The larynx as an immunological organ: immunological architecture in the pig as a large animal model

E Barker; K Haverson; C R Stokes; M Birchall; M Bailey

doi:10.1111/j.1365-2249.2005.02950.x

. 2006 Jan;143(1):6–14. doi: 10.1111/j.1365-2249.2005.02950.x

The larynx as an immunological organ: immunological architecture in the pig as a large animal model

E Barker ^*,^†, K Haverson ^†, C R Stokes ^†, M Birchall ^*,^†, M Bailey ^†

PMCID: PMC1809556 PMID: 16367928

Abstract

The larynx is a mucosal organ positioned at the divergence of the respiratory and digestive tracts. It is exposed to a wide variety of environmental components, including foreign antigens, tobacco smoke, laryngopharyngeal reflux and pollutants. The mucosal immune system generates either active immune responses or tolerance, depending on the nature of the antigen and we hypothesize that the larynx is important organ for immunological decision-making in the airway. Because the pig is an ideal large animal model in which to explore laryngological research questions, such as those relating to laryngeal transplantation, we investigated the normal mucosal immunology of the porcine larynx. Pig larynges and tracheae were processed and prepared for bright-field microscopy and quantitative, multiple-colour immunofluorescence histology using pig-specific monoclonal antibodies. There was an abundance of immunologically active cells within the mucosa of the larynx and trachea of both the newborn and adult animal. Specifically, major histocompatibility complex class II (MHC class II⁺) cells, CD4⁺ and CD8⁺ cells were identified, although regional differences in numbers were apparent: specifically, the supraglottis contained fewer immunologically relevant cells than other sites sampled. There was a significant correlation between the numbers of MHC class II⁺ and CD4⁺ cells indicating co-ordinate regulation and therefore functional local interactions. The presence of such an immunological structure suggests that the larynx may have important functions in respiratory immunology and that it may trigger strong alloresponses after laryngeal transplantation.

Keywords: allergy, larynx, pig, transplant

Introduction

Historically, the larynx has been considered as an inert mechanical valve. However, studies in man [1–3] and animals [4,5] show that the larynx does contain immunological tissue. A minor part of this has been reported to be present in the form of epithelial follicles (larynx-associated lymphoid tissue: LALT [1]) in an age- and disease-dependent manner [1,6]. However, as with studies of gut [7] and trachea [8], the majority of immunologically active cells appear to be present in a more diffuse, although still organized manner [3,4]. Relatively little analysis of this diffuse immunological architecture has been carried out to date and its function is still unclear. This diffuse architecture is responsive to environment because, in rats, experimental challenge with virus, with live or dead bacteria or in an experimental allergy model results in rapid increases in numbers of dendritic cells (DCs) above the steady state levels [9,10]. However, as mucosal immune systems in other sites express both active immune responses to pathogen-associated antigens and tolerance to harmless environmental antigens, the role of the DCs present in the steady state remains unclear.

The immunological activity of the laryngeal mucosa is likely to be crucial for determining the development and outcome of infectious, inflammatory and neoplastic disease. Involvement of the larynx can occur in allergic disease involving both upper and lower respiratory tracts. Within the upper respiratory tract, squamous cell carcinoma is the most frequent cancer. However, its occurrence is highly subsite-specific, the supraglottis and glottis having an incidence more than 30 times greater than the subglottis and trachea [11]. An association between tumour progression and the immunological architecture is suggested by the observation that higher levels of DCs within the epithelial compartment of excised tumours was associated with better post-surgical prognosis [12].

Progressive advances in immunology and surgery have made laryngeal transplantation an increasingly viable proposition for patients with irreversible laryngeal disease. One successful laryngeal transplant has been performed on a human subject [13], and the patient continues to do well in terms of speech and swallowing 7 years later ([14]; Strome 2005, personal communication). Limited re-innervation did occur in this patient, and the problem of functional restoration is under study in several human and animal models, including the pig [15–17]. However, several questions regarding long-term survival and immunosuppression need to be answered before routine clinical use can be advocated. As with all organ transplantation, the survival of a laryngeal allograft will be strongly dependent on its ability to trigger an alloresponse. The immunogenicity of a tissue has been correlated with its content of specialized antigen-presenting cells [18]. For example, it has been shown that by depleting ‘passenger’ DCs by ‘parking’ renal allografts in transient recipients, allorejection is reduced significantly when the graft is retransplanted [19]. If the laryngeal mucosa bears few immunologically active cells, direct allorejection is likely to be less important than indirect allorejection.

Thus, there are compelling reasons for investigating the immunological architecture of the larynx and the variation between subsites and between individuals [3]. Characterizing the immunological composition in the healthy larynx is a prerequisite for understanding the normal immunological functions of the larynx and how these change with pathology, such as infection and neoplasia. It is also necessary for measuring change due to ischaemia–reperfusion injury or allorejection following laryngeal transplantation. The pig is now an important preclinical model for laryngeal transplantation [4]. Here, we define in detail the immunological architecture of the normal pig laryngeal mucosa and discuss the clinical implications.

Materials and methods

Animals

All pigs were either Landrace–Large White hybrids or inbred Minnesota minipigs [20]. Five pig larynges were obtained from the departmental abattoir after electrical stunning and exsanguination but prior to the animals passing through the scalding tank. Larynges and tracheae were obtained immediately post-mortem from pigs from the animal resource unit, University of Bristol, killed using a captive bolt. Material was also obtained from two newborn piglets before suckling colostrum. Organs were opened using a dorsal longitudinal incision. The true and false vocal folds were injected subepidermally with a histological marking system (Wak-Chemie Medical GMBH, Steinbach, Germany), to allow appropriate orientation at a later stage. Each hemi-larynx was divided into two by an incision through the vestibule, separating the supra- from the subglottis. Three regions of the tracheal mucosa (at 25%, 50% and 75% of the distance between the first tracheal ring and the tracheal bifurcation) were identified and removed. These studies were performed with the approval of the Ethical Committee of the University of Bristol and under licence from the UK Home Office (PPL 30/1786).

Immunohistology

The samples were embedded in OCT (Tissue-Tek® Sakura, Finetek, CA, USA), snap-frozen, processed and prepared for conventional histology haematoxylin and eosin (H&E) and quanititative multiple colour immunofluorescence histology, as described previously by our group [21]. Briefly, frozen sections (4–6 µm) were incubated with a series of solutions including 5% goat and 5% pig serum, optimally diluted primary antibody and isotype-specific secondary fluorochrome conjugates (Southern Biotechnology Associates, Inc., Birmingham, AL, USA). Tertiary staining was achieved with incubation of 7-amino-4-methylcoumarin-3-acetic acid (AMCA) Avidin D (Vector Laboratories, Inc., Burlingame, CA, USA). Three washes of phosphate-buffered saline (PBS) were used between stages to remove unbound serum, antibody or conjugate. Nuclear staining was achieved with a 10-min incubation of either 0·08 mg/ml 4,6-diamidino-2-phenylindole (DAPI, Saint Louis, MO, Sigma) or 0·67 µg/ml propidium iodide (Sigma). Excess DAPI or propidium iodide was removed with PBS. Vectashield® mounting medium (Vector Laboratories, Inc.) was applied prior to the coverslip. The tissue sections were visualized using a combined red, blue and green filter, using a Leica DMR epifluorescence microscope equipped for differential interference contrast (DIC) (Leica, Milton Keynes, UK) and images captured using a high sensitivity colour camera (Colour Coolview; Photonic Science, Robertsbridge, UK) and image analysis software (Image Pro Plus; Media Cybernetics®, Silver Spring, MD, USA). DIC images of the same field were digitized concurrently. For each pig the tissue was prepared as outlined above and approximately 20 fields were captured from the supraglottis and 20 fields from the subglottis. Each field was counted three times in order to minimize counting error.

Monoclonal antibodies

Different epitopes were identified with the following monoclonal antibodies: major histocompatibility complex (MHC) class II (MSA 3) [22], CD3 (PPT 3) [23], CD4 (STH 293 and MIL 17) [24,25], γδ T cells (PPT 27) [26], CD25 (K231·3B2) [27], CD45 (K252·1E4) [28], CD45RC (MIL 15) [25] and collagen IV fibres (AD 4D12G12G10, A. J. Bailey, Bristol, unpublished data).

Cell isolation

Laryngeal epithelial cells were separated, allowing the intraepithelial leucocytes and epithelial cells to be characterized individually. The laryngeal mucosa was dissected free from the cartilage in a single layer and stitched into a sack with the epithelial surface outwards. The mucosal sack was incubated in RPMI (Dutch modification without l-glutamine, Gibco BRL, Gaithersburg, MD, USA), dithiothreitol (DTT) (Boehringer Mannheim GmbH) (1 mM) and indomethacin (0·15 mg/ml, Sigma) at 37°C and shaken vigorously for 30 min to remove mucus. The sack was then incubated in RPMI with trypsin (340 unit/ml, Sigma) for three 45-min washes. After each wash the supernatant was collected, ethylenediamine tetraacetic acid (EDTA) (2 mM) added, centrifuged and cytospun separately.

Statistics

Data were analysed using Wilcoxon's signed-rank test and Pearson's correlation coefficient (SPSS: SPSS Inc., Chicago, IL, USA).

Results

Haematoxylin and eosin

The typical histological appearance of the pig laryngeal mucosa using H&E is shown in Fig. 1a and b. The supraglottic mucosa in the adult pig was covered with stratified squamous epithelium. The subglottis was respiratory in type, covered with pseudostratified columnar epithelium. This architecture was consistent in newborn and adult animals (data not shown).

Expression of MHC class II in the upper respiratory tract

Figure 1(c,d) shows the adult supra- and subglottis, respectively. There were MHC class II⁺ cells both above and below the basement membrane in all sites of the larynx and trachea in newborn and adult animals. The results presented are from the adult animals. There were MHC class II⁺ cells both above and below the basement membrane in all sites of the larynx and trachea (Fig. 2). Although they occurred throughout the upper respiratory tract, their distribution was not uniform. The distribution suggested expression on leucocytes within the epithelium and this was confirmed by cytospins of laryngeal epithelial digests, which demonstrated clearly that there was no MHC class II present on the epithelial cells (Fig. 1e). In these isolated cell preparations, close proximity between the antigen-presenting cells (MHC class II⁺) and adjacent CD45⁺ cells were observed frequently. Despite the presence of MHC class II⁺ cells in both supra- and subglottis, there was a marked difference in the immunological structure of the stratified squamous and respiratory mucosa. Numbers of MHC class II⁺ cells were highly variable between adjacent areas in the same anatomical sites in the same animal (Fig. 3a). However, there were significant differences overall in the number of MHC class II⁺ cells in the lamina propria between laryngeal sites. The supraglottis contained significantly fewer MHC class II⁺ cells in the lamina propria than the subglottis (P < 0·001), proximal trachea (P < 0·001), mid-trachea (P = 0·004) and distal trachea (P = 0·016). In addition the subglottis contained significantly more MHC class II⁺ cells than the distal trachea (P < 0·05) (Fig. 3b). Within the epithelium there was considerably less variation between sites, although there was a significant difference between the supraglottis and the proximal trachea, the former epithelium containing significantly fewer MHC class II⁺ cells (Fig. 3c).

Fig. 2 — Expression of major histocompatibility complex (MHC) class II antigens in pig laryngeal and tracheal mucosa. Each row represents a different anatomical site: supraglottis (a, b and c), glottis (d, e and f); subglottis (g, h and i) and trachea (j, k and l). The first column (a, d, g and j) shows immunofluorescent staining. Blue, nuclear material stained with 4,6-diamidino-2-phenylindole (DAPI); green, MHC class II; red, specific staining of collagen type IV. The second column (b, e, h and k) shows the same field using differential interference contrast microscopy. The third column (c, f, i and l) represents the combined image using immunofluorescence and differential interference contrast microscopy.

Fig. 3 — Expression of major histocompatibility complex (MHC) class II antigens within the subglottic epithelium of the pig larynx. Sites with the same letter are not significantly different. (a) Each data point represents adjacent fields of mucosa from a single animal. (b, c) Expression of MHC class II antigens by cells in the subepithelial lamina propria and epithelium, respectively; each symbol and line represents results from a single animal.

T cell subsets in the upper respiratory tract

Figure 4a shows MHC class II⁺ cells in a dense band on either side of the basement membrane. This band also included CD3⁺ T cells, distributed diffusely both within and beneath the epithelium. In addition, occasional clusters of T cells appeared in the epithelium and lamina propria (Fig. 1f). Although both CD4⁺ and CD8⁺ T cells were present in all sites (Fig. 4b), the distributions were not the same. There were significantly more CD8⁺ than CD4⁺ T cells in the epithelium in the subglottis (P < 0·005), but there was no significant difference observed in the supraglottic epithelium. However, there were significantly fewer CD8⁺ cells within the epithelium (Fig. 5a) and lamina propria (Fig. 5b) of the supraglottis compared with any other site (P = 0·016). In addition, there were significantly more CD8⁺ cells in the lamina propria of the subglottis than in any other site.

Fig. 4 — Expression of major histocompatibility complex (MHC) class II and T cell antigens in the pig laryngeal mucosa. (a) Blue, nuclear material stained with 4,6-diamidino-2-phenylindole (DAPI); green, MHC class II; red, CD3. (b) Blue, nuclear material stained with DAPI; green, CD4 T cells; red CD8 T cells. (c) Blue, CD8 T cells; red, CD4 T cells; green, γδ TCR complex: the γδ TCR can be identified on cells singularly (x) or in combination with CD8 (y), as arrowed. (d) Blue, CD4; green, CD45RC; red. CD25: the CD4 T cells can be seen to be co-expressing CD45RC (x) and CD25 (y), as arrowed.

Fig. 5 — Expression of CD8 by T-cells in the epithelium (a) and lamina propria (b) of the pig larynx. Each symbol and line represents results from a single animal. Sites with the same letter are not significantly different.

In contrast, the number of CD4⁺ cells within or beneath the epithelium was not dependent on site (data not shown). In the lamina propria, CD4⁺ cells predominated in both the supra- and subglottis (P = 0·005). Double-positive CD4⁺CD8⁺ effector/memory T cells have been reported in the pig [29]: however, we observed that less than 2% of T cells within the laryngeal mucosa were double-positive. Despite the low numbers, there were significantly more double-positive cells in the subglottis than supraglottis both within (P < 0·002) and beneath (P < 0·002) the epithelium (data not shown).

Although pig T cells, in common with other artiodactyls, have been reported to exhibit γδ T cell receptors (TCR) more frequently than in the human and rodent, the great majority of cells present in the laryngeal epithelium and lamina propria were conventional αβ TCR cells. As with other cell types, there were more γδ⁺ cells in the subglottis compared to supraglottis (Table 1). In addition, there was a subset of cells present that expressed the γδ TCR independent of expression of other T cell co-receptors (Fig. 4c and Table 1).

Table 1.

Mean (± s.d.) of the densities of the different cell types (cells/mm²) in the pig laryngeal mucosa. The numbers are calculated from 10 pigs for the CD4, CD8 and major histocompatibility complex (MHC) class II data, and from five pigs for the CD8 γδ and γδ data.

	Epithelium		Lamina propria

	Supraglottis	Subglottis	Supraglotis	Subglottis
CD4⁺	522 (658)	552 (383)	1770 (2231)	2712 (2815)
CD8⁺	1133 (1345)	3462 (3276)	312 (426)	1822 (1728)
MHCII⁺	472 (367)	810 (393)	301 (103)	1047 (304)
CD8⁺γδ⁺	15·3 (15·5)	20·5 (13·3)	2 (2)	16 (9)
CD8^–γδ⁺	147 (77)	214 (64)	28 (17)	127 (133)

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The presence of higher numbers of both MHC class II⁺ antigen-presenting cells and CD4⁺ T cells in subglottis than supraglottis suggested that the numbers of the two types of cell might be regulated co-ordinately in order to optimize their interaction. Consistent with this concept, there was a clear positive correlation between the numbers of CD4⁺ T cells and MHC class II⁺ cells, not only in all sites of the larynx (Table 2) but also at the level of individual fields within sites. In addition, activation antigens were present on subsets of the CD4⁺ T cells within the larynx, as shown in Fig. 4d. The majority of the CD4⁺ T cells were CD45RC^– (80% and 95% in the epithelium and 95% and 72% in the lamina propria), although some CD4⁺CD45RC⁺ cells were present in all sites. Some of the CD4⁺ T cells in all sites also co-expressed CD25 (IL-2R), suggesting recent activation (Fig. 6).

Table 2.

Correlation between CD4 and major histocompatibility complex (MHC) class II in all sites of the pig larynx, using Pearson's correlation coefficient (10 pigs).

	r²	P-value
Supraglottis–epithelium	0·557	(< 0·01)
Supraglottis–lamina propria	0·221	(< 0·05)
Subglottis–epithelium	0·501	(< 0·01)
Subglottis–lamina propria	0·589	(< 0·01)

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Fig. 6 — Identification of CD4 antigens by cells in the epithelium and the lamina propria of the subglottis. Pooled data from five pigs showing co-expression of CD45RC and CD25 antigens on CD4⁺ T cells.

Discussion

These results show that the normal laryngeal mucosa contains the required machinery for antigen recognition and T cell activation (MHC class II and CD4⁺ T cells). There was evidence of local antigen recognition within the laryngeal mucosa (CD4⁺CD25⁺ T cells). These results suggest that the larynx is an immunologically active organ with the potential for rapid immunological responses. Organized LALT has been described previously in the pig, predominantly in the epiglottis [4] and the lack of such follicular structures in the supra- and subglottis reported here is likely to be a real observation. In the supra- and subglottis we demonstrated clusters of immunologically active cells interspersed with less dense areas of leucocytes, but the major immunological components were diffusely distributed.

Although not defined functionally, the MHC class II⁺ cells described here have a dendritic morphology, and it is likely that they are DCs, consistent with observations from the rat trachea [8,9,24]. The small intestinal mucosa also contains numerous MHC class II⁺ antigen-presenting cells of more than one type, with the potential for presentation to naive and primed T cells [30]. DCs may extend dendrites between enterocytes, allowing direct antigen sampling and bacterial trapping [31,32]; they may acquire antigens which have crossed the epithelium intact [33]. Following antigen acquisition, intestinal mucosal DCs migrate through afferent lymphatics to the mesenteric lymph nodes, where they can present antigen in T cell areas [34]. The lymphatic drainage of the larynx is complex, possibly reflecting the different embryological origin of the supraglottis and subglottis [35], but DCs from the larynx might be expected to migrate to the deep cervical chain of lymph nodes [36]. However, Constant [37] has shown that lung antigen-presenting cells present ovalbumin locally, and that very few ovalbumin (OVA)-loaded cells reach the local nodes. Thus, laryngeal antigen-presenting cells may acquire antigen in the mucosa and traffic to the deep cervical lymph nodes, where presentation results in relocalization of T cells to the laryngeal mucosa, or there may be direct, local presentation of antigens by antigen-presenting cells to CD45RC⁺ and CD45RC^– CD4⁺ T cells. The positive statistical and topographical correlation in the present study within fields of the laryngeal mucosa between MHC class II and CD4+ T cells suggests that, at least in part, there is direct, local immunosurveillance and recognition of antigen. The observation that the majority of CD4⁺CD45RC⁺ cells also express CD25 is consistent with the appearance of activated, naive cells within the mucosa, either as a result of local presentation or of rapid homing as they lose CD45RC after antigen-recognition in lymph nodes.

In studies of human and rat, laryngeal and intestinal epithelial cells expressed MHC class II [3,38,39]. The present results confirmed that pig laryngeal epithelial cells did not express MHC class II, as all the MHC class II staining co-localized with CD45. This is consistent with findings in pig gut epithelium [21]. In the present study, there was a change of epithelial type from stratified squamous epithelium proximal to the vocal folds to pseudostratified columnar epithelium distal to the folds, a pattern present from birth. In man, at birth, the mucosal surfaces are covered in respiratory epithelium, with the exception of the vocal folds which are stratified squamous [40]. In our studies in the pig, epithelial type also correlated strongly with leucocyte numbers. Our results suggest that the similarity in histological architecture and epithelial type between the subglottis and trachea also extends to the immunological architecture. The supraglottis contained many fewer cells than the other sites, perhaps consistent with the more protective stratified squamous epithelium. We hypothesize that this reflects either a difference in the type or level of response to antigenic exposure with epithelial type and subsite.

Clearly, many factors will impact on the initiation and promotion of tumours in the upper respiratory tract. However, squamous cancer of the airways in man is extremely rare below the vocal cords, where expression of MHC class II was highest in our study. Previous studies have demonstrated an association between high DC numbers in man and prognosis after resection of squamous cell carcinoma, consistent with a role for immunological surveillance. This adds significantly to other evidence that immunosurveillance is an important defence against laryngeal cancer, such as the association with human papilloma virus (HPV) infection [41], the better prognosis of tumours exciting a prominent inflammatory response and the response seen occasionally in combination treatment with cytokines (e.g. interleukin-2 [42]).

It is known that the immunogenicity of transplanted tissue correlates with the content of specialized antigen-presenting cells within it [18]. Hornick and Lechler presented evidence that the early immunogenicity of an allogenic tissue is dependent on the number of professional antigen-presenting cells (able to provide ‘signal 1’ and ‘signal 2’) to the recipient T cells. The 3-year survival data available for intestinal transplants suggest that mucosal organs have a poorer prognosis than organs such as the kidney (45% and 79%, respectively [43]). The results from other studies on the immunological architecture of mucosal tissues, together with the present results in the larynx, suggest that this is attributable to the high prevalence of antigen-presenting cells (MHC class II⁺) throughout mucosal tissues. This is of increasing importance as laryngeal transplantation becomes an increasingly viable therapeutic prospect due, in part, to the harnessing of modern neuroscience to the problem of functional laryngeal reinnervation [15,16]. Thus, when laryngeal transplantation is performed, the presence of these cells is likely to affect adversely survival of an allograft, confirming the need for studies of effective immunosuppression or specific immunomanipulation targeted to the local class II–CD4⁺ cell axis.

In summary, these results demonstrate that the larynx contains the required machinery for immunosurveillance and recognition of local antigens. It is yet to be determined whether the responses generated are geared primarily for an active immune response against pathogens or, alternatively, induction of tolerance to harmless environmental antigens. We hypothesize that this lymphoepithelial architecture may be involved in important immunological decisions, similar to those made by the gastrointestinal tract. Additionally, it is probable that a laryngeal allograft will trigger a strong alloresponse in the absence of significant immunomodulation.

Acknowledgments

Emma Barker was funded by the MRC and Martin Birchall by the Wellcome Trust (grant no. 061125/Z/00).

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PERMALINK

The larynx as an immunological organ: immunological architecture in the pig as a large animal model

E Barker

K Haverson

C R Stokes

M Birchall

M Bailey

Abstract

Introduction