Pulmonary appendix of the short-tailed shrew BlarinaA unique immunologic organ.код для вставкиСкачать
THE ANATOMICAL RECORD 266:184 –191 (2002) DOI 10.1002/ar.10056 Pulmonary Appendix of the ShortTailed Shrew (Blarina): A Unique Immunologic Organ WESLEY WILKIN PARKE* Robert E. Van Demark Institute of Anatomical Research, Anatomy Division of Basic Medical Sciences, University of South Dakota School of Medicine, Vermillion, South Dakota ABSTRACT The right bronchus of the short-tailed shrew, Blarina brevicauda, terminates in a nonrespiratory pulmonary appendix (PA) containing two bronchial extensions. The experimentally demonstrated ability of these structures to collect and peristaltically expel aspirated material was initially assumed to be a sufficient reason for their developmental persistance, but as bronchus associated lymphoid tissue (BALT) became a subject of immunologic interest in other species, a possible immunologic role for the concentrations of BALT observed in the shrew PA were investigated. As the BALT of the PA contained many well-differentiated plasma cells and numerous particle-containing macrophages, 6- paraffin sections were treated with an immunoperoxidase avidin-biotin preparation that chromogenically identified alpha chains of IgA in many of the PA plasma cells and their associated luminal secretions. Also, vascular injections revealed that the PA had a complex relationship with anastomotic sinusoids connecting the bronchial and pulmonary circulation systems, and scanning electron microscopy showed that the luminal epithelial surfaces of the PA were virtually identical to the scattered BALT aggregates in the bronchi of other animals. It thus appeared that these unique structures in the shrew are morphologically and topographically suited to receive aspirated antigens that induce secretory IgA production, while possibly providing other humoral and cellular immunologic products to the general circulation. Anat Rec 266: 184 –191, 2002. © 2002 Wiley-Liss, Inc. Key words: BALT; IgA; bronchial epithelium; Insectivora; pulmonary clearance mechanisms; antigen sampling The right lung of the short-tailed shrew, Blarina brevicauda, bears an extrapulmonary appendage that has no morphologic or apparently functional equivalent in any other mammal. In this mouse-sized Insectivore, two major derivatives of the right stem bronchus pass through the right caudal lung lobe and, uniting at the lung margin, terminate in a common pleural covering as a nonrespiratory appendix (Figs. 1 and 2A and B). Approximately 80% of the 106 specimens originally examined by Parke (1956) exhibited these structures as two muscular tubes directed medially along the caudal lung margin so that, in situ, they occupied the right costophrenic sinus. The remainder of the series showed these bronchial elements to be of unequal length or, less frequently, irregular in form. © 2002 WILEY-LISS, INC. A subsequent developmental study and a search of the related literature on lung malformations resulted in a comprehensive review (Parke, 1959) that indicated these shrew PA were homologous to aplastic bronchial abnormalities occasionally reported in other mammals. Since similarly sacculate bronchial anomalies are predisposed to pathologic conditions, as they are unable to effectively eliminate accumulations of secreted and aspirated matter, *Correspondence to: Wesley W. Parke, Ph.D., Division of Anatomy, USD School of Medicine, Vermillion, SD 57069. Fax: (605) 677-6381. E-mail: email@example.com Received 16 August 2000; Accepted 16 December 2001 Published online 15 February 2002 PULMONARY APPENDIX OF BLARINA 185 Fig. 1. Dorsal view of the lungs of a Blarina with a tracing of the tracheobronchial pattern, copied from a cast-corrosion specimen, superimposed. Note the larger terminal branches of the right stem bronchus that converge toward the caudal right lung margin to form the PA. This specimen was prepared for a previous study (Parke, 1968). Fig. 3. Thin-section view of the lumen and peribronchial tissue of a PA bronchus (from a South Dakota specimen) fixed in distention. A luminal mucuous plug (Muc) containing particle-laden macrophages (Mac) lies above the epithelium (Ep) and its surrounding muscular tissue, whereas deeper macrophages and plasma cells (PC) are indicated in the peribronchial lymphoid tissue. From a paraffin-embedded section cut at 3 and stained with a Masson’s trichrome preparation. Fig. 2. A: Paraffin section of a PA fixed in the noncontracted state, showing a large quantity of luminal mucous containing a profusion of naturally aspirated particulate matter and the location of the peribronchial lymphoid tissue. B: Paraffin section of a shrew PA fixed in a contracted state, showing the exceptionally heavy peribronchial musculature that peristaltically expels the luminal contents. Both sections in this figure were derived from New Jersey specimens, cut at 5 and stained with Harris’s hematoxilin and eosin. a rationale for the persistence of the PA in this one species was sought. Unfortunately, the earlier investigations took place prior to the general recognition of the immunologic potential of bronchus associated lymphoid tissue (BALT), so efforts were first directed toward the function of the unusually heavy peribronchial musculature and the probability that this pulmonary appendix (PA) might serve as an enhancement to the nonspecific lung clearance mechanisms. An experimental study of carbon-black aerosol inhalations in a series of recently captured specimens did indicate that these bronchial extensions received, accumulated, and effectively expelled particulate aspirated matter, and it was assumed that this function provided sufficient reason for their evolutionary development and retention (Parke and Wetzel, 1968). However, as numerous publications of the last four decades have focused attention on the pulmonary BALT and its role in providing more specific humoral and cellular protection to both the lung and the general circulation in other mammals, a series of shrew lungs were examined to ascertain whether these unusual pulmonary appendages in Blarina might also function as unique immunologic organs. Unfortunately, it is very difficult to maintain live Blarina under the laboratory conditions now mandated for typical experimental small mammals, as they will not live communally nor tolerate disturbances such as repetitive cage cleanings. Thus, the scope of this investigation was confined to demonstrating, in a qualitative sense only, that these 186 PARKE Fig. 4. Sectional view of a quadrant of a PA bronchus from a South Dakota specimen processed with the avidin-biotin immunoperoxidase technique. The chromogen-labeled IgA alpha chains mark the locations of plasma cells (PC) superficial and deep to the peribronchial muscula- ture (Mus), and show IgA in the lymphoepithelial cells (EP). This 6--thick paraffin section was not counterstained, but histologic detail was produced by a phase-contrast technique for photomicrography. structures may serve to assist the more specific aspects of pulmonary immunologic defenses. In all the previous publications concerning these bronchial extensions of Blarina, they were referred to as “bronchial diverticula.” Critical semantic analysis has suggested that this is not an accurate designation since these structures are not lateral outgrowths, as the term would imply, but are terminal bronchial elaborations. As the two bronchial elements are found in a single enclosing sac of visceral pleura, and, as indicated in this report, they are structurally and functionally analogous to the cecal termination known as the “appendix” in the gut of other mammals, the term “pulmonary appendix” (PA) will be subsequently applied to the whole pleura-encased extrapulmonary complex in Blarina. tem (BALT) is noted for the production of the immunoglobulin, IgA. Early research in lung immunobiochemistry showed that an extensive range of cross-reactivity to antibodies raised against this immunoglobulin occurs in many other mammals (Vaerman et al., 1969; Vaerman, 1973), and particularly against human IgA in another Insectivore, Erinacea europa (Vaerman and Heremans, 1971). Therefore, detection of this specific immunoglobulin appeared to be the simplest and most direct way to qualitatively indicate this phase of the suspected immunologic potential of the Blarina PA. Despite the lack of a stain specific for plasma cells per se, the Masson trichrome preparation permitted the positive identification of these cells in the peribronchial lymphoid aggregates in the shrew PA by their characteristic clock-faced chromatin distribution within the nucleus and the prominent eccentric golgi apparatus. Fluoroscein conjugated rabbit antihuman IgA serum, specific for alpha chains, was initially applied to Blarina PA paraffin cross-sections. However, this preparation proved to be too comprehensive, as it not only labeled plasma cells but indiscriminately labeled other lymphoid cells and leucocytes as well. Since a greater specificity was desired, an immunoperoxidase technique employing an avidin-biotin peroxidase complex in a commercial preparation (Lymphoscan Kit; Biomedia Corp., Foster City, CA) produced precisely for the IgA alpha heavy chain was selected. This product utilized the remarkable affinity that avidin has for biotin, which is approximately one million times more efficient than the reaction of most antibodies for their corresponding antigens. The covalent attachment of the activated forms of biotin to biologically active proteins, particularly antibodies, permits the spe- MATERIALS AND METHODS The lungs from a series of 16 Blarina were used in this phase of investigation. All were procured from an approximately 2-km2 region of wooded land in Clay County, South Dakota, by a grid setting of commercial snap traps baited with a mixture of hamburger and peanut butter. The traps were tended and rebaited every 4 hours for 1 day a week over a 4-week period to ensure freshness of the material. For the histologic specimens, the entire thoracic pluck was removed at the trap site and immediately immersed in Bouin’s solution for fixation. After 48 hours of fixation, the PA containing lung segments selected for general histologic examination were paraffin-embedded, sectioned, and processed with either Harris’s hematoxilin and eosin or Masson’s trichrome stain (Fig. 3). The mucosa-associated lymphoid tissue in both the vertebrate digestive system (GALT) and the respiratory sys- 187 PULMONARY APPENDIX OF BLARINA sectional surfaces of remarkable detail (Fig. 7). Then four specimens were split longitudinally to expose their epithelial surfaces (Fig. 8). Both sets of SEM specimens were critical-point dried, gold-coated to 30 – 40 nm, and photographed by SEM. RESULTS Fig. 5. In situ view of the diaphragmatic surface of the shrew right caudal lung lobe after removal of the diapragm. Note that the PA (large black arrow) occupies the right costophrenic sinus, and the vascularly congested sinusoidal areas (small arrows) flank its emergence from the caudal margin of the lung. cific detection of labeled antibodies by using various tracing probes associated with avidin (Wilcheck and Bayer, 1984). As applied to the paraffin sections of the Blarina PA, it chromogenically labeled structures containing IgA with a deep amber color. Although a counterstain was recommended and tried, it often obscured the desired labeling. Thus, a phase-contrast view of the processed slides, in spite of some loss of structural detail, proved to be the best method of observing the reactive locations. The use of a green filter further enhanced the contrast of the amber color and permitted the positive identification of the labeled material in black and white photographs (Fig. 4). Because the developmental sequences of the Blarina PA indicated that it was arterially supplied by the bronchial arteries, and a superficial examination of the structures showed hypervascularization in the lung tissues adjacent to their extrapulmonary commencement (Figs. 5 and 6), three trapped specimens were not field-dissected, but were brought back to the laboratory intact and received intraaortic injections of a mixture of India ink and latex to illustrate the vascular relationships. The injected lungs were then cleared by a previously published technique (Parke and Michels, 1965), transilluminated, and photographed under low magnification. Since the specific histomorphology of BALT has been well described in other mammals, the scanning electronmicroscopy (SEM) of four examples of the shrew PA was accomplished as follows: The bronchi of two freshly fixed specimens of the PA were subjected to a freeze-fracture preparation in which they were immersed in a mixture of liquid nitrogen and ethanol; they then were simply fractured between the grips of two forceps, producing cross- The Masson trichrome staining of the South Dakota series of shrew lungs revealed numerous plasma cells in the BALT of the PA immediately external to the muscular layer, and often just external to the mucosa. The accumulated mucous within the PA lumina, as well as its peribronchial BALT, consistently contained numerous macrophages readily identified by their phagocytized inclusions (Fig. 3). The sections that received the avidin-biotin peroxidase cytochemistry (ABC) treatment for the identification of the IgA alpha heavy chains showed that many, but not all, of the well differentiated plasma cells contained IgA and, in addition, the epithelial cells and luminal mucous immediately adjacent to the positively labeled PCs also showed positive staining for IgA alpha chains (Fig. 4). Consecutive serial sections permitted the tracing of the arterial supply of the PA from the intrapulmonary lung parenchyma distally into the extrapulmonary appendages. As would be expected because of their nonrespiratory character, pulmonary artery terminals did not extend beyond the lung margin, and the extrapulmonary bronchi of the PA were supplied exclusively by continuations of the bronchial arteries; however, their corresponding veins were drained by extensions of the pulmonary veins. In the freshly killed specimens, an area of congested lung tissue was consistently noted surrounding the origins of the PA bronchi just as they left the lung margin (Fig. 5). In serial sections this was seen to be an area of vascular sinusoids (Fig. 6A). The specimens receiving the latex-India ink injections demonstrated that this was a region of a triple vascular anastomoses, for when the injected medium descended the relatively large bronchial arteries it filled these sinusoids, and, by retrograde flow from them, filled the regional terminals of both the pulmonary arteries and the pulmonary veins (Fig. 6B). The functional nature of these sinusoidal areas is unclear, but it is obvious that their presence provides the PA tissues with a generous source of blood-borne inclusions as well as an afferent route for immunologic products that might be generated in the PA for later release to the general circulation. The SEM photomicrographs of freeze-fracture preparations provided a clear view of the lymphatic vessel relationships of the epithelium of the PA. The lymphatics serve as an entirely afferent component of the vascular system, and their role as the most likely primary structural mechanism for the transendothelial passage of particulate and dissolved substances to the deeper lymphatics, and hence to the general circulation, in mucosa associated lymphoid tissues (MALT) has long been recognized. A recent publication by Azzali and Arcari (2000) well illustrated how these “intraendothelial channels” in GALT serve for the transendothelial passage of lymphatic cells to the deeper vessels. As the selected section of the freeze-fractured PA in Fig. 7 shows, the collecting lymphatics here, as in the graphically detailed counterparts in the Peyer’s Patches in rabbits, form a system of well defined channels that are shown coursing in the internal aspect of the basement membrane and communicating 188 PARKE Fig. 6. A: A 4- section of the dilated proximal bronchi (1) of a shrew PA. Note that the muscular layer is not as pronounced as it is in the extrapulmonary extensions, and the surrounding vascular sinusoids are indicated (2). B: A shrew PA specimen that was injected with a mixture of latex and India ink via the aorta. The injection medium followed the systemic circulation through the bronchial arteries (3), and by the triple anastomoses of the sinusoids back-filled the larger branches of the regional pulmonary veins (4) and pulmonary arteries (5). Only branches of the bronchial arteries extend along the PA bronchi. The specimen was cleared in methylsalycilate and photographed under low power by transillumination. with a similar plexus external to the lamina propria (LP) that drains into the intramuscular and more external lymphatics of the BALT. The SEM analysis of the luminal aspect of the PA tissues produced some interesting results. Under a relatively low (⫻1,000) magnification, the surface characteristics of the PA epithelium were quite uniform throughout the entire extrapulmonary length of these structures. The conventional ciliated bronchial epithelium cells were relatively few in number, as the lymphoepithelial cells typical of BALT predominated in the views of the epithelial “pavement” of the PA. These cells showed numerous microvilli on their luminal surfaces, with deep crevices between the cells that were often filled with a mucous exudate. In fact, the view provided in Fig. 8 is virtually identical to the photomicrographs furnished by Bienenstock and Johnston (1976) and Racz et al. (1977) in their reports showing SEM preparations of lymphoepithelial surfaces of rabbit BALT. function was suspected. Klein (1875) noted the lymphoid aggregates in the tracheobronchial system and stated that they were morphologically identical to the lymph follicles described in tonsils and intestines. However, it was not until nearly a century later that Bienenstock and his associates (1973), in an extensive restudy of the pulmonary lymphoid aggregates, originated the term “bronchus associated lymphoid tissue” (BALT). Subsequently, the terms “gut associated lymphoid tissue” (GALT) and the more inclusive concept, “mucosa associated lymphoid tissue” (MALT) were established as semantic retroformations (Sminia, 1996). BALT is distributed throughout various vertebrate groups, including mammals, birds, and some reptiles. Sminia and associates (1989) noted that the degree of its development within certain species seemed to be directly dependent upon the extent of the antigen load of that particular group. Since BALT collectively refers to the entire distribution of lymphoid aggregates throughout the lung, Sminia and coworkers (1989) introduced the term “bronchus associated lymphoid unit” (BALU) to designate the individual, topically discrete lymphoid masses. The histology of BALT has been most intensively studied in the rat (Simecka et al., 1986; Plesch, 1982) and the rabbit (Bienenstock et al., 1973), where it can be detected on the exposed epithelial surfaces of the opened bronchi as whiter-appearing patches that at first may seem to be randomly distributed, but are most consistently found near the acute angles of bronchial bifurcations. Plesch (1982), while noting that the number of BALUs in an DISCUSSION As the entire epithelial linings of the vertebrate digestive and respiratory tracts are continuously exposed to ingested and aspirated antigens, their extensive association with the lymphoid components of the immune system is quite understandable. The scattering of lymphoid cells throughout the body’s mucosal surfaces, and their discernible concentrations into lymphoid aggregates, such as Peyer’s Patches, were known long before the extent of their PULMONARY APPENDIX OF BLARINA Fig. 7. SEM view of a PA bronchus section produced by the freezefracture technique. The luminal mucous (Muc) is seen on the surface of the epithelium (E). Note the lymph channels on both sides of the lamina propria (LP) that lies internal to the peribronchial musculature (Mus). These lymph channels are believed to aid the return of particle-laden macrophages from the lumen to the peribronchial lymphoid tissue. SEM magnification ⫻1,000. animal’s tracheobronchial tract appears to be an interspecific variable, estimated that the respiratory system of a rat may contain 30 –50 units. Since it is now firmly established that BALT is a complex association of immunologically related cellular types, its demonstration as the major histologic component within the highly muscular, nonrespiratory PA of Blarina gives this unique structure a much greater physiological significance than its originally surmised role as simply an assistant to the general lung defenses provided by the mucociliary and peristaltic pulmonary clearance mechanisms. The above-described demonstrations of IgA and the IgAproducing plasma cells of the lymphoid tissue concentrated within the shrew PA, in addition to the SEM and histologic photomicrographs, strongly suggest that it is an immunologic organ. Admittedly, no quantitative judgments may be made from the evidence presented here, since the individual immunologic status of such randomly acquired wild specimens must be remarkably variable. Nor has a sufficiently in-depth histologic analysis been made upon which to base qualitative conclusions concerning other humoral and cellular immunologic functions. Yet, some reasonable assumptions may be derived from 189 Fig. 8. An SEM photomicrograph of the lymphoepithelial surface of an extrapulmonary bronchus of a South Dakota Blarina PA. Note that the typical ciliated bronchial epithelial cells are sparsely distributed among the microvilli-covered cells that form the epithelial “pavement” which is characteristic of a typical BALT surface. This type of epithelial cell composition is found throughout the extrapulmonary extent of the PA bronchi and is identical to that photographed in the BALT of other small mammals. The deep epithelial grooves run parallel to the extrapulmonary bronchial axes. SEM magnification ⫻1,000. the information presented in this report and related previous publications (Parke, 1956, 1959; Parke and Wetzel, 1968) when it is considered against the evidence derived from the published studies on other animals. These are discussed below. The Blarina PA has been observed to receive aspirated antigenic substances, both naturally and experimentally, and periodically expel them by peristaltic and mucociliary actions. Thus, these particular blind bronchial extensions are able to avoid the pathologic consequences characteristic of anomalous homologous developments that occasionally occur in other mammals. However, they still receive a continuous turnover of aspirated antigens since the fossorial nature of this shrew constantly exposes them to a “high antigen load.” These aspirated particles, while in the lumina of the PA bronchial extensions, are brought into approximation to the BALT that is the characteristic peribronchial tissue for their entire extrapulmonary length. In this temporary retention of the aspirated matter, numerous observed macrophages enter the accumulated mucous and phagocytize its particulate matter. Although 190 PARKE the actual process has not been observed in the shrew PA, it appears that these particle-laden cells gain access to the deeper layers of the BALT, presumably aided by the illustrated transepithelial lymphatics, and there present the antigenic material to the receptive lymphoid cells. Thus, IgA-producing plasma cells elaborate the specifically generated secretory antibodies found in the luminal mucosal secretions while other assumed cellular and humoral responses may be readily relayed to the general circulation by way of the complex vascular associations demonstrated at the proximal attachments of the PA. There is no intention here to imply that any of the tissue components within the bronchial extensions of the shrew PA have been found (to date) to be unique to this structure. Rather, they are an anatomic and topographic arrangement of a type of histologic organization that is relatively disseminated elsewhere, but is here apparently concentrated to enhance effectiveness. This situation shows a developmental, morpological, and functional relationship similar to that which the more consolidated GALT in the vermiform appendix shows to the scattered collections of Peyer’s Patches in the intestines. Like the human vermiform appendix, the PA in the shrew has lost most of the original major functions of the tubular structures from which it was derived, but has greatly retained and amplified its immunologic role. After the antigen sampling function of the avian Bursa of Fabricious was defined, there was considerable speculation about which tissue organization in the nonavian vertebrates provided an equivalent function. Fichtelius et al. (1968) named the collective GALT of the Peyer’s Patches and the cecal appendix as their favorite candidate for this assignment, and it makes an interesting theoretical postulation that the unusual sequence of developmental events, as described by Parke (1959), positioned the shrew PA to ideally receive aspirated matter into its paired lumina. Because of the great antigenic load environmentally placed on the tracheobronchial system of the shrew, the PA, as an antigen sampling device, may constitute an opportunistic expression of an exceptional version of a bursa equivalent in this one species of mammal. As such, this unique phyletic development provides a good example of the concept of “exaptation” (Gould, 1977), in which the ontological expression of preexisting structures (air-conducting bronchi) is modified to perform a novel function. The recalcitrance of the Blarina toward being a compliant laboratory animal is quite unfortunate. If these hyperactive, solitary, venomous, and nearly totally fossorial mammals could be induced to produce captive laboratory colonies, there is little doubt that their unique tracheobronchial organization would be, by now, the subject of a more extensive literature and a considerable source of additional knowledge concerning the immunological capacities of the vertebrate respiratory system. It is hoped that this report will stimulate further attempts to investigate the true nature of the very remarkable PA in this one group of mammals. TAXONOMIC NOTATION The short-tailed shrew, Blarina, for the practical purposes considered here, constitutes a monospecific genus of the family Soricidae (Order: Insectivora) with a single species, brevicauda. It inhabits virtually all of the eastern United States, from southern Canada to Florida, and as far west as the proximities of the 100° meridian that roughly coincides with the commencement of the highplains prairie. Although insular and otherwise isolated populations form various subspecies, sampled specimens from the extremes of the generic geographic range all show the presence of the PA. Their closest relative, the small short-tailed shrew (Cryptotis parva), is also a monospecific genus that is almost coextensive with Blarina in its distribution. Although it has a much less obligatory fossorial habit and a generally smaller body size, Cryptotis resembles the Blarina enough that the determination of two fewer upper rear premolars (and now, the complete lack of anything that resembles the PA) in Cryptotis may be required for a definitive differentiation. Blarina is almost exclusively fossorial in habit, establishing extensive runs under the forest leafmold or vegetative thatch. These malodorous and vicious little mammals have a very high metabolic rate and must daily consume their body weight in other small vertebrates, invertebrates, and some vegetable matter. Their submandibular glands produce a venomous saliva, and they readily subdue and eat small mammals of equivalent size. They can tolerate exposure to light for only a brief time, and if unable to establish a reclusive habitat they soon die in extreme agitation. 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