Histological and immunohistochemical characterization of mucosa‐associated lymphoid tissue and antigen‐presenting cells in trachea and lung of cattle

The presence of bronchus‐associated lymphoid tissue (BALT) and its structural components has been described in different healthy animal species and in animals with diseases of the respiratory tract. In contrast to normal mammals, BALT is absent in healthy human adult lungs, but has been found in the lungs of children. The histological characteristics of organized mucosa‐associated lymphoid tissue (MALT), its subsets of immune cells and their in situ distribution in the lung of healthy subadult and adult cattle shows close similarities with BALT in humans and other animal species such as sheep, horses and pigs. This study clearly demonstrates that organized MALT also occurs in the tracheal mucosa of cattle. The absence of tracheal MALT and BALT in calves suggest that these structures are not constitutive. In the mucosa of bovine trachea, bronchi and bronchioli, MHC II+ and CD11c+ dendritic cells (DCs) are located in the epithelium and in the lamina propria mucosae. These DCs are already present in calves soon after birth. Examination of tangential epithelial sheets shows that in the bovine tracheal epithelium, like in man and rat, a dense network of MHC II+ and CD11c+ DCs exists and that their number is considerably higher than in conventional transverse sections. In the bovine tracheal and bronchial epithelium, MHC II+ DCs are extending their dendrites towards the lumen indicating that these DCs possibly are involved in sampling of luminal antigens. The presence of significantly higher numbers of MHC II+ DCs in the tracheal and bronchial/bronchiolar mucosa of older cattle in than in calves possibly results from local stimulation with exogenous antigens during postnatal life. Detection of DCs expressing the costimulatory molecules CD80 and CD86 in calves and cattle suggests maturation of DCs, which is most likely induced by stimulation with exogenous antigens.


| INTRODUC TI ON
Mucosa-associated lymphoid tissue (MALT), which is located along the surface of mucosal tissues such as the respiratory airways of rodents, farm animals and horses, plays an important role in the local immune response (Liebler-Tenorio & Pabst, 2006).In healthy human adult lungs, however, BALT is not present (Pabst & Gehrke, 1990), but was found in the lungs of about one third of young children who died from sudden infant death syndrome or other causes (Tschernig et al., 1995).In the respiratory tract of cattle and other animal species, MALT, that is, nasopharynx-, larynx-and bronchus-associated lymphoid tissue (BALT) is described (Anderson et al., 1986a;Barman et al., 1996;Chen et al., 1989;Huang et al., 1990;Liebler-Tenorio & Pabst, 2006;Mair et al., 1987Mair et al., , 1988;;Meek et al., 2022).Information about the phenotypes of immune cells in respiratory MALT, however, is limited and restricted to immunoglobulin containing cells in the mucosa of the tracheobronchial tree of healthy cattle (Anderson et al., 1986b) and to MALT and immune cells including dendritic cells (DCs) in the nasopharynx of healthy cattle (Meek et al., 2022).In the tracheal mucosa of healthy sheep and horses (Chen et al., 1989;Mair et al., 1987Mair et al., , 1988)), lymphoid aggregates or lymphoid nodules, respectively, are described (Chen et al., 1989;Liebler-Tenorio & Pabst, 2006;Mair et al., 1987Mair et al., , 1988) but descriptions about the occurrence of MALT in the bovine trachea, to the best of the author's knowledge, are not available.
Bovine respiratory disease (BRD) is caused by multiple factors including stress, viral agents such as bovine respiratory syncytial virus and bovine herpes viruses, and bacterial agents (McGill & Sacco, 2020).In the respiratory tract, like in other tissues, MALT induces immune responses to antigens sampled from the mucosal surfaces (Cesta, 2006;Liebler-Tenorio & Pabst, 2006).The first line of defence against microbial pathogens involved in BRD is the innate immune system of the respiratory tract (McGill & Sacco, 2020).
Beside epithelial cells, antigen-presenting cells (APCs), that is, DCs and macrophages, have a main function in innate immune responses (Mellman & Steinman, 2001;Summerfield et al., 2015).DCs are the most important professional APCs and in the lungs, like in other nonlymphoid tissues, there is a pool of immature sessile DCs, so-called steady state DCs (Summerfield et al., 2015;Zanna et al., 2021).
These resident DCs undergo a maturation process upon sampling of pathogens.Thereafter, the maturated DCs migrate to regional lymph nodes where they present the antigens to T cells which is essential for the induction of effective responses of T and B lymphocytes (Kawasaki et al., 2022;Summerfield et al., 2015;Zanna et al., 2021).
In the respiratory tract of humans and different animal species, the distribution of DCs has been described (Heier et al., 2008(Heier et al., , 2011;;Holt et al., 1990;McNeilly et al., 2006;Tschernig et al., 2001Tschernig et al., , 2006)).Except from an immunohistochemical study in tissues including trachea and lungs of 8-9-month-old cattle (Romero-Palomo et al., 2013), little is known about the occurrence and distribution of DCs in the mucosa of the bovine respiratory tract.
The aims of this study were to characterize the histological structure of MALT and the topographical distribution of T and B lymphocytes and APCs, that is, macrophages expressing CD68 and cells with dendritic morphology expressing MHC class II (MHC II + DCs), CD11c, CD80, CD86, and to quantify MHC II + DCs in the tracheal and pulmonary mucosa of conventionally raised calves and cattle.

| Animals
Trachea and lung samples were obtained from 32 cattle of various age, sex and breed (Table 1).The 15 calves of group A were euthanized or deceased because of diseases unrelated to the respiratory tract and had been sent to the Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany, for necropsy.The 10 cattle aged 6 or 7 months of group B had served as control animals in a non-invasive experiment at the Friedrich-Loeffler-Institute, Jena, Germany not related to this study.The experiments were conducted in accordance to the German Animal Protection Law and with approval by the Animal Protection Commission of the Friedrich-Loeffler-Institute (Jaeger et al., 2007;Reinhold et al., 2008).The seven adult cattle of group C had been slaughtered in an abattoir in Gleidingen, Lower Saxony, Germany.The 32 animals were not known to have any previous history of respiratory disease and their respiratory tract had no macroscopical or histological alterations.No animals were killed for the purpose of this study.

| Tracheal tissue samples
From all animals, one transverse, full-thickness section of the lateral wall of the tracheal ring from the proximal, mid and distal trachea (Figure 1) was fixed in 10% neutral buffered formalin (NBF) and routinely embedded in paraffin.From nine animals of group A (nos. 2-7, 9, 10 and 12) and all animals of group C a further transverse section of the three tracheal localizations was embedded in OCT compound (Tissue Tek, Sakura-Finitek, Vogel GmbH & Co KG, Gießen, Germany), snap-frozen in isopentane/liquid nitrogen and stored at −80°C prior to use.From animals of group B, frozen tissue samples were not available.
From the nine animals of group A and all animals of group C an additional sample from the three tracheal localizations was collected for the preparation of longitudinal frozen sections (tangential tracheal epithelial sheets) according to Holt et al. (1988Holt et al. ( , 1990) ) for examination with antibodies to MHC II and CD11c (Table 2).

| Lung tissue samples
From each of the 32 animals, two lung samples were taken (Figure 1).All samples were fixed in 10% NBF and routinely embedded in paraffin.
From all 15 animals of group A and from all seven animals of group C, one sample of lung tissue including the segmental bronchus of the right anterior cranial lung lobe (localization L2a) (Figure 1) was embedded in OCT compound, snap-frozen in isopentane/liquid nitrogen and stored at −80°C prior to use.From the animals of group B, frozen tissue samples were not available.

| Histological examination
From NBF-fixed tissue samples, paraffin sections (2-3 μm) were cut and stained with haematoxylin and eosin (H&E).Slides were evaluated regarding distribution of lymphoid tissues in the different compartments, that is, lamina epithelialis and lamina propria mucosae of trachea, bronchi and bronchioli, with a standard binocular light microscope (Carl Zeiss 670; Carl Zeiss, Jena, Germany; field of view area, 40× magnification: 0.16mm 2 ).Quantification of the total number of lymphoid aggregates and nodules in tracheal and bronchial/bronchiolar mucosa was performed by counting them per transverse section of the three tracheal and the two lung localisations (Appendix S1).
TA B L E 1 Anamnestic data of animals included the study.

| Immunohistochemistry
Paraffin-embedded tracheal and lung samples were incubated with unlabelled mono-and polyclonal antibodies to MHC II, CD3 + and CD20 + lymphocytes, CD68 + macrophages and CD31 + endothelial cells according to routine protocols as described in Table 2.After incubation with biotinylated secondary antibodies (goat anti-mouse IgG or IgM, goat anti-rabbit IgG, respectively) (Vector Laboratories Inc., Burlingame, CA, USA) diluted 1:200 in PBS/Tween with 1% BSA, the avidin-biotin complex (ABC, Vector Laboratories Inc.) was applied according to the manufacturer's instructions.Thereafter, slides were incubated with 3,3´diaminobenzidine as chromogen.

| Evaluation of the distribution of T and B lymphocytes, macrophages and MHC II + DCs in lung and trachea
The distribution of cells immunolabelled with antibodies to CD3, CD20, CD68 and MHC II in paraffin sections of trachea and lung as well as the distribution of MHC II + , CD11c + , CD80 + and CD86 + DCs in frozen transverse and longitudinal sections of the tracheal and lung tissue was analysed using a standard light microscope (Carl Zeiss 670).
For evaluation of the numbers of CD3 + , CD20 + and CD68 + cells in the tracheal and bronchial/bronchiolar mucosa a semiquantitative scoring was performed by counting the number of positively stained cells in 5 fields at 400× magnification (Tables 3 and 5).  4 and 6;

Quantification of MHC II
Shapiro-Wilk test revealed lack of normal distribution in the majority of data sets.Therefore, Kruskal-Wallis test with consecutive Dunn's multiple comparison testing was applied to evaluate differences in MHC II + DC counts between the three age groups using GraphPad Prism version 9 for Windows, GraphPad Software, San Diego, California USA.Similar testing was applied to data sets of lymphoid aggregates and nodules in the three examined anatomic tracheal sites of the different age groups.For the respective values in the two lung sites, Wilcoxon rank sum test was used.For all comparisons, p values lower than 0.05 were assumed as statistically significant.

| Distribution of lymphoid tissue and of T and B lymphocytes, plasma cells and macrophages in the trachea
Details of the histological and immunohistochemical findings are summarized in Table 3.
The tracheal epithelium of the animals of the three different age groups was infiltrated with lymphocytes (Figure 2), plasma cells and round CD68 + macrophages.In the lamina propria mucosae of the animals of group A (Figure 2a-c) and in the animals of group B and C (Figure 2d-i) a mild-to-moderate diffuse, often perivascular and periglandular infiltration with lymphocytes, plasma cells and round CD68 + macrophages was present.Single CD68 + cells showed a dendritic morphology.In the presence of lymphoid aggregates and/or lymphoid nodules, the infiltration located between the lymphoid structures had a broader, band-like appearance.
In all animals of group B and C, solitary organized lymphoid nodules were found (Figure 2d-f).Additionally, varying numbers of nonorganized lymphoid aggregates of predominantly T cells and few B cells were present (Figure .2g-i).Beside primary follicles, secondary follicles with germinal centres (Figure 2d) and mitotic figures were present.
In the centres of secondary follicles few T lymphocytes, numerous B lymphocytes (Figure 2e, f) and few CD68 + macrophages were found.
In the parafollicular areas, which consisted of T cells and single B cells (Figure 2e, f), CD31 + high endothelial venules (HEVs) were detected.
The epithelium above large lymphoid nodules of the cattle of group B and C was often attenuated, had fewer goblet cells than the adjacent epithelium, was focally non-ciliated and infiltrated with predominantly B lymphocytes and few T lymphocytes (Figure 2d-f).4 and Figures 3 and 4.
In transverse, full-thickness paraffin and frozen sections of 10 animals aged between 2 h and 14 days of group A, single MHC II + DCs were detected in the tracheal epithelium (Figure 3a).In all calves, MHC II + DCs were present in the lamina propria mucosae (Figure 3a).In the sections of the youngest calves of this group aged between 1 h and 4 days, only single MHC II + DCs were present in the superficial lamina propria mucosae.In the lamina propria mucosae of three calves aged 9 or 10 days, an increase of MHC II + DCs was noted and in calves aged between 14 and 52 days, a dense network of numerous MHC II + DCs was present.In all cattle of group B and C, like in calves aged between 14 and 52 days of group A, single MHC II + DCs were present in the epithelium and a dense network of DCs was found in the superficial lamina propria mucosae (Figure 3b).Additionally, MHC II + lymphocytes were present in the epithelium and in the lamina propria mucosae of all animals.
Quantification and statistical analysis of DCs immunolabelled for MHC II revealed that their number was significantly higher in the older animals of group B and C (p = 0.005 or p < 0.0001) than in the calves of group A. No significant differences were detected between group B and C (p = 0.255; Table 4 and Figure 4).The mean count of   In nearly all animals, a fine granular and/or homogenous cytoplasmic MHC II positivity was seen in approximately 25%-50% cells (group A and B) or more than 50% (group C) of the tracheal epithelial cells (Figure 3a, b).
The results regarding occurrence, distribution and number of CD11c + and CD80 + DCs in frozen, transverse sections of animals of group A and C were similar as the distribution of MHC II + DCs in paraffin and frozen transverse sections (Figure 3c-f).In all animals of both age groups, only single CD86 + DCs were present in the upper layers of the lamina propria mucosae.
Evaluation of longitudinal frozen sections (tracheal epithelial sheets) of animals of group A and of group C showed that, in contrast to transverse sections, a considerably higher number of both MHC II + and CD11c + (Figure 3e) intraepithelial DCs forming a dense network was present.

| Distribution of lymphoid tissue and of T and B lymphocytes, plasma cells and macrophages in the lung
Details of the histological and immunohistochemical findings are summarized in Table 5.
In the bronchial and, less frequently, in the bronchiolar epithelium of all animals lymphocytes, plasma cells and round CD68 + macrophages were present.Single CD68 + cells showed a dendritic morphology.were detected.Lymphoepithelium or dome areas were not found.

In calves of group
Lymphoid aggregates and nodules were counted in the two sampled localizations.No aggregates or nodules were found in group A. Numbers of aggregates and nodules revealed a prominent count range for all localizations in both groups.The numbers within the two examined localizations were summed up and reported as group means.In group B, the mean sum was 4.65 lymphoid aggregates and 1.30 lymphoid nodules while group C showed an average sum of 3.43 lymphoid aggregates and 1.29 lymphoid nodules (Table 5).No statistically significant differences were detected between the two sample sites of group B and C (Wilcoxon rank sum test; p > 0.05).
In the interalveolar septa of nearly all calves of group A, single T lymphocytes were present.In the interalveolar septa of cattle of group B, single T cells, and in animals of group C, single to few disseminated T lymphocytes were seen.

| DISCUSS ION
The presence of BALT and its histomorphological characteristics have been described in many different animal species (chicken, rabbit, mouse, rat, farm animals and monkeys) (Pabst & Gehrke, 1990;Liebler-Tenorio & Pabst, 2006;Pabst, 2022).Interestingly, in contrast to normal mammals, BALT is absent in healthy lungs of adult humans but has been found in children (Tschernig et al., 1995).In this investigation, organized lymphoid structures, that is, BALT, were found in subadult and adult cattle, but were not present in calves aged between 1 h and 52 days.These findings confirm and extend those of a previous report (Anderson et al., 1986a).Absence of BALT in calves strongly suggests that it is not constitutive in cattle.
The results of this investigation, to the best of the author's knowledge, for the first time demonstrate that MALT also occurs in the tracheal mucosa of healthy subadult and adult cattle.The absence of lymphoid tissue in the trachea of calves examined in this study suggests that tracheal MALT, like BALT, is not constitutive.So far, tracheal MALT structures, that is, lymphoid nodules, are only described in healthy adult horses, and, together with laryngeal lymphoid tissue, have been designated as larynx-and trachea-associated tissue (LTALT) (Mair et al., 1987(Mair et al., , 1988)).
The histological and immunohistochemical results of this study show that the mucosal immune system in trachea and lung of healthy cattle has two main compartments.One of them consists of subepithelial MALT structures with B cell follicles and parafollicular T cell areas with HEVs.The second compartment comprises the surface epithelium with intraepithelial T cells and the lamina propria mucosae with T and B lymphocytes and plasma cells.Intraepithelial CD3 + lymphocytes were already present in the epithelium and in the lamina propria mucosae of the tracheal and bronchial/bronchiolar mucosa of calves soon after birth as described in children (Heier et al., 2011;Stoltenberg et al., 1993).These findings indicate that the structural and cellular components of the bovine airway mucosa develop early in life, possibly due to local contact with environmental factors.
Furthermore, our results demonstrate that in the lamina epithelialis and in the lamina propria mucosae of the bovine trachea and lung APCs, that is, MHC II + and CD11c + DCs and CD68 + macrophages, are present.The histological features of bovine tracheal MALT and BALT, their topographical distribution and the composition of their cellular constituents closely resemble descriptions of human BALT (Brandtzaeg et al., 2008;Brandtzaeg & Pabst, 2004), which has mucosal inductive sites, that is, MALT with B-cell follicles, and mucosal effector sites, that is, immune cells populating the epithelium and the lamina propria mucosae.
In humans and animals, formation of BALT can be induced under a range of disease states such as inflammatory reactions and infections (Foo & Phipps, 2010).The results of Anderson et al. (1986a) and of the present investigation suggest that development of MALT in the trachea and lung of conventionally raised cattle is stimulated by contact with antigens of microbial pathogens from the environment.In contrast to Anderson et al. (1986a), who found a higher number of lymphoid nodules in the anterior lung lobes, we did not see such differences between the tissue samples from the anterior and posterior lung lobes.A methodological reason for this finding could be that Anderson et al. (1986a) performed a systematical histological investigation on a total of 24 lung sites while in this study only one sample each was examined from the two lung lobes.Our study did not reveal any differences regarding the number of lymphoid nodules between the three samples from the proximal, mid and distal trachea and we cannot exclude that this finding is due to a similar methodological reason as discussed for the lung.Sampling of antigens from mucosal surfaces in the respiratory tract occurs through specialized 'microfold' or 'membrane' (M) cells, which are located in the follicle-associated epithelium (Cesta, 2006;Pabst & Tschernig, 2010).The absence of lymphoepithelium in the lungs of cattle examined in this study is in agreement with the results of Anderson et al. (1986a).In that study, lymphoepithelium was only seen in the lungs of cattle with pneumonia indicating that stimulation with a respiratory pathogen could be responsible for its development (Anderson et al., 1986a).In the tracheal mucosa of healthy cattle examined in this investigation, as described in the trachea of healthy horses (Mair et al., 1987) and in the nasopharynx of 8-month-and 8-13-month-old healthy cattle (Anderson et al., 1986a;Meek et al., 2022) (Mair et al., 1987).
Antigen presentation in the respiratory tract is mediated by APCs, that is, DCs and macrophages, which take up antigens through endocytosis or phagocytosis, respectively (Kawasaki et al., 2022).In this study, putative DCs, that is, MHC II + and CD11c + cells with dendritic morphology, were present within the airway epithelium and in the lamina propria mucosae of trachea and lung of all cattle of the three age groups.The topographical distribution of these putative DCs closely resembles descriptions of DCs in tracheal and/or bronchial mucosa of humans, rats, sheep and pigs (Bimczok et al., 2006;Heier et al., 2008Heier et al., , 2011;;Holt et al., 1988Holt et al., , 1990;;McNeilly et al., 2006;Tschernig et al., 2001Tschernig et al., , 2006)).Furthermore, our results confirm and extend findings about the occurrence of MHC II + DCs in trachea and lungs of cattle as described by Romero-Palomo et al. (2013).
As described in the trachea of pigs, rats and children, MHC II + DCs in bovine tracheal and bronchial epithelium extended their dendrites towards the lumen (Bimczok et al., 2006;Heier et al., 2011;Holt & Schon-Hegrad, 1987;Jahnsen et al., 2006).This finding suggests that DCs in the tracheal and pulmonary mucosa of cattle are involved in sampling of luminal antigens.
Our results in tangential epithelial sheets (Holt et al., 1988), to the best of our knowledge, for the first time demonstrate the existence of a dense intraepithelial network of MHC II + and CD11c + DCs in the bovine tracheal epithelium like in the respiratory epithelium of humans and rats (Holt et al., 1988(Holt et al., , 1990)).Interestingly, this network of MHC II + and CD11c + DCs is already present in calves soon after birth as described for neonatal rats (Nelson et al., 1994) suggesting that DCs are constitutive in the respira- that the bovine tracheal epithelium is populated by a considerably higher number of MHC II + and CD11c + DCs than it is visible in conventional transverse sections.Obviously, antigen-presenting DCs in the bovine trachea are located in a strategic position as an intraepithelial network of sentinel cells for inhaled antigens.Quantification and statistical evaluation of MHC II + DCs in the tracheal and bronchial/bronchiolar mucosa revealed that their number was significantly higher in cattle aged 6-7 months and 2 years than in calves.Age-associated numerical increase of DCs expressing MHC class II antigen in the epithelium of the tracheal mucosa of rats was demonstrated by Nelson et al. (1994).Functional studies in rats showed that inhalation of microbial stimuli leads to numerical increases of DCs into the tracheal epithelium in adult rats whereas such response is markedly attenuated in newborn rats and does not reach adult equivalence until 1-week postweaning (Nelson & Holt, 1995).Possibly, the numerical increase of MHC II + DCs seen in the tracheal and bronchial/bronchiolar mucosa of the older cattle examined in this study is induced by local stimulation with inhaled exogenous antigens during postnatal life.
DCs are a heterogeneous population of cells with certain phenotypical and functional properties and have been divided into different subsets (Bošnjak et al., 2022;Kawasaki et al., 2022;Summerfield et al., 2015).The presence and distribution of DCs expressing MHC II and CD11c in the bovine tracheal and bronchial/bronchiolar mucosa suggests that these cells are monocyte-derived DCs representing a resident population of immature sessile, so-called steady state DCs, which only occur in non-lymphoid tissues (Summerfield et al., 2015).
In the bovine tracheal and bronchial/bronchiolar mucosa of calves and adult cattle, DCs expressing the costimulatory molecules CD80 and CD86 were found.After antigen uptake, accumulation of MHC II molecules occurs on the cell surface of immature DCs, which is followed by a maturation process.During maturation, DCs upregulate costimulatory molecules, which is important for the activation of T lymphocytes, and show extension of long processes (Mellman & Steinman, 2001).The presence of DCs expressing CD80 and CD86 in the airway mucosa of conventionally raised, healthy cattle indicates maturation of immature DCs as discussed for the human tracheal mucosa (Tschernig et al., 2006), which most likely is induced by stimulation with inhaled antigens.
In the tracheal and bronchial/bronchiolar mucosa of all examined cattle, macrophages were detected with an anti-human CD68 antibody (clone EBM11), which reacts with bovine macrophages in lungs and in different lymphoid tissues including lymph nodes (Ackermann et al., 1994).These results suggest that APCs in the mucosa of bovine trachea and lungs consist of both, DCs and macrophages as described for human bronchial and nasal mucosa (Heier et al., 2008(Heier et al., , 2011;;Jahnsen et al., 2004).In this study, single CD68 + cells, which were located in the lamina epithelialis mucosae and lamina propria mucosae of the bovine trachea and lung, had a dendritic morphology.Because macrophages, like DCs, can show a dendritic morphology and share phenotypical similarities such as expression of MHC II, CD11c, CD80 and CD86, it is difficult to differentiate DCs from macrophages (Summerfield et al., 2015).Further characterization of APCs in tissue sections of the tracheal and bronchial/bronchiolar mucosa of cattle would require the availability and application of additional cell markers for bovine DCs and macrophages and detection methods on tissue sections such as multicolour immunofluorescence techniques.
In the calves and cattle of this study, positive cytoplasmic immunolabelling for MHC II was seen in tracheal and bronchial epithelial cells as reported for bronchial and/or tracheal epithelial cells of humans, rats and horses (Banks et al., 1999;Wosen et al., 2018).These findings indicate that MHC II + bovine airway epithelial cells play a potential role as non-professional APCs.
In summary, in addition to BALT, organized MALT also occurs in the bovine trachea and their absence in calves suggests that these structures are not constitutive.In the tracheal and pulmonary mu-

a
Frozen tracheal (both transverse full-thickness and longitudinal mucosal samples) and pulmonary samples (localisation L2a: segmental bronchus) of the animals of group A and C were incubated with blocking serum (normal goat serum diluted 1:5 in PBS) labelled with monoclonal antibodies to MHC II, CD11c, CD80 and CD86 (Table 2).Thereafter, endogenous peroxidase activity was inhibited with 0.5% H 2 O 2 in ethanol.After incubation with secondary biotinylated antibodies (goat anti-mouse IgG, Vector Laboratories Inc.) and ABC (Vector Laboratories Inc.), immunoreaction was visualized with 3-amino-9-ethyl-carbazol (AEC) as chromogen (Biologo, Dr. Hartmut Schultheiss e.K., Kronshagen, Germany).After counterstaining with Mayer's haematoxylin, sections were coverslipped with Kaiser's glycerol gelatine (VWR™ International GmbH).As negative controls, tracheal and lung sections (NBF-fixed or frozen) were incubated with either ascites fluid from non-immunized BALB/c mice (Cedarlane, Ontario, Canada) or normal rabbit serum (Sigma Aldrich Chemie GmbH, Steinheim, Germany) at appropriate immunoglobulin concentrations.NBF-fixed, paraffin-embedded and frozen bovine lymph node tissue served as positive control.F I G U R E 1 Diagram of the bovine lower respiratory tract showing the tissue sampling sites.T1: Proximal trachea; T2: Mid trachea; T3: Distal trachea; L1: Small bronchus, bronchioli and lung parenchyma of right anterior lung lobe (animals of group A, and C); L2a: Segmental bronchus of right anterior cranial lung lobe including adjacent bronchioli and lung parenchyma (animals of group A and C); L2b: Small bronchus, bronchioli and lung parenchyma of right posterior cranial lung lobe (animals of group B); L3: Major bronchus including adjacent bronchioli and lung parenchyma of right caudal lung lobe (animals of group A, B, C).TA B L E 2 Primary antibodies and summarized protocols applied on paraffin and/or frozen sections.Tissue Tek® O.C.T.-Compound, Vogel MedTec GmbH & Co. KG, Gießen.

+
DCs was performed by counting positively labelled cells in five randomly chosen fields at 400× magnification in the lamina propria mucosae of transverse sections of the three tracheal localizations and in the lamina propria mucosae of transverse sections of bronchi and bronchioli of the two lung localisations in a blinded manner.The number of MHC II + DCs was expressed as mean number of cells (Tables

3. 2 |
Distribution and numbers of MHC II + DCs and MHC II + epithelial cells, and distribution of CD11c + , CD80 + and CD86 + DCs in the trachea Details of the topographical distribution of DCs and the numbers of MHC II + DCs are summarized in Table
A, only in the oldest animal aged 52 days, single, focal, small, non-organized, peribronchial and -iolar lymphoid aggregates with few T and single B lymphocytes were found.In all animals of group B and C, peribronchial and -bronchiolar lymphoid aggregates mainly consisting of T cells (Figure5a-c) with fewer B cells were present.The majority of cattle also had BALT structures, which were composed of solitary, lymphoid nodules with primary and secondary follicles (Figure5d-i).The latter had a small central pale zone with F I G U R E 3 DCs in paraffin and frozen sections of bovine tracheal tissue immunohistochemically labelled with antibodies to MHC II, CD11c and CD80.Intraepithelial MHC II + cells with one of them showing a dendritic morphology and apparently extending its processes into the epithelium (arrow) in a 14-day-old animal.MHC II + DCs are present in the superficial lamina propria mucosae close to the basement membrane (dashed line).There is a fine granular to diffuse cytoplasmic labelling of epithelial cells.(a, 400×), paraffin section.MHC II + DCs in the epithelium (arrows) and in the superficial lamina propria mucosae (dashed line: basement membrane) of a 2-year-old animal.There is a fine granular to diffuse cytoplasmic labelling of epithelial cells (b, 400×), paraffin section.CD11c + DCs in the superficial lamina propria mucosae (dashed line: basement membrane).(c, 400×).28-day-old animal, frozen section.CD11c + DCs in two different 2-year-old animals.Beside single intraepithelial cells (arrow), there is a dense network of such cells in the lamina propria mucosae of a transverse frozen section (dashed line: basement membrane) (d, 400×).A tangential frozen section demonstrates a dense network of CD11c + cells in the surface epithelium (e, 640x).Single CD80 + DCs in the superficial lamina propria mucosae (dashed line: basement membrane).(f, 400×).2-day-old animal, frozen section.mitotic figures, numerous B cells, few T cells and CD68 + macrophages.The epithelium overlaying the nodules was infiltrated with T and B cells.The parafollicular areas consisted of T cells and fewer B cells(Figure 5e, f, h, i).Additionally, CD31 + high endothelial venules (HEVs)

+
DCs and MHC II + epithelial cells, and distribution of CD11c + , CD80 + and CD86 + DCs in the lung Details of the topographical distribution of DCs and the numbers of MHC II + DCs are summarized in Table 6 and Figure 7. Intraepithelial MHC II + DCs were only seen in the bronchial epithelium of three calves of group A, in three of 10 animals of group B, and in all cattle of group C. In the bronchi of all animals, the majority of MHC II + DCs was present in the superficial lamina propria mucosae (Figure 6a, b).Some MHC II + DCs were extending their processes into the epithelium (Figure 6b).In the majority of animals of group A and in all cattle of group B and C, MHC II + DCs were detected in the lamina propria mucosae of the bronchioli.In animals of group B and C, a dense network of MHC II + DCs was located around the lymphoid aggregates and nodules of the BALT.Quantification and statistical analysis of DCs immunolabelled for MHC II revealed that their number was significantly higher in the older animals of group B and C than in the calves of group A (p = 0.0003 or p = 0.0002; Table 6 and Figure 7).In animals of group A, an average of 5.6 MHC II + DCs was counted, while in group B a mean cell count of 17.0 and in group C a mean number of 20.2 was detected.No statistical differences were detected between group B and C. The bronchial epithelial cells showed a fine granular and/or homogenous cytoplasmic positivity for MHC II in nearly all animals of group A (approximately 25%-50% of cells) and in all animals of group B and C (more than 50% of cells) (Figure 6a, b).Additionally, MHC II + lymphocytes were seen in the epithelium and in the lamina propria mucosae.The results regarding occurrence, distribution and number of CD11c + and CD80 + DCs in the superficial lamina propria mucosae of bronchi and bronchioli of the animals of group A and C were similar to those for MHC II + DCs.In animals of group C, an increased number of CD11c + DCs was present around the BALT.Compared to animals of group C, only in approximately 50% of the animals of group A, CD80 + DCs were seen and their number was markedly lower than in animals of group C. In the animals of both age groups, only single CD86 + DCs were found in the superficial areas of the bronchial and bronchiolar lamina propria mucosae.

F
Numbers of MHC II + DCs in the lamina propria mucosae of the trachea in animals of three different age groups (group A, calves; group B, 6-7-month-old cattle; group C, 2-yearold cattle).The number of MHC II + DCs increased in an aged dependent manner.However, no statistically significant differences were detected between group B and C. Box plot including minimal and maximal values, median and quartiles.Kruskal-Wallis test with Dunn's multiple comparison testing (***p ≤ 0.005; ****p ≤ 0.001).
, lymphoepithelium overlying organized lymphoid tissue was present.Possibly, stimulation of cells belonging to the local mucosal immune system, that is, resident APCs, at the proximal sites of the respiratory tract by inhaled microbial antigens and/or the resident flora is involved in the development of lymphoepithelium and mucosal lymphoid tissue.The epithelium overlying the tracheal lymphoid nodules of the healthy, subadult and adult cattle examined in this study had histological features indicating the presence of follicle-associated epithelium (lymphoepithelium) as described in the trachea of healthy adult horses tory immune system of cattle.Examination of such longitudinal frozen sections (tangential epithelial sheets) clearly demonstrated F I G U R E 6 DCs in paraffin sections of bovine lung tissue immunohistochemically labelled with antibodies to MHC II.MHC II+ DCs in the superficial bronchial lamina propria mucosae of a 2-day-old animal (a, 400×) and of a 2-year-old animal (b, 400) (dashed line: basement membrane).One of these cells, which is located close to the basement membrane, is extending its processes into the epithelium (b, arrow).There is focal fine granular apical (a) and diffuse fine granular and cytoplasmic (b) positivity for MHC II of epithelial cells.F I G U R E 7 Numbers of MHC II + DCs in the lamina propria mucosae of the lung in animals of three different age groups (group A, calves; group B, 6-7-month-old cattle; group C, 2-yearold cattle).The number of MHC II + DCs increased in an aged dependent manner.However, no statistically differences were detected between group B and C. Box plot including minimal and maximal values, median and quartiles.Kruskal-Wallis test with Dunn's multiple comparison testing (****p ≤ 0.001).
cosa, intra-and subepithelial MHC II + and CD11c + DCs are already present in calves and increased numbers of MHC II + DCs are present in older cattle.Intraepithelial MHC II + DCs are extending their dendrites towards the tracheal and bronchial lumen suggesting that they are involved in sampling of luminal antigens.The presence of DCs expressing costimulatory molecules (CD80 and CD86) indicates maturation of DCs most likely due to stimulation with exogenous antigens.

Animal no. Age Lamina epithelialis mucosae Lamina propria mucosae Mean count of MHC II +
Detection of dendritic cells in the tracheal mucosa in cattle of different age groups and quantification.Abbreviations: +, MHC II + dendritic cells (MHC II + DCs) detected; −, not detected; n.e., not examined.Lymphocytes, plasma cells, macrophages and lymphoid tissue in the bronchial/bronchiolar mucosa in cattle of different age groups.
TA B L E 4 TA B L E 5Note: Number of immune cells: (+), single cells detected (1-5 cells/400× magnification); +/++, low-to-moderate infiltration of cells (approximately 40-60 cells/400× magnification); −, not detected.a Numbers of lymphoid aggregates and nodules were counted in two sections and displayed as group mean with range of values.