electron microscopy and in the nonnal human lung are found only within the airway epithelial LC, and

Biology of airway dendritic cells

then in smaller numbers than in rodent epidermal LC.'^ The situation in rodents is somewhat different in that Birbeck granules do not appear to be present in the DC of the rodent airways. Why only certain populations of DC appear to possess Birbeck granules is not clear. Within sections of lung and airway tissue the relatively large size and pleomorphic morphology of these cells has constituted a significant impediment to their accurate determination and quantitation. In practice most sections, cut either transversely or parallel to the lumen of the airway and immunostained for Ia antigen, either fail to show any evidence of Ia or only succeed in sectioning isolated portions ofthe arborizing processes ofthe 'dendritic' cell, creating a misleading picture. Recent studies from this laboratory have approached the problem of visualizing and quantifying the DC population of the airway epithelium. It was reasoned that, for isolated airway segments, a tangential plane of section would provide some sections passing through the nuclei ofthe epithelial cells in a plane almost parallel to the underlying basement membrane. Appropriate staining of these sections should provide an en face view of the intra-epithelial cell population which would expose the full extent ofthe epidermal network of cells as is commonly seen in staining of epidermal sheets for LC. A schematic representation of the distribution of the epithelial DC population is presented in Fig. I and from this we can gain an immediate appreciation of how tangential sectioning of the tissue results in a plan view of the DC distribution. Immunoperoxidase staining of these tangential airway sections for la has indeed revealed an extensive network of intra-epithelial DC identical to that found in the epidermis of both rat and human

Figure 1 DC distribution in tracheal epithelium. Schematic representation of trachea. The tangential plane of section is represented by the dashed line and the insert shows distribution of DC as visualized by immunohistochemical analysis.

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tissue.'^'^'"^^ Figure 2 shows a representative tangential section of tracheal epithelium immunostained for Class II with the mAb Ox6 and allows an appreciation of the interlocking network formed by these arborizing cells. With conventional transverse sections of airway these cells are not recognizable as DC. and have been mislabelled in the literature as Ia* epithelial cells or monocytes; however, within these sections there are few if any ED2 positive macrophages and hence from our results it is clear that virtually 100% ofthe intra-epithelial Ia staining can be attributed to the DC population. The tangential sectioning technique also provides an excellent means of visualizing the cellular changes occurring within the epithelium during inflammation and we will touch on this subject later in the discussion. Using this method we were able to perform a detailed analysis of the numbers of DC within the epithelium of the rat airways and found a higher density of DC on the dorsal surface of the airways compared with the ventral surfaces (881 per mm- compared to 675 per mm-).-' If we are correct in our belief that these epithelial DC are critical to the processing and presentation of antigens impinging on the airways, then that result would be consistent with this, and with the notion that the higher dorsal numbers reflects the greater amount of material likely to be deposited on the dorsal surface ofthe airway as a result of the aerodynamics of inhalation.'

Figure 2 Immunohistochemical appearance of rat tracheal DC network. Tangential sections of rat tracheal tissue were stained for MHC class Il-bearing celts with tbe 0x6 mAb and visualized using a peroxidase conjugated sheep anti-mouse second antibody. Insert shows a resident DC at higher magnification.

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Further evidence in support of DC numbers being influenced by environmental factors was evident when epithelial DC numbers within each airway generation were compared.^' Again, numbers were highest in the upper airways (600-800 per mm-) and decreased rapidly further down the respiratory tree, reaching 75 per mm^ in the peripheral lung, suggesting that higher numbers are necessary in the upper airways to cope with the increased antigen exposure. In an effort to minimize exposure to atmospheric dust these studies were performed on specific pathogen free (SPF) animals housed on low dust chaff bedding; however, on occasions when pine woodshavings were used as bedding, patches of epithelial staining were seen in which there was an increased density of DC and the expression of Ia appeared more intense. The obvious conclusion from this observation was that inhaled particulate pine dust was able to initiate a local DC response in the immediate area of impact. In a follow-up study, exposure to an aerosol of LPS resulted in an increase of DC numbers from 600 per mm- to approximately 1000 per mm-^ after 24 h.-"* To date, conventional theory would describe DC as a rather static and quiescent population; however, most of these data have derived from studies of the epidermal LC population and our studies of airway DC suggest, for the first time that, depending on their location, DC may be a more dynamic population than previously thought and may be active players in the pathogenesis of the airway inflammatory response.

Ontogeny The first few weeks of life represent a critical window in which the young of most species are exposed, for the first time, to a large array of foreign and potentially damaging antigenic materials. This is also a period during which we know there is an increased susceptibility to infectious disease and to increased sensitization to inhaled antigens. Given the paramount importance of immune mechanisms in these processes, it is entirely logical and reasonable to consider the role of the DC networks within the airways during this period. In the rat, studies by Van Rees^^ first demonstrated Ia positive cells with weak acid-phosphatase activity and slender cytoplasmic processes within the lung on day 16 of gestation. In this study the la positive cells were always located within the mesenchymal tissue between the epithelial tubules of future alveoli and in perivascular or peribronchial tissue. Following birth the number of these cells increased gradually, reaching maximum numbers 21 days after birth; however, no Ia positive free alveolar cells were ever seen. Similar studies by McCarthy^*^ also demonstrated la positive DC within the lung mesenchyme at day 15 and this was followed by their appearance within the airway epithelial lining at day 17, a time at which the airways are not exposed to environmental material. Birbeck granules were absent from these cells which were phenotypically Ia", C l l b ^ . Ox41 , 0x43 . W3/13-. W3/25" and 0 x 8 " . Interestingly, those Ia* cells within the epithelium ofthe airways remained Cl Ib , suggest-

ing that they are either a different cell population to the mesenchymal DC or that local micro-environmental factors as controlling expression of this marker. At day 20 of gestation the pulmonary DC were only 40% as effective as adult ceils in an MLR reaction. While this study found that adult numbers of Ia" DC were present within 5 days after birth, recent work from our laboratory^' has highlighted the responsiveness of these cells to environmental stimuli during this period and has extended the above studies. By staining tangential sections of infant rat trachea with the Ox6 antibody, we were able to show that in animals bom and housed in an environment in which the dust levels are minimized, airway Ia^ DC are usually not detectable until 2-3 days after birth, and adult staining pattems are only evident after weaning at day 21 (Fig. 3a). In contrast, staining with the mAb Ox62 showed that large numbers of Ox62* DC are present in fetal, infant and adult rat airway epithelium. Co-staining experiments showed that 0x62* DC expressing Ox6 are rare in the neonate but increase steadily such that by weaning approximately 65% ofthe Ox62* DC are also Ia"^. Anatomically. Ia* DC first appear at the base of the nasal turbinates. which is the first area ofthe airway exposed by inhalation to environmental antigen and again this pattem of expression is consistent with the notion that maturation of the airway DC network is dependent on inflammatory stimuli. Densitometric analysis of Ox6 staining on individual nasal epithelial DC supported this notion and showed that the mean intensity of Ia expression per cell 2-3 days after birth was comparable with that of epidermal LC from adjacent facial skin. In contrast, the intensity of Ia on tracheal DC remained low for the first week after birth and then rose rapidly to attain adult levels of expression shortly after weaning (Fig. 3b). In addition, the rate of postnatal appearance of DC expressing high levels of Ia was significantly increased by i.p. administration of IFN-y and retarded by aerosol exposure to the steroid fluticasone propionate. While neonatal DC expressed low levels of Ia they did, however, express adult levels of class I MHC and this may hint at a difference in the ability of neonates to respond to inhaled pathogens by either CD8 or CD4 dependent mechanisms. From these studies it is clear that the generation of a local dedicated AP system in the neonatal respiratory tract is driven by local factors and can be modulated either positively or negatively by extemal stimuli. The fufl implications of these observations in terms of immune capacity remain to be determined. Turnover

As discussed earlier, our current understanding of DC biology envisages a model in which function will alter depending on location and stage of life-cycle. Thus, antigen uptake and processing ability in peripheral locations gives way to presenting capacity in central lymph nodes and. in the process, provides a transfer mechanism for antigen. If this model is correct, we might expect that net flow of DC from peripheral to central locations is dependent to some extent on the steady state antigenic exposure of the peripheral tissue and that this in tum may set the

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base line tumover rate for DC in that tissue. However, most of the available data conceming DC tumover is based on studies oflymphoid organs or skin, that is locations where exposure to immunizing amounts of antigen is infrequent. We have examined the tumover of DC situated in the airway epithelium as a situation in which there is constant antigenic exposure (see earlier). Based on previous studies which suggest that DC in peripheral tissues are maintained via a seeding of bone marrow precursors, we attempted to interrupt this supply of incoming precursors by eradicating bone marrow with exposure to whole body irradiation of 1000 rads. These experiments clearly showed that the resident airway DC population declined by 85% over the following 72 h. By employing congenic rats expressing allotypic variants of CD45 which are detectable with mAb we were able to able to repopulate the bone marrow and monitor the rates of repopulation at peripheral sites such as the airway epithelium. These studies demonstrated quite clearly that the airway DC have a

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half-life of approximately 2days.^^ This contrasted strongly with epidermal LC with a half-life of 15 days or longer. Figure 4 illustrates the comparison in changes of DC density occurring in the trachea, lung and skin following irradiation. As DC in the gut wall have also been shown to have a half-life comparable to that of the airway DC^^ these data suggest that DC populations associated with mucosal surfaces are cycling at a faster rate than those in other tissues and this may be reflective of their increased requirement to handle larger and more consistent antigenic loads.

Inflammation Our initial experiments aimed at determining changes in airway DC during inflammatory responses involved brief aerosol exposure to bacterial LPS,-^ which resulted in a transient, approximately 50% increase in the density of DC during the 24-48 h period following exposure. To carry this approach one step further we exposed normal PVG rats to a 60 min aerosol of heat killed MorcLxella catarrhalis organisms.^^"^ This bacterium was chosen because of its known ability to induce a rapid influx of neutrophils and to induce a purulent tracheitis in humans. Immunostaining of tracheal tissue taken immediately after bacterial exposure demonstrated that the earliest detectable cellular response within the tracheal tissue is the recmitment of putative class II MHC complex-bearing DC precursors. These small round intensely class II positive cells initially arrived in advance of the neutrophil

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Figure 3 Postnatal development of the airway intraepithelial DC network, (a) Numerical changes in DC population after birth. Data shown are mean ± s.d. derived from five to seven animals in each age group; at least 200 cells were counted for each animal, (b) la expression on individual airway epithelial DC in rats. Frozen sections of tracheal epithelium were immunoperoxidase stained with mAb Ox6. and the intensity of staining determined on randomly selected DC; data shown arc mean ± s.d. derived from three litters in each age group and seven adult animals.

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Figure 4 Comparative rates of decline in the density of epidermal Langerhans cells (D), lung parenchymal DC (•) and tracheal epithelial DC (O) following whole body irradiation. Data were initially derived as mean number of DC per unit of surface area (for tracheal epithelium and epidermis) or per microscope field (for lung) by using groups of three to four animals per time point and normalized to respective day 0 control figures for presentation.

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influx; however, unlike the neutrophils which rapidly passed through the epithelium into the lumen of the trachea, the DC precursors remained in the epithelium. The numbers of DC precursors within the epithelium reached a maximum within 1 h after exposure, reaching approximately three times the steady state number. Within the ensuing 24 h period, this number remained constant; however, within 8 h of epithelial residence the DC began to alter their rounded shape and change towards a more pleomorphic form reminiscent of veiled cells. By 24 h most of these cells had developed into mature highly branched DC. Thus, it appears that following the influx of DC precursors into the epithelium, local environmental factors begin to induce their differentiation into mature functional DC. Forty-eight h after exposure, local lymph nodes were removed and depleted of resident macrophages and B ceUs. The remaining Ia positive cells were taken to represent DC migrating from the inflamed epithelium, and both the parathymic and intemal jugular nodes demonstrated a 200% increase in DC over this period. This build up in the local nodes was maximal at a time when the epithelial DC numbers were in decline and apparently represents a movement ofthe recruited DC from the local site of inflammation to the regional lymph nodes. Figure 5 illustrates the numerical changes in DC number which occurred in the epithelium and compares this with the change in neutrophils and macrophages in the lumen of the trachea. To summarize, these results suggest that during the course of an intense and acute inflammatory response in the conducting airways, active DC 'surveillance' within the lining epithelium is amplified and consequently results in an increase in the traffic of these cells from the epithelium to the lymph nodes.

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