Griinhagen [1887] denied the fat transport theory. Schafer [1912] differentiation of leucocyte types, Hardy & Wesbrook [1895] first made

Transcription

1 I J. Physiol. (1938) 93, I-9 6I :6I2.II2.II THE ROLE OF LEUCOCYTES IN FAT ABSORPTION BY E. H. LEACH From the University Laboratory of Physiology, Oxford (Received 24 January 1938) IN 1867 Arnstein noted the presence of fat in leucocytes of the intestinal epithelium. Although the amount of fat in them increased after fat feeding, he did not attribute to them any important role in fat absorption. In fresh preparations he observed migration of these cells from the epithelium to the lumen of the intestine. Zawarykin [1883] and Schafer [1885] came to the conclusion that leucocytes transport fat from the lumen to the lacteals. In a careful study of the intestinal epithelium, R. Heidenhain [1888] described a greater diversity of types of leucocytes. He showed that some contained true fat droplets, while others contained inclusions which blackened with osmium tetroxide, were insoluble in ether and stained with acid fuchsin. Both he and Griinhagen [1887] denied the fat transport theory. Schafer [1912] admitted that some of the droplets, which he had regarded as fatty in nature, were not true fat. He did not describe the distribution of the latter. Fat transport was now stated to take place from the epithelial cells, not from the lumen of the intestine, by means of lymph corpuscles. He still referred to lymph corpuscles and leucocytes synonymously, although the various types of leucocytes both of the blood and intestinal epithelium had already been accurately described. He regarded the fat transport theory as applicable to mammals, in which the most frequently occurring type of leucocyte of the intestinal epithelium is the lymphocyte. Consequently it is the latter which is usually referred to in connexion with the fat transport theory. Although Zawarykin [1883] and R. Heidenhain [1888] made some differentiation of leucocyte types, Hardy & Wesbrook [1895] first made an accurate study of them. In the drawings and description of microscopical preparations by Thanhoffer [1874] and nearly all subsequent workers in this field (including Schafer) it is often possible to recognize the various types of leucocytes of the intestinal epithelium. Mottram et al. [1922] subjected rats to radium emanations and found that in such PH. XCIIT. >x

2 2 E. H. LEACH animals, which presumably suffered from lymphopenia, no fat absorption occurred. For reasons unstated, they drew the conclusion that, although lymphocytes play a role in fat absorption, it is not that of fat transport. The object of the present work was to determine whether any or all of the types of leucocyte of the intestinal epithelium which have now been recognized take part in fat absorption. Fat absorption in normal frogs has therefore been studied and in addition the effect of benzene-which causes leucopenia-on fat absorption in the rabbit has been examined. METHODS Spring and summer frogs weighing g. were used. Some of these had been kept at a temperature of 40 C. for periods up to 28 days. Olive oil (about 0-1 c.c.) was dropped into the mouth, and the animals were killed at intervals after feeding. Portions of various segments of the intestine were fixed in Heidenhain's "Susa" [Heidenhain, 1916] and in absolute alcohol. Sections of the Susa-fixed material were stained by haematoxylin-eosin and Masson's iron hsematoxylin-ponceau 2R-acid fuchsin-light green [Masson, 1923]. Alcohol-fixed material was stained by methyl green-pyronin. Other material was fixed in 1 p.c. osmium tetroxide or 1 p.c. osmium tetroxide and 1 p.c. chromic acid for 7 days, washed for 7 days, then embedded in gelatine, hardened in formaldehyde, and cut frozen. Some sections were counterstained in Scharlach R. Rabbits were given a daily subcutaneous injection of 1-10 c.c. of "benzole cryst." (of Harrington) until the blood leucocyte count was reduced to about c.mm. Then 5-15 c.c. of olive oil were fed by stomach tube to normal and benzene-poisoned animals. The faeces were collected daily before and after the fat feeding. Each 24 hr. portion was separated by hand, acidified with hydrochloric acid, dried at 1100, powdered and weighed. A sample of each was continuously extracted with light petroleum in a Soxhlet apparatus for 3 hr. The percentage of petroleum-soluble substance associated with the non-fat residue was calculated for the 2 days before, and for several days after, the fat feeding. Hence by subtracting the total amount of petroleum-soluble substance that would have been associated with the non-fatty residue of the fwces after fat feeding from the total fat of the faeces, the total of the unabsorbed olive oil was obtained. The greater part appeared in the feces between 24 and 48 hr. after the feeding. After 2-5 days, when little excess fat was present in the faeces, olive oil was again fed and the animals killed 7 hr. later by a blow on the head. A complete post-mortem examination was made. Small pieces from the middle of the small intestine were cut open and fixed in 5 p.c. formaldehyde in 0-9 p.c. sodium chloride solution for 24 hr., and then embedded in gelatine. Sections were cut at 15,., stained in Sudan III and mounted in Farrant's medium. Control material was fixed in "Susa" and embedded in paraffin. Sections were stained by routine methods. RESULTS Fat absorption in frogs The epithelial cells became elongated during fat absorption owing to the large amount of fat that they contained (PI. I, figs. 1, 2). The maximum deposit was usually at the 12th hour after feeding. The size of

3 . ; THE JOURNAL OF PHYSIOLOGY, VOL. 93, No. 1 0 I0100P i To ~_ MI~~~~~~~k PLATE I face p. 2

4 LEUCOCYTES AND FAT ABSORPTION the droplets was much smaller in the animals which had been kept at a low temperature. The presence of a few fat droplets in the epithelial cells even at the end of a fast of at least 28 days supports the view of Rony et al. [1933] that a continuous excretion and reabsorption of fat occurs in the intestine. From sections stained by routine methods it is possible to distinguish three types of leucocytes in the intestinal epithelium. (1) Lymphocytes. These are present between the epithelial cells and in the subepithelial connective tissue. The number found during the various stages of fat absorption did not differ from that present during starvation. (2) Eosinophitls. Although occurring in the epithelium, especially near the basal border, these are more numerous in the subepithelial connective tissue. They decrease in number during fat absorption and can easily be recognized by their closely packed refringent granules. (3) Phagocytic cells. These are large (diameter 5-20,t.). The nucleus is flattened or crescentic and peripherally placed. One or more large vacuoles are present in the cytoplasm. Various cell inclusions are visible, some of which appear to be red or white blood corpuscles in various stages of digestion. The ferrocyanide reaction reveals the occasional presence of iron-containing granules. The cells found near the base of the epithelium are small, those near the striated border larger. Some cells can be seen migrating through the striated border (P1. I, fig. 3) and other large ones within the lumen of the intestine. During fat absorption, a decrease occurs in the number of these phagocytic cells. After vital staining with trypan blue, the dye was identified in these cells, indicating that they may be members of the reticulo-endothelial system. Sections of material fixed with osmium tetroxide-chromic acid showed no black or brown granules in the lymphocytes. The oxyphil granules of the eosinophils stain light brown. Neither the number nor the intensity of staining of these granules varies during fat absorption. Within the phagocytic cells can be seen black droplets of varying size and also large brown inclusions corresponding to the R.B.c. fragments. The black droplets are present even after the animal has starved at least 28 days. The number and size of these droplets present within a phagocytic cell increased during fat absorption although the total number of these cells decreased (P1. I, figs. 1, 2). Very rarely, cells containing black droplets can be found in the subepithelial connective tissue or lining the walls of the lacteals. They are much smaller than the phagocytic cells of the epithelium

5 4 E. H. LEACH Sections of material fixed in osmium tetroxide alone show a slightly different appearance. A heavy, general, brownish-black staining obscures much detail. The oxyphil granules of the eosinophils are dark brown and, if the lighting is far removed from critical illumination, they appear black. After treatment with ether for 12 hr., the black droplets within the phagocytic cells disappear but the dark brown granules of the eosinophils are unaffected. In some control material, fixed in formaldehyde and stained in Scharlach R, red staining droplets can be seen in the epithelial cells and in the phagocytic cells. The oxyphil granules of the eosinophils stain light yellow. Effect of benzene on fat absorption The benzene-injected animals became very weak. "90 p.c. benzole" and chemically pure benzene were found to be as toxic as "benzole cryst." and no more potent in leucotoxic activity. Thiophene and dioxane are both chemically related to benzene and the former is a constant impurity in " benzole ". Neither was found to act as a leucotoxin when administered in non-fatal doses. TABLE I w.b.c.'s/c.mm. Olive oil fed Olive oil Benzene Serial no. blood c.c. absorbed, p.c. injected, c.c. F.A F.A F.A F.A F.A In differential W.B.C. counts of other benzene-poisoned rabbits, it was found that the reduction in the lymphocyte count was roughly proportional to the reduction in the total w.b.c. count. The lymphoid tissue of lymph glands and appendix showed very marked depopulation. Few normal, mature lymphocytes were visible either here or in the blood. From sections of other organs, the results of Selling [1916] were confirmed. In sections of the intestine of the normal fat-fed animal F.A. 5, much fat was visible within the epithelial cells and in the lacteals but none within the lymphocytes of the epithelium. In the benzene-poisoned animal F.A. 7 there was much less fat present in the epithelial cells and slightly less in the lacteals. In the control sections, it was found that the number of lymphocytes in the intestinal epithelium had not been markedly diminished by the benzene poisoning. However, the nuclei of the lymphocytes both here and in the blood had become definitely pycnotic.

6 LEUCOCYTES AND FAT ABSORPTION 5 DISCUSSION It does not appear likely, from the experiments with frogs, that lymphocytes are involved in fat transport, as no significant change has been noted in the relative number present during the various conditions, and no fat could be identified in them by the methods used. Winiwater [1930] found that prolonged feeding on exclusively fatty diet does not increase the number of lymphocytes in the intestinal epithelium of mice. Goldmann [1933], using special methods, was unable to find fat in lymphocytes although he identified it in all the other types of leucocytes. Again, in the experiments of Clark & Clark [1917], the cells which took up the fat droplets were never identified as lymphocytes or even as leucocytes. Although Schafer attributed fat transport activities to lymph corpuscles, it does not appear certain from his drawings that true lymphocytes were engaged in this activity at all. The results of the experiments on rabbits, which at first sight support the fat transport theory of lymphocytic activity, in reality tend to disprove it. During fat absorption, neutral fat is laid down within the epithelial cells whenever the rate of entry of fat into these cells is greater than the rate of removal from them [No 11, 1910]. In the benzene-poisoned animals the rate of entry of fat must be diminished because, after administration of fat, more fat appears in the faeces of these subjects than in the fa%ces of normal animals. The greatest decrease in this fatabsorbing capability was 30 p.c. in animal F.A. 7. If the rate of removal of fat from the epithelial cells had been interfered with to a greater extent than this, more fat would have been laid down within these cells than in a normal animal. As the amount of such fat was found to be less than normal, the interference with the rate of removal must have been less than 30 p.c. If the lymphocytes of the intestinal epithelium play any part in fat transport, it would have been expected that the reduction in the rate of removal of fat would have been large owing to the pycnotic change in their nuclei and the small reserves of lymphocytes in blood and lymphoid tissue. Schafer [1885] has also suggested that the lymphocytes carry the fat direct from the lumen of the intestine to the lacteals where they disintegrate. The experiments described above would confirm this theory, were it not that no fat was visible in the lymphocytes of the epithelium. In the benzene-poisoned animal F.A. 7, 2 g. of fat were absorbed. If this amount is transported by lymphocytes which disintegrate after

7 E. H. LEACH migration to the lacteals, so large a destruction should have led' to a rapid depletion of the lymphocytes of the intestinal epithelium; for in such an animal the reserve of lymphocytes in the blood and lymphatic organs was much decreased. No such depletion was, however, observed. Mottram et al. [1922] considered that their experiments indicated that lymphocytes play a role in fat absorption other than that of fat transport. Presumably they were convinced that failure of fat absorption in their rats was due to the depletion of lymphocytes in the lymphoid organs and not to a more general effect of the radium emanations. A similar conclusion cannot be drawn from the experiments described here. Although only three benzene-poisoned rabbits were used, it appears from Table I that the failure of fat absorption bears a nearer relationship to the total amount of benzene injected than it does to the lymphopenia induced. So the failure of fat absorption is probably due to a more general toxic effect of benzene poisoning and no role need be attributed to lymphocytes in fat absorption. The second type of leucocyte to be considered is the eosinophil. These occur in a much higher proportion in the blood of the frog than in that of mammals. The granules of these cells do not stain with Scharlach R and so are probably not true lipide in nature. In preparations fixed in osmium tetroxide together with chromic acid, the granules are yellow, but with osmium tetroxide alone they are brown, and if the lighting does not approximate to critical illumination they appear to be black. It is interesting to note that both Zawarykin and Schafer used osmium tetroxide alone and apparently from the drawings and description regarded these granules as absorbed fat. Griinhagen [1887] employed Flemming's solution and did not regard these granules as fat. Heidenhain [1888], using osmium tetroxide only, described black granules in such cells but did not regard them as fatty in nature as they were insoluble in ether and stained with acid fuchsin. It appears that Schafer [1885] was referring to these eosinophils when he stated that the fat-containing cells became smaller as they migrated from the surface through the epithelium. His larger forms near the surface would then correspond to what are here described as phagocytic cells. Schafer [1912] admitted that some of the osmium-stained granules of leucocytes were, as He ide n ha in stated, insoluble in ether and possibly not of true lipide nature. From the experiments here described, it appears that such ether-insoluble granules occur mainly in the eosinophils and the ether-soluble ones only in the phagocytic cells. Hence it is unlikely that eosinophil cells are concerned in fat transport.

8 LEUCOCYTES AND FAT ABSORPTION The phagocytic cells of the epithelium were first described by Arnstein [1867]. He noted that they contained fat droplets and observed them migrating in fresh preparations from the epithelium into the lumen of the intestine. R. Heidenhain [1888] described degenerating red and white blood corpuscles within them. Hardy & Wesbrook [1895] pointed out that contained disintegrating eosinophils may give them the appearance of modified eosinophils. Bacteria [Ruffer, 1890], iron [Macallum, 1894], methylene blue [Champy, 1911] and vital dyes [v. Mollendorf, 1925] have all been noted within them. There is little accord as to the direction of migration, and such cells may well be concerned in either absorption or secretion. The result of the experiments here recorded are best interpreted as indicating that the phagocytic cells migrate through the epithelium, becoming larger as they approach the striated border; they take up fat in addition to other particulate matter and discharge it when they disintegrate within the lumen of the intestine. But the only direct evidence of migration is that of Arnstein [1867] who actually saw them entering the lumen. There is no evidence for the assumption that they are the precursors of any type of subepithelial cell, lymphocyte or eosinophil, which Schafer both in his description and drawings implied. The nature of these cells is discussed fully by Patzelt [1936]. The suggestion offered to account for the occurrence of fat in these cells is that they take up fat, just as v. M6llendorf [1925] considered that they take up vital dyes, owing to their phagocytic activity. This phagocytosis and migration into the lumen would involve a very minor inefficiency in fat absorption. It must be remembered that, in addition to fat, vital dyes, iron, disintegrating blood corpuscles and possibly other products of excretion will be removed coincidently. Fat was occasionally seen in subepithelial cells, probably histiocytic and definitely not lymphoid in nature. Zawarykin [1883] described similar cells containing large fat droplets even in Meissner's plexus. Farkas & Thanhoffer [1933], using Flemming's fixative, described a similar distribution. Weiner [1932] also noted such cells. Macallum [1894] found iron and fat, and Wassilj eff [1925] trypan blue in them. Onozaki [1936] has found them recently in the colon of the dog. Actually such fat-containing cells may be seen not only in the connective tissue of small and large intestine but also in that of skin, stomach and submaxillary gland. It is unlikely that these histiocytic cells, relatively small in number, could play any large part in fat absorption. It is not unlikely that they are precursors of the phagocytic cells which have also been shown to take up vital dyes. 7

9 E. H. LEACH SUMMARY 1. No fat can be detected in lymphocytes of the intestinal epithelium of the frog. 2. The oxyphil granules of the eosinophil leucocytes may easily be mistaken for fat droplets. 3. The phagocytic cells are the only leucocytes of the epithelium which contain fat. This fat is carried back into the lumen by migration of these cells. 4. Subepithelial cells, probably histiocytic in nature, may contain fat. 5. Benzene poisoning decreases the fat-absorbing capability of rabbits and decreases the number of lymphocytes in the blood stream and lymphatic organs. 6. Because no fat could be identified within lymphocytes and because in the benzene-poisoned animals there was no accumulation of fat within the epithelial cells or depletion of lymphocytes of the mucosa, it is concluded that lymphocytes do not transport fat from the epithelial cells or from the lumen of the intestine to the lacteals. 7. The failure of fat absorption appears to be due to a general toxic effect of benzene poisoning. 8. There is no evidence that any type of leucocyte plays an important part in the transference of fat from the epithelium to the lacteals. My thanks are due to Dr H. M. Carleton for having suggested this problem and for much help and criticism, also to Prof. Sir Charles Sherrington, Prof. R. A. Peters and the late Prof. Dreyer for permission to work in their respective departments, and to Dr R. B. Fisher for help with facal extractions. REFERENCES Arnstein, C. (1867). Virchow8 Arch. path. Anat. 39, 527. Champy, C. (1911). Arch. Anat. micr. 18, 55. Clark, E. R. & Clark, E. L. (1917). Amer. J. Anat. 21, 421. Farkas, G. & Thanhoffer, L. v. (1933). Z. Biol. 93, 560. Goldmann, J. (1933). Veirchows Arch. path. Anat. 287, 587. Grunhagen, A. (1887). Arch. mikr. Anat. 29, 139. Hardy, W. B. & Wesbrook, F. F. (1895). J. Physiol. 18, 490. Heidenhain, M. (1916). Z. wi88. Miikr. 33, 232. Heidenhain, R. (1888). Pfiug. Arch. 43, Suppl. vol. Macallum, A. B. (1894). J. Phy8iol. 16, 268. Masson, P. (1923). Tumeur-Diagnostics Hi8tologique8, p Paris: A. Maloine et Fils. Mollendorf, W. v. (1925). Z. ZeUforsch. 2, 129. Mottram, J. C., Cramer, W. & Drew, A. H. (1922). Brit. J. exp. Path. 3, 179.

10 LEUCOCYTES AND FAT ABSORPTION 9 Noll, A. (1910). Pftiug. Arch. 136, 208. Onozaki, T. (1936). T6hoku J. exp. Med. 29, 224. Patzelt, V. (1936). Handbuch der Mikroskopischen Anatomie de8 Men8chen, 3, Berlin: Julius Springer. Rony, H. R., Mortimer, B. & Ivy, A. C. (1933). J. biol. Chem. 102, 161. Ruffer, A. (1890). Quart. J. micr. Sci. 30, 481. Schafer, E. A. (1885). Int. M8chr. Anat. Physiol. 2, 6. Schafer, E. A. (1912). Textbook of Micro8copic Anatomy, Quain'8 Anatomy, 2, pt. 1, p London: Longmans, Green and Co. Selling, L. (1916). Johns Hopk. Hosp. Rep. 17, 83. Thanhoffer, L. v. (1874). Pftuig. Arch. 8, 391. Wassiljeff, A. (1925). Z. ZeUfor8ch. 2, 257. Weiner, P. (1932). Z. mikr. anat. For8ch. 30, 193. Winiwater, H. de (1930). C.R. Soc. Biol., Pari8, 104, 121. Zawarykin, T. (1883). Pfliig. Arch. 31, 231. EXPLANATION OF PLATE I Fig. 1. Lower part of intestine of normal frog 12 hr. after fat feeding. Fixation 1 p.c. osmium tetroxide-i p.c. chromic acid. Three phagocytic cells (P) containing fat clearly distinguishable. Fig. 2. Upper part of intestine of normal frog 24 hr. after fat feeding. Same fixation. Epithelial cells now heavily loaded with fat. Two phagocytic cells (P) distinguishable, one of which contains fat droplets. Fig. 3. Lower part ofintestine of frog, which had previously been kept at a low temperature, 5 hr. after fat feeding. One phagocytic cell (X) leaving epithelium. Another (P) and other unmarked ones containing various inclusions.