Results

DC in humans are a very heterogeneous population of cells, and their histological identification has to combine certain phenotypic characteristics with specific morphological criteria. In order to identify DC subsets in bronchial tissue we therefore applied a monoclonal antibody specific for HLA-DR to delineate all APC in combination with monoclonal antibodies to various DC and macrophage markers. HLA-DR+ cells were found in both the epithelium and lamina propria in all specimens examined. Most of these cells co-expressed CD11c, a marker for DC in the mouse. However, a large proportion of this HLA-DR+CD11c+ population also expressed macrophage markers CD68 and RFD-7, demonstrating that CD11c was not a suitable marker to identify DC in human bronchial tissue. However, a smaller proportion of the HLA-DR+ cells co-stained with monoclonal antibodies specific for CD1c. This marker is expressed on dermal DC and has previously been used to identify DC in bronchial tissue. Interestingly, CD1c is also expressed at high levels on most of the myeloid DC precursor subset in circulation characterised as lin^negCD11c+ HLA-DR+.13 In peripheral blood CD1c is also expressed on a subset of B cells. However, CD1c+ cells in bronchial tissue were invariably negative for the pan B cell marker CD20, and also for CD14 and CD68, confirming that they were distinct from B cells, monocytes, and macrophages. CD1c+HLA-DR+ cells were found scattered both in the epithelium and lamina propria. In the epithelium in particular, they often displayed typical pleomorphic dendriform morphology (fig 1a), confirming that this population was indeed DC. Interestingly, at baseline the number of CD1c+HLA-DR+ DC was significantly lower (p=0.009) in patients being treated with topical steroids (n=3) than in steroid naïve patients (fig 2), which agrees with previous human studies on allergic asthmatics7 as well as similar studies in rats.14

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Figure 1

Three-colour immunofluorescence staining for (A) CD1c and (B) CD123 (both Cy3, red) combined with HLA-DR (Alexa 488, green), and laminin (Cy5, blue) in cryosections of (A) challenged or (B) unchallenged bronchial mucosa, respectively. (A) CD1c+DR+ dendritic cells and (B) CD123+DR+ dendritic cells are shown by their mixed (yellow) colour. Some are indicated by filled and open arrows in lamina propria and epithelium, respectively. Epithelial basement membrane is indicated by arrow heads. (A) Note intraepithelial CD1c+HLA-DR+ cells displaying typical dendriform morphology. Most bronchial vessels are HLA-DR+ (mixed green and blue, some with luminal *). (B) Note one CD123+DR+ vessel (*).

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Figure 2

Density of lamina propria CD1c+HLA-DR+ dendritic cells (numbers/mm²) in cryosections of bronchial mucosa from patients with allergic asthma before and after challenge as indicated. Cell counts from the same individual are connected by lines. Medians are indicated by horizontal lines. Numbers refer to patient numbers in table 1. Patients receiving steroids (up to 24 hours before challenge) are indicated.

To examine how DC subsets responded to local allergen challenge, all seven individuals underwent bronchial challenge with house dust mite allergen which resulted in an EAR in six patients, four of whom also experienced an LAR (table 1). There was a dramatic increase (almost threefold) in the number of CD1c+HLA-DR+ DC in the lamina propria 4–5 hours after the challenge (fig 1a and 2), while a nearly twofold increase (1.7 v 3.0 cells/mm basement membrane, fig 1a) occurred in the epithelium. The number increased in both the steroid naive patients and those treated with topical steroids, although the increase was more pronounced in the former (fig2). There was no significant correlation between the magnitude of the LAR and the change in DC numbers.

We have recently shown that another DC subset, designated plasmacytoid DC (P-DC), occurs in small numbers in patients with silent nasal allergy and increased dramatically in response to allergen challenge.11 In this study, however, P-DC was almost absent from the bronchial mucosa both before and after challenge. Only specimens from one patient contained scattered P-DC with no difference between baseline values and those after challenge (fig 1b). To ascertain whether the recruitment of DC was accompanied by other leucocyte subsets we screened the sections for the presence of tissue eosinophils, a common feature of allergic airway inflammation. Interestingly, in those patients receiving steroids no eosinophils were detected before challenge, but they were invariably present after provocation; in one patient high numbers of eosinophils were found. In the steroid naive patients all specimens contained variable numbers of eosinophils, but there was no significant difference compared with baseline. This suggests that recruitment of DC and eosinophils may be regulated independently, and this possibility should be addressed in follow up studies.

Discussion

We have shown for the first time that CD1c+HLA-DR+ DC rapidly (within 4–5 hours) accumulate in human bronchial mucosa in response to allergic inflammation. This finding is in agreement with the kinetics of DC trafficking during the early phase of inflammatory reactions in rat airways.6 It is likely that these incoming DC are derived from circulating Lin^negCD11c+HLA-DR+ myeloid DC precursors. Freshly isolated Lin^negCD11c+HLA-DR+ myeloid DC precursors from peripheral blood are potent stimulators of both CD4+ and CD8+ T cell proliferation, suggesting that this DC subset can act as a potent APC without prior activation signals.15 It is therefore tempting to speculate that these DC newly recruited into the airways following bronchial challenge with allergen can participate directly in the late phase of local allergic reactions. The lack of correlation between the change in DC numbers and the magnitude of LAR in this study does not rule out this possibility, but rather emphasises the fact that allergic symptoms result from a complex series of interactions between various immune cells—for example, APC, T cells, mast cells, eosinophils—and other tissue elements.

The finding that P-DC were virtually absent from the lower airways contrasts with our recent findings in upper airway allergy.11 This could imply that recruitment of this DC subset is restricted to certain regions within the airway tree. However, bronchial tissue from one patient contained some P-DC, which suggests that these cells also have the ability to traffic to the lower airways. It is therefore more likely that these cells have a delayed recruitment compared with CD1c+HLA-DR+ DC and, if so, this opens up the possibility that distinct DC subsets play different functional roles at different time points in the allergic process.

Ideally, a non-atopic control group should have been included in this study to confirm the immunological specificity of these responses. However, the inclusion of control subjects was not accepted by the ethical committee.

While this study is based on a relatively small number of patients, the consistency of the overall findings strongly suggests that the rapid population dynamics of human airway DC during inflammation closely parallels that previously described in animal models. Given the key role of these cells in regulating T cell activation to inhaled antigens in experimental animals, a more detailed study of their functional phenotype in human airways—particularly in asthmatics—would appear to be warranted.