Bronchus associated lymphoid tissue in the lungs of cattle: relationship to age

Research in Veterinary Science /986.4/.2//-220

Bronchus associated lymphoid tissue in the lungs of.. cattle: relationship to age M. L. ANDERSON*, P. F. MOORE, Department ojPathology, D. M. HYDE, Department ojAnatomy, D. L. DUNGWORTH, Department oj Pathology, College oj Veterinary Medicine, University of California, Davis, California 95616, USA

The occurrence and morphological features of bronchus associated lymphoid tissue (BALT) were examined by light and electron microscopy in the nonpneumonic left lungs of five age groups of cattle from neonate to aged adult. The amount of BALT was correlated with age. It was absent in neonatal lungs and increased progressively with age until it declined in aged adult cattle. Within age groups the occurrence and morphology of BALT was variable. Widely scattered lymphoid aggregates were the predominant morphological type of BALT in the bovine lung. Follicular development in BALT foci was less frequent and quite variable within individual lungs. Morphologically distinct Iymphoepithelium overlying BALT foci could not be demonstrated in non-pneumonic bovine lungs. BOVINE respiratory disease is a common and economically important problem that has multiple causative factors (Yates 1982). The current methods of immunisatiun for some of the respiratory disease pathogens have not been uniformly successful and some vaccinations may have an adverse effect on the animal when respiratory infections occur (Martin 1983). Respiratory immunity to certain infectious agents is best correlated with a local immune response and when the lung is appropriately stimulated it can provide substantial local cellular and antibody mediated immune responses (Kaltrieder 1976). Lymphoid tissue within the lung contributes to these immune responses and a better understanding of bovine pulmonary lymphoid tissue is important to the development of effective methods to stimulate local respiratory immunity. The term bronchus associated lymphoid tissue (SALT) has been used to describe the lymphoid tissue associated with the airways of the lung (Bienenstock et al 1973). Morphological studies of SALT in laboratory animals have been performed by a number of investigators (Bienenstock et al 1973, 'Present address: Veterinary Diagnostic Laboratory. University of California. 18830 Road 112. Tulare. California 93274. USA

Chamberlain et a11973, Fournier et aI1977, Racz et al 1977, Plesch 1982). These studies have shown that well developed SALT foci have a complex structure containing a lymphoid follicle with adjacent mixed lymphoid cells and venules with high endothelium. The respiratory epithelium overlying SALT has been referred to as lymphoepithelium due to its unciliated, flattened appearance and the infiltration of lymphoid cells. The functions of SALT are not fully established but its roles in sampling luminal antigens for the initiation of local immune responses and as a source of immunoglobulin A (IgA) immunoblasts for mucosal secretory defence have experimental support (Bienenstock et a11982, McDermott et aI1982). The objectives of this report are to describe the occurrence and morphology of SALT in the nonpneumonic lungs from various age groups of cattle. These results are compared to SALT in pneumonic calf lungs and to the literature pertaining to SALT in laboratory animals. Materials and methods

Animals Lungs were obtained from clinically normal cattle that were conventionally raised in both university and commercial herds. The initial selection of lungs for histopathological study was based on the absence of gross lesions. The lungs from five age groups of cattle were sampled. Table I contains the number of lungs sampled in each group. The neonatal group consisted of calves under four days old. The four-, eight- and 18-month-old groups each contained lungs of cattle of those approximate ages. The adult group consisted of aged, cull dairy cows over three years old.

Lung sampling The left lung was infused via the left principal bronchus with fixative at 30 em pressure. The routine fixative was 10 per cent neutral buffered formalin. A modified Karnovsky's fixative was used on selected

211

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M. L. Anderson, P. F. Moore, D. M. Hyde, D. L. Dungworth

TABLE 1: Selection of non-pneumonic lungs from grossly normal lungs from five age groups. Rejection on the basis of histological lesions ranged from 25 of 32 (78 per cent) in fourmonth-old calves to one of six (17 per cent) in aged adult cows

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Total sampled

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11 32 22 8 6

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79

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lungs for additional ultrastructural studies (Nowell 1971). The lungs were allowed to fix for 24 to 48 hours before sampling. The lung was systematically sampled at a total of 24 sites for histological examination. Six samples each 2'5x2'OxO'5 em in size were taken from each of four lung lesions (Fig I). The distal trachea, right principal bronchus and a tracheobronchial lymph node were also sampled.

Histological examination Lung tissue from all animals was examined histologically for evidence of pneumonia. Lungs which had cellular exudation into airspaces, focal cellular infiltration of alveolar parenchyma or diffuse peribronchial/peribronchiolar cellular infiltrations at any sample site were excluded from the study. The remaining 31 non-pneumonic left lungs were used for the study of the structure and distribution of HALT (Table I). Organised lymphoid tissue foci associated with the tracheobronchial tree were referred to as HALT and

FIG 1: Diagram of systematic sampling used on infused whole left lungs for histological examinations. Each region contained six samples 2'5 x 1·0 x 0'5 em centred on a major lobar bronchus spaced equidistant from the proximal to the distal extremity of the lobe. ACl Anterior cranial lobe, PCl Posterior cranial lobe, VCl Ventral caudal lobe, Del Dorsal caudal lobe

classified as lymphoid aggregates or lymphoid nodules based on histological criteria. Lymphoid aggregates were defined as focal accumulations of small mononuclear cells and histiocytic cells with no evidence of follicular organisation. The lymphoid nodules were defined as organised structures with a centrally located lymphoid follicle containing larger, paler staining lymphocytes and macrophages. Adjacent to the follicle along the lateral and inner (luminal) margins was a zone of small lymphocytes and other mononuclear cells. Three airway categories were established in order to classify the location of lymphoid tissue within each histological section of lung. The centrally located major lobar bronchus was the large bronchus category. The small bronchus category included a variable number (one to seven) of segmental and subsegmental bronchi. The last category included all the bronchioles present within each histological section. The number and types of HALT foci occurring within the various airway categories in each histological section were recorded. Significant differences between HALT foci by region and age were calculated using a computer-based statistical program (HMD?) (Dixon 1981).

Ultrastructural examination Transmission electron microscopic examinations were performed on selected samples of lung. Samples were chosen from sites identified as having HALT by histological examination. Tissues were post-fixed in osmium tetroxide, dehydrated in acetone and infiltrated and embedded in epon-Araldite mixture (Polysciences). Sections of I /-1m were cut for light microscopy and stained with toluidine blue. Sites containing lymphoid tissue were selected for thin sectioning. Thin sections were mounted on formvar coated slotted grids and stained with uranyl acetate and lead citrate. Samples for scanning electron microscopic study of airway surfaces were selected from lung regions found to have the greatest amount of HALT development by histological examination. Samples of airway and adjacent lung parenchyma were approximately l'Ox l-Ox 0'5 em in size. Samples were dehydrated in alcohol, infiltrated with amyl acetate, subjected to critical point drying, mounted on stubs and plated with gold. Examination of 90 samples of lung airway from non-pneumonic lungs were performed from all age groups with BAL T. Airways containing nonciliated epithelial patches thought to represent Iymphoepithelium were photographed. These regions were excised and embedded in plastic for step serial sectioning and histological examination to verify the presence of underlying lymphoid tissue.

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FIG 3: Occurrence of the two histological categories of BALT foci compared in each age group in which BALT was observed. Foci with histological evidence of lymphoid follicles (Iymhoid nodules) were a relatively small percentage of total BAL T and varied with the total occurrence of BALT in each group

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Examination of HALT in pneumonic lungs Six grossly normal lungs from four-month-old calves with chronic lymphoid bronchitis and bronchointerstitial pneumonia ('cuffing' pneumonia) were selected for study. The sampling and examination methods were the same as those used in the nonpneumonic lungs. The criteria for quantification of lymphoid tissue foci was changed for the pneumonic lungs because of extensive lymphoid proliferation which resulted in peribronchial and peri bronchiolar lymphoid cuffs. These cuffs were recorded as a single focus. Results

variable amount of lymphoid tissue was identified in the lungs of all older cattle. There was considerable variability in the total number of SALT foci sampled within each of the age groups. The mean number of HALT foci increased with increasing age to a maximum in the 18-month group and then declined in the adult group. There were significant differences in the mean number of SALT foci between groups (P
Occurrence of HAL T Lymphoid tissue was not observed in the tracheobronchial tree in any of the neonatal calves (Fig 2). A

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The location of lymphoid tissue within the wall of

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M. L. Anderson, P. F. Moore, D. M. Hyde, D. L. Dungworth

.:». FIG 4: Example of a lymphoid nodule within the lamina propria of a large lobar bronchus illustrating the usual location of SALTfoci within larger bronchi (magnification x !OO)

the airway appeared to be related to the size and structural complexity of the wall. In larger airways which had a wide lamina propria and prominent smooth muscle layers, the HALT foci were predominantly located directly beneath the basement membrane of the epithelium (Fig 4). In smaller

bronchi the location of HALT foci was variable, although they were frequently encountered beneath a narrow band of smooth muscle within the submucosa (Fig 5). Foci within the walls of bronchioles typically extended from the epithelium to the adventitia (Fig 6) and were often observed at sites of airway bifurcation. However, the sampling method used precluded a systematic analysis of the relative frequency of BALT at these sites. Lymphoid aggregates and nodules were widely distributed throughout all airway levels and in all regions of the lung. Evidence for preferentiallocalisation to a particular airway size was lacking. All regions of the lung contained BALT but there was a tendency for occurrence and follicular development of HALT to be greater in the cranial lobes than in the caudal lobe. The regional differences in amount of HALT compared among all cattle were not statistically significant but consistently exhibited this pattern (Fig 7). In the four- and 18-month-old groups the dorsal caudal lobe had significantly less (P
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Lymphoid aggregates consisted of variably sized, discrete collections of small mononuclear cells with densely staining nuclei and scant amounts of cytoplasm (Figs 5, 6). Ultrastructural examination

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FIG 7: Regional occurrence of BALT within the left lungs of all cattle, There was a consistent tendency for the dorsal caudal region to have the least lymphoid nodules and lymphoid aggregates

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revealed a mixed population of cells which were predominantly lymphocytes. Scattered among the lymphocytes were large electron lucent cells often with dendritic cytoplasmic processes extending between adjacent lymphocytes. These cytoplasmic processes were occasionally observed to be associated with reticular fibres. They were presumed to represent a mixed population of reticulum cells and could not be further classified on the basis of ultrastructural morphology. Lymphatics were frequently identified adjacent to aggregates (Fig 8). The lymphoid nodule was structurally more complex than the lymphoid aggregate. The central feature was a lymphoid follicle which exhibited a variable morphology similar to follicles in peripheral lymphoid tissues. Primary and secondary lymphoid follicles were observed. Secondary follicles contained a central pale zone with mitotic figures and scattered macrophages containing phagocytosed cellular debris. Lymphocytes and dendritic reticulum cells were also observed within this zone. Peripheral to the follicular region along the lateral and luminal margins there was a zone which varied in prominence and contained a mixed population of small lymphocytes,

plasma cells and macro phages scattered among connective tissues and vascular structures (Fig 9). Venules with high endothelium were common in these cellular areas and frequently had lymphocytes emigrating through the endothelium (Fig 10). The cells along the luminal side of the follicle extended to

216

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FIG 10: High endothelial venule (HEV) within the parafollicular area of a lymphoid nodule. Lymphocytes (Ll are passing through the endothelium. Plasma cells (Pc) are also present (magnification x 16001

the overlying epithelial basement membrane and occasionally infiltrated the epithelium. Particular interest was given to the examination of the airway epithelium for evidence of non-ciliated Iymphoepithelium overlying BALT foci. Light microscopic and ultrastructural studies did not provide clear evidence of lymphoepithelium overlying BALT foci in the lungs of non-pneumonic cattle. Infiltration of the overlying epithelium was observed but the columnar, ciliated nature of the epithelium was retained. Step-serial sections were performed on selected samples in an unsuccessful attempt to identify attenuated non-ciliated epithelium within the sample. Scanning electron microscopy was also employed since larger areas of lung airway surface could be examined for Iymphoepithelium. Lymphoepithelium could not be demonstrated in nonpneumonic lungs by this method. In an effort to determine if lymphoepithelium could be identified in other sites of ciliated respiratory epithelium a study of the nasal mucosa was performed in the eight-month-old calves. Samples of mucosa from the posterior nasal septum in the naso-

FIG 11: Scanning electron micrograph of a non-ciliated epithelial patch in the bovine nasopharyngeal mucosa. Non-ciliated cells contain surface microvilli (bar represents 25 I'm)

Lymphoid tissue in the bovine lung

217

pneumonic lungs. Pneumonic lungs had more BALT foci which were distributed in a regional pattern (Fig 13). Significant increases were associated with the cranial lobe, particularly the posterior cranial lobe which contained more than half of the total lymphoid tissue sampled in the lung. Lymphoid tissue was predominantly associated with bronchioles which contained over 65 per cent of the total amount. These small airways were typically encircled by large confluent lymphoid tissue cuffs which contained follicles. These cuffs appeared to compress the surrounding alveolar parenchyma and narrow the airway lumen (Fig 14). The overlying epithelium was 55

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pharyngeal region from these calves were processed similarly to lung tissue. Histologically all samples contained extensive development of subepithelial lymphoid tissue with focal infiltration and alteration of the ciliated epithelium overlying lymphoid follicles. Scanning electron microscopic examination of the mucosal surface revealed multiple discrete nonciliated epithelial patches morphologically compatible with Iymphoepithelium (Fig II). These patches were excised and embedded in plastic. Sections of the epithelium revealed that it was attenuated and infiltrated with lymphocytes through an interrupted basement membrane (Fig 12). BAL T

in pneumonic lungs

The lymphoid tissue in the lungs of four-month-old calves with histological lesions of enzootic pneumonia exhibited quantitative and morphological differences from BALT in four-month-old non-

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FIG 13: Regional occurrence of BAlT compared in two groups of grossly normal lungs from four-month-old calves. Lungs with histological lesions of proliferative bronchointerstitial pneumonia ('cuffing') exhibited increased regional occurrence and development of lymphoid tissue foci. Significant differences lP
M. L. Anderson, P. F. Moore, D. M. Hyde, D. L. Dungworth

218

non-ciliated regions throughout the length of the affected airway. The larger, more proximal bronchi had discrete foci of hyperplastic lymphoid nodules in which a more localised area of infiltrated, nonciliated epithelium (Iymphoepithelium) could be related to the underlying lymphoid tissue by histological and scanning electron microscopic examinations (Fig 15, 16). Discussion

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FIG 14: Example of hyperplastic lymphoid tissue surrounding an affected bronchiole in the lung of a four-month-old calf with regional proliferative bronchointerstitial pneumonia (magnification x 32)

infiltrated with lymphocytes and mixed inflammatory cells. Scanning electron microscopic examination of the surface demonstrated widespread and irregular

The results of this study indicate that HALT has a variable occurrence in the non-pneumonic bovine lung and consists predominantly of widely scattered Iymphoreticular aggregates which develop after birth. The development of lymphoid follicles within HALT foci was an inconsistent feature. The presence of Iymphoepithelium overlying HALT could not be demonstrated in non-pneumonic lungs. Pneumonic lungs had larger amounts of and more developed HALT in which lymphoepitheliurn was readily demonstrated. The use of the term HALT to refer to the lymphoid tissue in the bronchial tree of cattle should be clarified. References to HALT have described it as a Iymphoepithelial nodule with a Iymphofollicular structure intimately associated with an area of specially adapted epithelium (Bienenstock et al 1982, Jericho 1982). In this study the broader usage of the term is based in part on studies of HALT in the rat

Lymphoid tissue in the bovine lung

which suggest that the various morphological types of lymphoid tissue from simple clusters of mononuclear cells to Iymphoepithelial nodules represent different stages of development of a single entity (Gregsen et al 1979a). The lyrnphoepithelial component of the HALT structure has been shown to be an inconsistent feature in adult rats (Chamberlain et al 1973, Gregsen et al 1979b, 1979c). In view of these observations it is reasonable to refer to all lymphoid tissue associated with the bronchial mucosa as HALT. The use of conventionally raised animals of unknown exposure to respiratory pathogens and other pulmonary insults was unavoidable. The screening procedure of gross examination followed by extensive histological sampling allowed the authors to obtain non-pneumonic lungs from cattle of a range of ages in sufficient numbers for meaningful comparisons to be made. However, it is important to note that conventionally raised laboratory animals have increased occurrence and development of HALT than germ-free or specific pathogen free animals (Giddens et a11971, Gregson et aI1979b). The results presented here were undoubtedly influenced by the previous environment of the animal but do give information concerning the usual development of HALT in healthy bovine lungs. The occurrence of HALT foci within each group of

219

animals varied significantly according to the age of the cattle. The neonatal calves had no lymphoid tissue within the lungs. These results are in agreement with studies in neonatal rats, rabbits and pigs (Jericho 1970, Bienenstock et a11973, Gregson et aI1979a). An explanation cannot be definitely established for the age associated increase in HALT occurrence in the four-, eight- and 18-month-old groups followed by a decrease in adult cattle. Age has been correlated with the size and morphological activity of certain peripheral lymphoid tissues of cattle (Lubis et a11982) and sheep (Reynolds and Morris 1983). However, because of the varying environments of each of the age groups it is possible that differences in the intensity of local stimulation may be more responsible for the observed differences than age alone. The occurrence and degree of follicular development of HALT was quite variable within each of the age groups, perhaps due to differences in local stimulation and environmental conditions. It would be necessary to use specific pathogen free cattle to reduce these influences. However, the overall low frequency of lymphoid follicles, which were found in only about IS per cent of HALT foci, the complete absence of follicular development in some lungs and the increased occurrence in pneumonic lungs suggest that follicles are dynamic structures which occur as a result of local antigenic stimulation. Two morphological categories, the lymphoid aggregate and lymphoid nodule, were used to define HALT for descriptive and quantitative purposes. There was a continuous spectrum of morphological complexity, from the small lymphoreticular cell cluster to the more complex lymphoid tissues associated with follicular HALT, suggesting that these categories simply represent differing development of the same entity. There was no indication that the degree of organisation of HALT was decreased in the distal airways as has been reported in the rat (Fournier et al 1977). Well developed lymphoid nodules in the bovine lung had some of the zonal organisation and structural features of rabbit HALT as characterised by Racz et al (1977) in which a follicular area with an overlying dome area and a lateral parafollicular area were established as specific zones. The cell population within the bovine follicular and parafollicular areas were quite similar to the rabbit with the exception that interdigitating reticular cells were not detected around high endothelial venules. In addition, a morphologically distinct dome area was not apparent in the bovine since similar cell populations and high endothelial venules were observed in the parafollicular regions on both the luminal and lateral sides of the follicle. The inability to demonstrate Iymphoepithelium by the methods employed indicates that this feature is at

220

M. L. Anderson, P. F. Moore, D. M. Hyde, D. L. Dungworth

least rare in the normal bovine lung. Some descriptions of BALT have emphasised the Iymphoepithelium indicating that it is a normal component of the BALT structure (Bienenstock et a11973, 1982). However, in addition to the failure of some investigators to consistently demonstrate Iymphoepithelium mentioned previously, there is experimental evidence that its presence is related to antigenic stimulation (Gregson et al 1979c). In these experiments the degree of BALT hyperplasia and Iymphoepithelial development in rats was related to the degree of local antigenic stimulation. It was noted that in control animals only large BALT follicles possessed Iymphoepithelium. They concluded that Iymphoepithelium was present as an adaptive response to antigenic stimulation of the underlying lymphoid tissue. The demonstration of Iymphoepithelium overlying BALT foci in the pneumonic calf lungs in the present study may have a similar antigen driven immunological development. Whether the presence of Iymphoepithelium represents an immunologically induced change of the overlying epithelium or a direct effect on the epithelium by a respiratory pathogen with subsequent lymphoid tissue response is unresolved. Morphologically well developed Iymphoepithelium is present in the nasopharynx in association with extensive submucosal lymphoid tissue. Such a site in the respiratory tract would presumably receive large amounts of surface stimulation from inhaled particle deposition and resident flora and would appear to be a favourable site for sampling the antigenic composition of the environment. Immunocompetent cells from these organised lymphoid tissues might conceivably migrate to other mucosal sites including the tracheobronchial tree for effector functions. The bovine lung appears to be relatively deficient in BALT development when compared to the rabbit and rat. The authors' examinations of lungs from conventionally raised rabbits and rats are in agreement with the literature reviewed by Bienenstock et al (1982) that BALT follicles and Iymphoepithelium are well developed features in the lungs of these species. It is not established whether the lack of Iymphoepithelium and the relatively simple structure of BALT in the healthy bovine lung are morphological evidence of a poorly developed local immune system or whether the morphological appearance of BALT simply reflects the degree of local antigenic stimulation present in each lung. The large amount of variability of BALT in the nonpneumonic lungs would suggest that BALT occurrence and development is influenced by external factors. The great degree of lymphoid tissue proliferation seen in the lungs of calves with cuffing pneumonia emphasises the potential for local inflammation to induce lymphoid tissues within airways. These results

were obtained from conventionally raised animals which would presumably have greater BALT development than pathogen free animals; whether BALT is absent in the unstimulated bovine lung, however, cannot be answered by this study. The results here suggest that the lymphoid tissues present in the tracheobronchial tree of cattle are dynamic structures, occurring in response to local antigenic stimulation or inflammation, and that information concerning BALT structure and function derived from laboratory animal models should be applied cautiously to the bovine. Acknowledgements The authors would like to thank Ms M. Flores for the preparation of this manuscript and Ms P. Curtis and Dr A. Mariassy for their excellent technical assistance. References BIENENSTOCK, J., JOHNSTON, N. & PEREZ, D. Y. E. (1973) Laboratory Investigation 28, 686-692 BIENENSTOCK, J., McDERMOTT, M. R. & BEFUS, A. D. (1982) Bulletin Europeen de Physiopathologie Respiratoire 18,153-177 CHAMBERLAIN, D. W., NOPAJAROONSRI, C. & SIMON, G. T. (1973) American Review of Respiratory Disease 108, 621-631 DIXON, W. J. (1981) BMDP Statistical Software. Los Angeles, University of California Press. pp 105-115 FOURNIER, M., VAl, F., DERENNE, J. PH. & PARIENTE, R. (1977) American Review of Respiratory Disease 116, 685-694 GIDDENS, W. I., WHITEHAIR, C. K. & CARTER, G. R. (1971) American Journal of Veterinary Research 32,115-129 GREGSON, R. L., DAVEY, M. J. & PRENTICE, D. E. (l979a) Laboratory Animals 13, 231-238 GREGSON, R. L., DAVEY, M. J. & PRENTICE, D. E. (1979b) Laboratory Animals 13, 239-243 GREGSON, R. L., DAVEY, M. J. & PRENTICE, D. E. (l979c) British Journal of Experimental Pathology 60, 471-482 JERICHO, K. W. F. (1970) Research in Veterinary Science II, 548-552 JERICHO, K. W. F. (1982) Research in Veterinary Science 32, 206-212 KALTREIDER, H. B. (1976) American Review of Respiratory Disease 113, 347-379 LUBIS, I., LADDS, P. W. & REILLY, L. R. (1982) Research in Veterinary Science 32, 270-277 McDERMOTT, M. R., BEFUS, A. D. & BIENENSTOCK, J. (1982) International Review of Experimental Pathology 23, 47-112 MARTIN, S. w. (1983) Canadian Veterinary Journal 24, 10-19 NOWELL, J. A. (1971) American Review of Respiratory Disease 103,313-328 PLESCH, B. E. C. (1982) Advances in Experimental Medicine and Biology 149, 491-497 RACZ, P., JENNER-RACZ, K., MYRVIK, Q. N. & FAINTER, L. K. (1977) Journal of the Reticuloendothelial Society 22, 59-83 REYNOLDS, R. D. & MORRIS, B. (1983) European Journal of Immunology 13, 627-635 YATES, W. D. G. (1982) Canadian Journal of Comparative Medicine 46, 225-263

Accepted January 28, 1986