distribution of iron in the gastrointestinal tract of the common vampire bat: evidence for...

THE ANATOMICAL RECORD 198:183-192 (1980)

Distribution of Iron in the Gastrointestinal Tract of the Common Vampire Bat: Evidence for Macrophage-Linked Iron Clearance

DAVID MORTON AND WILLIAM A. WIMSA'M' Division of Biological Sciences, Cornell University, Ithaca, New York 14853 (D.M., W A . W J , and Department of Biological Sciences, Wright State Uniuersity, Dayton, Ohio 45435 (D.M.)

ABSTRACT Iron in the tissues of the digestive tract of the common vampire bat (Desmodus rotundus) has been studied using histochemical, electron microscopic, and autoradiographic methods. This animal is an obligate sanguivore and has a daily intake of dietary iron BOO times that of man. The amount and distribution of tissue iron is not affected by either a single blood meal or starvation but does reflect the degree of siderosis of each animal's liver and spleen. By 7 days after the injection of a trace amount of 55Fe into the peritoneal cavity, labelled siderotic macrophages are present both on the serosa and within the walls of the stomach and intestine. In the lower intestine, such cells can be derived from the germinal centers of Peyer's patches. Siderotic macrophages are often situated in the lamina propria under areas of siderotic epithelium. Label is also present in the apical cytoplasm of mucosal epithelial cells, a region of abundant siderosomes. The ultrastructure of these organelles is extremely variable. Accumulations of membranous whorls and stacks, "stippled bodies," ferritin molecules, and larger "ferruginous" complexes are bounded by one or a number of membranes. Iron is excreted when these epithelial cells are desqua- mated into the gut lumen. Similar Prussian blue-positive granules are present in the feces. Unlike other glandular cells, the parietal cells of the fundic caecum are siderotic. Their cytoplasm contains abundant siderosomes and ferritin with accumulations of amembranous ferritin bodies in the intracellular canalicular spaces. Prussian blue-positive granules are present in the lumens of fundic glands.

The common vampire bat (Desmodus rotundus) is unique among mammals in being an obligate sanguivore and therefore must han- dle unusually large quantities of ingested hemoglobin iron. Captive vampire bats con- sume approximately 15.5 ml of defibrinated blood per day (Wimsatt and Guerriere, '62). The mean hemoglobin content of bovine blood is 11.5 gm/lOO ml (Albritton, '521, and hemoglobin is approximately 0.34% iron (Comroe, '65). Thus, the mean iron content of a vampire's average daily dietary blood intake is 6.1 mg from hemoglobin alone. Comparison of the daily mean iron consumption of humans, 0.25 mgkg body weight (Rush et., '66), and the vampire, 203.3 mgikg body weight (30.0 gm bat), reveals an BOO-fold difference.

Preliminary observations have demonstrat- ed the presence of large accumulations of iron in the gastrointestinal tissues (Morton, '76).

This was not the situation in other organs, except for those with expected hemopoietic or iron storage functions. The objectives of the present study were both to describe the distribution of this iron and to investigate its significance relative to iron balance.

The anatomy of the gastrointestinal tract is unusual in that it has been adapted to accom- modate the ingestion of large quantities of blood (Huxley, 1865; Park and Hall, '51; Rouk and Glass, '70; Hart, '71; Forman, '72). A relatively small bore esophagus joins a re- duced pyloric stomach. To the bat's right, it gives rise to the duodenum, and to the left a large sac-like fundic caecum. The latter is the main depository for ingested blood and dis-

Received March 7, 1979; accepted March 10, '80. Reprint requests should be addressed to David Morton, Department

of Biological Sciences, Wright State University, Dayton, Ohio 45435.

0003-276X/80/1982-0183$02.00 0 1980 ALAN R. LISS, INC.

184 D. MORTON AND W. A. WIMSATT

tends during a meal to fill the peritoneal cavity. The division of the intestine into small and large segments is not obvious at the gross level, although their microanatomy is distinc- tive.

METHODS

The 23 male and ten non-pregnant female vampire bats used in this study were collected in the state of Oaxaca, Mexico, during Janu- ary 1969, January 1971,.and March 1973. They were maintained in specially designed cages (Wimsatt et al., '73) and were fed defibrinated bovine blood ad libitum for 6 days of each week. Bats were usually sacrificed by etherization, although five experimentally shared animals were first ensanguinated.

For light microscopy, the liver, spleen, stomach, and intestinal tract from each bat were fixed in 10% phosphate buffered formalin for 24 hours and embedded in glycol methacrylate (Feder and O'Brien, '68). Sections were cut a t 2 microns and placed on glass slides. The whole gastrointestinal tracts were often "jelly- rolled" to allow for a complete longitudinal profile in one section. A section of liver and spleen from the same animal was routinely added to the slide for parallel processing. Sec- tions were treated with the Prussian blue (hereafter referred to as PB) reaction for iron (Hutchinson, '531, counterstained in 0.1% nuclear fast red (Kernechtrot, Chroma, 1A402) in 5% aqueous aluminum sulfate and mounted with Permount or cellulose tridecanoate.

The identification of iron was further sub- stantiated with three additional histochemical tests: dinitroresorcinol (Humphrey, '351, chlor- ate hematoxylin (von Kutlik, '701, and batho- phenanthroline (Hukill and Putt, '62). Nega- tive controls were prepared by extracting iron from sections with either 5% oxalic acid, 3.7N H,SO, or 1% sodium dithionite in 0.1 M acetate-HC1 buffer, pH 4.5 (Lillie et al., '63). Details of the use of these methods on sections of tissue embedded in glycol methacrylate have been reported in an earlier paper (Mor- ton, '78).

For electron microscopy, 1-ml cubes of the fundic stomach, duodenum, ileum, and colon from four bats were fixed in ice-cold 5% glu- taraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2% hours, rinsed in ice-cold buffer, and postfixed in WO aqueous OsO, overnight at 4°C. This material was then rinsed in ice- cold distilled water and postfixed in 2% aqueous uranyl acetate for 20 minutes. Tis- sues were then dehydrated in acetone and

embedded in Epon 812lAraldite 506. Thick sections were exposed to the PB reaction, and thin sections were usually stained with WO aqueous uranyl acetate and lead citrate (Rey- nolds, '63). As a further check of the iron- containing status of cells and their organelles and inclusions, unstained thin sections were compared to parallel thin sections in which iron had been extracted with dithionite.

The large amounts of iron found in the digestive tract could be affected by the ingestion of a single blood meal. Nineteen animals were sacrificed at intervals of zero (five bats), 1 day (five bats), 2 days (two bats), and 3 days (two bats) after feeding overnight. In addition, five bats were starved for 1 day, fed for 1 hour, and sacrificed either 1 hour (three bats) or 6 hours (two bats) after withdrawal of the experimental meal. After tissues were processed either for light or electron microscopy, the relative amount of iron in cells and organs was evaluated both by direct microscopic observation and from photomicrographs.

In order to study the effects of further body iron loading, 2 ml of iron dextran (ImferonR, Merrell) containing 100 mg of iron was administered intraperitoneally to two vampire bats. %o control bats were likewise injected with sterile distilled water. After 12 days, the aforementioned tissues, along with the lungs, were processed for light microscopy.

To test the hypothesis that a t least some gastrointestinal iron is derived from the body, ten vampire bats were administered intraperitoneally with 50 pCi of 55Fe (New England Nuclear, 3.4 pg of iron as "Fe-citrate in 0.05 ml sterile 1% acetic acid). Pairs of bats were sacrificed after intervals of 3 ,7 , 14, 21, and 75 days. Tissues were processed for light microscopy. Sections on glass slides were coated with K.5 Ilford photographic emulsion diluted 1:3 with 2% glycerol in distilled water. These preparations, along with appropriate controls, were exposed for 3 months. Autoradiograms were developed in Kodak D-19, fixed in Kodak acid fixer, and stained with nuclear fast red. Autoradiographic and histochemical compar- isons were made using two additional sets of slides bearing parallel serial sections. One set was exposed to the PB reaction and stained in nuclear fast red, and the other was stained in nuclear fast red alone.

RESULTS

Close comparison between vampire bats subjected to various feeding and starvation regimens failed to demonstrate any correla-

IRON IN GASTROINTESTINAL TRACT OF VAMPIRE BAT 185

tive relationships. However, the total iron profile of any given bat reflected the relative siderosis of its liver and spleen, i.e., low to high concentrations of iron in the digestive tract were always linked to similar conditions in these organs. The location and quantity of iron appeared not to be affected by the sex or weight of individual animals. Unfortunately, information on the age of these bats was not available, as they were captured in the wild.

All the organs of the gastrointestinal tract of each vampire bat contained iron which varied in amount and cellular distribution along the various segments. However, the esophagus was iron-free except for a rare macrophage in the lamina propria. Also, in the least siderotic bats, the first few centimeters of the proximal duodenum and both the car- diac and pyloric stomach contained little or no iron. Iron was most abundant in the fundic caecum proximal to the esophageal-gastric- duodenal junction, decreasing gradually toward its blind end and disappearing complete- ly in the opposite direction toward the pylorus. Iron reappeared a few centimeters down the duodenum, increasing slowly to a peak in the ileum and decreasing again in the lower intestine. The majority of this iron was present in the mucosal epithelium and siderotic macrophages.

Most mucosal epithelial cells continued PB- positive granules in their apical cytoplasm (Figs. 1, 3-51’. The ultrastructure of these granules was heterogeneous, but, in general, they were membrane-bound vacuoles enclos- ing varying amounts of both ferritin (approximately 6 nm) and other larger “ferruginous” complexes (possibly clumped iron cores offer- ritin) unevenly distributed in a matrix of different electron densities. They were bounded by one or more unit membranes. Multiple membranes in many of the least siderotic vacuoles were created by a single membrane wrapped a number of times around the iron- rich matrix. The innermost leaflet of such membranes was often reflected internally, forming smaller membranous spirals (Fig. 10). Multiple limiting membranes and internal membranous spirals in the more siderotic vacuoles appeared compressed, forming myelin- like membranous lamellae and whorls, re- spectively (Fig. 9, arrow). Molecules of ferritin were often lined up between these tightly packed membranes. This description matches Richter’s definition of siderosomes first pro- posed in 1957 (’78), and this term will be used for the remainder of the paper where the

identity of PB-positive granules has been checked by electron microscopy.

In addition to their presence in siderosomes, molecules of ferritin and some larger “ferruginous” complexes were scattered throughout siderotic mucosal epithelial cells, primarily in the cell sap, mitochondria, and elements of the lysosomal system, and occasionally in various other organelles. At the light microscopic level, their presence correlated with a diffuse staining with the PB reaction.

In areas where the contents had not been disturbed, mucosal epithelial cells were observed both desquamating into and in the lumen (Figs. 1, 4, arrows). PB-positive material was present in the forming feces in the lower intestine (Fig. 8).

Gastrointestinal glandular epithelium rarely contained iron, except for the parietal cells of the fundic glands (Fig. 12). However, occa- sional PB-positive granules were evident in fundic mucous neck and chief cells, pyloric and Brunner’s glands, and intestinal goblet and Paneth cells, especially in tissues from the most siderotic bats. ?tvo forms of parietal cell PB-positive granules were identified. The first was identical to the siderosomes described previously and was located in the cytoplasm, which was rich in ferritin and larger “ferruginous” complexes (Fig. 13). The second was observed only in the intracellular canaliculi. These amembranous granules were composed of an irregular accumulation of ferritin and larger “ferruginous” complexes or perhaps the iron cores of ferritin (Fig. 14). No evidence of exocytosis was seen. PB-positive granules were observed in the ducts of fundic glands.

Siderotic macrophages were present in the connective tissues of the digestive tract in areas under siderotic mucosal epithelium (Fig. 1). Such cells were concentrated in the cores of the most siderotic villi of the ileum (Fig. 3) and in and around Peyer’s patches in the lower intestine. Within the germinal centers of these lymphatic nodules, siderotic macrophages were oriented in a concentrically arranged gradient, with the least and most siderotic centrally and peripherally located, respective- ly (Fig. 5). Central cells were usually without

se, serosa si, siderotic macrophages ba, bacteria gc, germinal center ic, intracellular canaiiculus st, “stippled” body

of iron dextran.

Abbreviations

‘All figures are of tissues from bats that did not receive an injection

186 D. MORTON AND W. A. WIMSATT

Fig. 1. Mucosa of the fundic caecum, LM X1000. The horizontal bars represent a distance of ten micrometers.

Fig. 2. Serosa of the fundic caecum. LM X780.

Fig. 3. Longitudinal section of the upper half of a villus of the ileum. The amount of both granular and diffuse PB staining in the cytoplasm of the epithelial cells increases toward the tip. The arrow indicates an intense thin line of PB staining under the striated border. LM X1940.

Fig. 4. Transverse section of portions of four villi of the ileum. LM XI700


Fig. 5. Lymphatic nodule of the ileum. LM X540.

Fig. 6. Siderotic macrophage from the middle of a germinal center. LM X3250.

Fig. 7. Siderotic macrophage from the periphery of a germinal center. LM X3250

Fig. 8. Forming feces in the colon. Both single and groups of PB-positive granules are scattered throughout this field between other debris and bacterial aggregations. LM X380.

188 D. MORTON A N D W. A. WIMSATT


granules, although their cytoplasm was PB- positive (Fig. 6). Cells occupying more peripheral positions embodied an increasing number of relatively small PB-positive granules. In the outermost layers, they contained fewer but larger, irregularly shaped inclusions, apparently created by a coalescence of these smaller granules (Fig. 7). Supernumary het- erochromatic nuclei were common in these cells, and centrally situated cells were observed engulfing lymphocytes (Fig. 6, arrow).

Autoradiograms from 7 days and on showed a concentration of silver grains over siderotic macrophages and the apical cytoplasm of siderotic musosal epithelial cells (Fig. 16). In Peyer’s patches, siderotic macrophages in the germinal centers had increasing accumulations of radioiron, as they were more peripherally situated (Fig. 17).

Groups of siderotic macrophages identical to those described above in peripheral positions in germinal centers were often observed between the apical and lateral aspects of these centers and the mucosal epithelium. More- over, massed groups of similar cells were located directly under patches of siderotic mucosal epithelial cells (Fig. 5). A bridge of diffuse PB-positive tissue connected these latter two areas. All these siderotic macrophages contained basophilic inclusions which appeared to be degenerating engulfed lymphocytes.

The mesothelial cells of the serosa often contained relatively small but intensely PB- positive granules. Siderotic macrophages were also seen adhering to and among these cells. There existed a gradient down the serosa with the highest concentration of both these cell types arrayed along the fundic caecum and their numbers decreasing down the intestine. In the more siderotic bats, large numbers of

siderotic macrophages were observed in the gut wall between bundles of smooth muscle cells (Fig. 2) and in the submucosa. Autora- diograms from 7 days and on showed large amounts of radioiron in these cells (Fig. 15).

Injection of iron dextran into bats resulted in increased numbers of siderotic macrophages in the gastrointestinal tract, liver, and spleen. Normally, these cells were rarely observed in the lung, but in iron-loaded bats, they were numerous in the connective tissue around blood vessels and ventilatory ducts. PB-positive granules appeared in many other cells that were not usually siderotic in uninjected bats. These included enterochromaffin, chief, and Paneth cells of the gastrointestinal mucosa, fibroblasts in the gastrointestinal connective tissues, smooth muscle cells of the splenic capsule and trabeculae, and occasionally in lymphocytes of the splenic white pulp and intestinal lymphatic nodules.

DISCUSSION

The large amounts of iron seen in the liver, spleen, stomach, and intestine of the common vampire bat must initially enter the body through absorption of dietary iron in excess of need. Body iron may be delivered back to the gastrointestinal tract either by migratory macrophages or via the circulation to resident macrophages. At the very least, radioactivity is present in siderotic macrophages within the gut wall by 7 days after injection of a trace amount of radioiron into the peritoneal cavity. This iron, some portion of which is in the form of ferritin, is apparently transferred from siderotic macrophages in the lamina propria to mucosal epithelial cells, where it is bound or it binds into siderosomes. Iron is effectively excreted as these cells desquamate into the gut lumen. This study neither precludes the

Fig. 9. Siderosomes in mucosal epithelial cell of the fundic caecum. Membranous whorls are present in the most mature siderosomes but are obscured by large amounts of iron. TEM X46,300.

Fig. 10. Siderosome in mucosal epithelial cell of the ileum. TEM ~72,500.

Fig. 11. Unstained siderosome in mucosal epithelial cell of the fundic caecum. Molecules of ferritin and larger iron complexes are clearly visible in the preparation, which was not stained after thin sectioning. The two “stippled” bodies may represent osmiophilic droplets of lipid. TEM x 123,100.

Fig. 12. Parietal cell of a simple acinar gland in the mucosa of the fundic caecum. LM ~4,500.

Fig. 13. Siderosomes in a parietal cell. TEM X83,700.

Fig. 14. Amembranous accumulation of iron cores of ferritin in intracellular canaliculus of a parietal cell. TEM ~83,700.

Fig. 15 Serosa of the fundic caecum from a bat sacrificed 7 days after administratian of radioiron. LM X 830.

Fig. 16. Mucosae of the lower ileum from a bat sacrificed 7 days after administration of radioiron. LM X830.

Fig. 17. Germinal center in a lymphatic nodule of the ileum of a bat sacrificed 7 days after administration of radioiron. The horizontal bar represents a distance of ten micrometers. LM x 830.


direct transfer of iron from the blood to the mucosal epithelium, nor the incorporation of newly absorbed iron into either the absorptive cells or macrophages.

Elements of similar mechanisms have been found in other iron replete mammals. The intraperitoneal injection of iron dextran into rats stimulates a mobilization of peritoneal siderotic macrophages which subsequently undergo serosal "absorption" through the muscularis into the connective tissues of the gut wall (Thirayothin and Crosby, '62). These cells are also present between smooth muscle bundles in patients with transfusion siderosis (Capell et al., '57). Although never seen in the vampire bat, the excretion of iron by diape- desis of macrophages across the mucosal epithelium is a recurring observation (Hochhaus and Quincke, 1896; Crosby e t al., '63; Astaldi et al., '66).

The positional interrelationship between siderotic mucosal epithelium and siderotic macrophages was first reported by Macallum (1894) in the digestive tracts of iron-fed guinea pigs. Similar associations are described in the duodenum (Gillman and Ivy, '47; Hukill and Putt, '62; Lillie et al., '63) and jejunum (Lillie et al., '63) of dietary siderotic guinea pigs, in the duodenum (Astaldi et al., '66) and jejunum (Astaldi et al., '66; Theron and Mekel, '71) of humans with hemochromatosis and Bantu siderosis, and in the jejunum (Richter, '74) and colon (Gillman et al., '59) of dietary siderotic rats. Interestingly enough, in the latter study, the involvement of the duodenum was mini- mal.

The scarcity of siderosomes in the mucosal epithelial cells of the duodenum of the vampire bat relative to the sections of the poste- sophageal digestive tract may reflect the partial segregation of absorptive and excretory activities a t the organ level. Radioisotopic studies have indicated that the duodenum is the principle site of inorganic iron absorption in rats (Manis and Schacter, '64; Brown and Rother, '63; Wheby and Crosby, '63; Wheby et al., '64; Howard and Jacobs, '72) and man (Noyes and Jordan, '64; Wheby, '66) and of hemoglobin iron in guinea pigs (Conrad et al., '66). Inorganic iron absorption in rats decreas- es in the lower intestinal tract and is negli- gible in the stomach (Wheby and Crosby, '63).

Duodenal and other mucosal siderosomes probably function as a sink for excess epithelial cell iron, whether derived from endoge- nous sources or absorption. Electron microscopic autoradiograms have indicated the incorporation of a small portion of absorbed

radioiron into ferritin containing cytoplasmic organelles in normal, iron overloaded, and X- linked anemic mice, but not in iron-deficient animals (Bkdard et al., '71, '73).

Evidence suggests that additional iron is secreted by the parietal cells of the fundic caecum. Both oxyntic and chief cell siderosis have been reported in humans with accumu- lating iron deposition from multiple blood transfusions (Capell et al., '57) and patients with alcoholic and idiopathic hemochromatosis (Zeitoun and Lambling, '67). The observation of amembranous ferritin bodies in the intracanalicular space of the parietal cells of the vampire bat resembles the description of clusters of electron-dense particles of iron in the hepatic canaliculi and bile of rats fed an iron-loaded, high fat, and protein deficient diet and subsequently returned to a standard diet (Bradford et al., '69). I t is of note that actual visual evidence of exocytosis was not seen in this or the present study.

The specific elements of the siderotic state of Peyer's patches can be interpreted t o represent different stages of iron transfer. Mac- rophages differentiating in germinal centers accumulate iron as they move radially. This movement is probably due to the pressure of lymphopoiesis. Mature siderotic macrophages may migrate from the deeper lymphatic tissue and pile up in groups under the luminal epithelium. Alternatively, these groups could represent the remnants of old germinal centers that have been pushed to the periphery of the lymphatic nodules by new ones.

The excretion of iron in mammals has been generally considered to be relatively unimpor- tant in maintaining iron balance when compared to iron absorption. However, some excess absorption does occur if the diet contains high levels of either heme or inorganic iron. Under equivalent conditions, as the experimental oral dose increases, the absolute amount of iron absorbed is increased, while the percentage of iron absorbed is decreased (Bothwell and Finch, '57). Either a more effi- cient mucosal barrier to excess heme iron absorption or functional iron excretion, or both, would be useful, if not essential, adap- tations for a sanguivore. In the vampire bat, compensatory iron loss linked to epithelial cell turnover in the rnucosa of the gastrointestinal tract, perhaps along with parietal cell secretion, does remove some excess body iron. Although there may be quantitative differ- ences, the qualitative similarities between these mechanisms and both the iron histo- chemistry of the digestive organs of experi-

192 D. MORTON AND

mentally iron-overloaded mammals and the pathology of iron overload conditions in humans suggest a more general importance for the excretion of iron through the digestive tract.

ACKNOWLEDGMENTS

This research was partially supported by NSF Research Grant GB 6435X (to W.A. Wim- satt) and based on a portion of a dissertation submitted in partial fulfillment of the require- ments for the Ph.D. degree, Division of Bio- logical Sciences, Cornell University, Ithaca, New York 14853.

The authors thank Dr. Mandayam V. Par- thasarathy for the use of a unique ultrastructural facility and Mr. Anthony L. Guerriere for his technical assistance on this project.

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