J. Physiol. (1977), 270, pp. 473-488 With 6 text-ftgure8 Printed in Great Britain
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ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT ACROSS THE SMALL INTESTINE OF THE NEW-BORN PIG BY K. A. BURTON AND M. W. SMITH From the Agricudltural Research Council Institute of Animal Physiology, Babraham, Cambridge CB2 4AT
(Received 3 January 1977) SUMMARY
1. Measurements of porcine and human IgG transport across the small intestine of the new-born pig have been made in vivo and related to the amount of endocytosis taking place. 2. The amount of immunoglobulins transported, following intraduodenal administration to conscious animals, is directly related to the degree of endocytosis which these proteins produce. Administration of protein in amounts sufficient to cause a maximal endocytotic response causes saturation of the protein transporting process. 3. Absorption of small amounts of human IgG from the small intestine can be accelerated by the addition of sow colostrum or porcine IgG in quantities sufficient to stimulate endocytosis. These effects disappear when the amount of human IgG administered is itself sufficient to fully stimulate endocytosis. 4. Preferential transport of porcine over human IgG can be demonstrated when both are given as a single solution to individual pigs. The degree of preference is, however, small in relation to the total amount of immunoglobulin transported. 5. The initial formation of eindocytotic vacuoles in the pig intestine seems, unlike the situation in rats and mice, to provide a major route for the trans-cellular movement of macromolecules including immunoglobulins. Present evidence suggests that, though sow colostrum can on occasion stimulate such transport, it does so merely by acting as a proteincontaining solution to increase the amount of endocytosis taking place. INTRODUCTION
The route by which immunoglobulins (IgG) cross the intestinal epithelium of new-born animals has recently been questioned (Jones &
K. A. BURTON AND M. W. SMITH Waldmann, 1972; Rodewald, 1973). It was originally thought that the large protein-filled vacuoles, formed in the mucosa following the ingestion of colostrum, represented part of a single pathway for the transcellular transport of proteins. Endocytosis was assumed to be a non-selective process, selectivity in transport being achieved subsequently through the specific binding of homologous immunoglobulins to receptors on the surface of the vacuolar membrane and the proteolytic digestion of unbound protein (Brambell, 1966). The need to make these multiple assumptions disappeared once it became established that immunoglobulins could enter the mucosa by more than one route. It was this second route of transport, involving initial binding to the microvillar membrane followed by packaging and transport within small coated vesicles, which accounted for the specific transport of homologous proteins. Uptake of immunoglobulins into the vacuolar system was shown, on the other hand, to be nonselective and there was no subsequent transport of protein from these vacuoles into the intercellular spaces (Rodewald, 1973). The idea that immunoglobulin entry into vacuoles was a terminal event was borne out by more recent experimental findings showing negligible amounts of homologous immunoglobulins to be absorbed across the distal ileum of the neo-natal rat (Morris & Morris, 1974). Although this newer hypothesis proved satisfactory for rats and mice, it failed to account for the non-selective transport of immunoglobulins seen to take place in pigs and other animals. It could be that coated vesicles are absent from the mucosa of these animals or that, if present, they lack the ability to preferentially transport homologous proteins. It could also be that coated vesicles are present and functional, but that their effect is lost through the simultaneous transport of large amounts of immunoglobulins and other proteins through an open-ended, nonselective, vacuolar system. The present work attempts to distinguish between some of these possibilities by measuring the in vivo transport of immunoglobulins across a restricted area of the new-born pig intestine under different conditions. The amount of vacuole formation at the end of these experiments was also estimated and related to the overall efficiency of protein transport in this tissue. Both pig and cow colostrum contain factors capable of increasing the rate at which immunoglobulins cross the intestine of new-born animals (Balfour & Comline, 1962; Hardy, 1970). A secondary aim of the present work was to see whether sow colostrum could facilitate the absorption of human immunoglobulin across the pig intestine. Facilitation did occur, the amount correlating well with the ability of the added compound to stimulate endocytosis. The implication of these findings to the action of accelerator substances in general is discussed. 474
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 475 METHODS Animal8 Piglets were removed immediately after birth. They were not allowed to suck the sow. Parturition was induced on day 112 of gestation by the previous I.M. injection of prostaglandin analogues (ICI 80996 or 79939). Normal gestation period in the pig is 114 days. All piglets used came from the Babraham herd of Large Whites. They were operated on within 1 hr of collection and kept warm subsequently through the use of a thermostatically controlled electric blanket. Intravenous glucose was given both before and during the experiment (see below). Operative procedure Each animal was anaesthetized with Fluothane (Imperial Chemical Industries Ltd, Macclesfield, Cheshire) for cannulation of its jugular vein (FG nylon i.v. cannula, outside diameter 1-34 mm, Portex Ltd, Hythe, Kent). The cannula was positioned finally so as to lie in or adjacent to the heart. A small mid line incision was next made immediately distal to the sternum to expose the stomach. The stomach was moved with blunt forceps to reveal the duodenum which was then cannulated with non-toxic polyvinyl tubing (Portex Ltd, Hythe, Kent). Both i.V. and intraduodenal cannulae were then strapped to the back of the animal. Heparin (25 i.u.) and glucose (2 ml. 50 % (w/v) solution) were injected i.v. and the animals left to recover consciousness. Solutions of immunoglobulins, made up in sow colostrum or in balanced saline (Krebs & Henseleit, 1932) at 370 C, were finally injected into the duodenum over a 5 min period. The volume of solution injected was 10 ml. in all cases. Piglets used in these experiments had a mean body weight of 1-29 + 0 04 kg (mean + s.E. for thirty-two animals). Nine 1 ml. samples of blood were removed during the experiment, an equal volume of 50 % (wlv) glucose being added on each occasion to keep the fluid volume constant. Experiments took either 8-5 or 9*5 hr to complete. The duodenal cannula was covered with adhesive plaster following administration of protein solution to avoid any possibility that the cannula might catch or move relative to the animal. The amount of jugular cannula left free was also kept to a minimum for a similar reason. In fact the piglets spent the whole of the experimental period either asleep or lying quietly on the electrically heated blankets. It was often possible to obtain blood samples without waking the animal. The whole procedure was tolerated without obvious signs of discomfort. Determination of immunoglobulin Blood taken for analysis was spun at top speed on a micro-centrifuge (Quickfit Instrumentation, Stone, Staffs), for 6 min. The separated plasma was stored at 40 C and analyses completed within the next two days. Determination of immunoglobulins was by electrophoresis through agarose containing specific antibodies to the proteins (Laurell, 1966), immunoglobulins in the samples being first coupled to albumin with glutaraldehyde to confer a net negative charge on the molecule. Part of the assay system came as a kit from ICL Scientific, Euclid, California, 92708, but the specific antisera to porcine and human IgG were prepared in sheep locally using conventional means. These antisera were absorbed against pooled samples of serum taken from unsuckled, new-born piglets before being incorporated into 1 % (w/v) agarose-buffer medium. The final concentration of antiserum in agarose was kept as low as possible to increase the sensitivity of the assay. Unknown samples were run alongside standards and the height of precipitin rockets compared directly. The height of precipitin rocket obtained was reproducible for any given standard, the
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error over the concentration range 25-800 /zg ml.-' varying from 2-7 to 5-5 % (means + s.E. of nineteen and eleven observations respectively).
Quantification of endocyto8i8 The small intestine, dissected out at the end of each experiment, was divided into ten segments, the middle piece from each segment being fixed in formal saline (4 % (v/v) formaldehyde in isotonic saline). Sections cut at 4 ,um were then triple-stained for protein using Heidenhain's Azan reagent and the degree of vacuolation assessed by counting the number of vacuolated cells present per section of villus. It was decided to adopt a rating system 0-4 to represent the degree of endocytosis taking place. A rating of 0 meant that no vacuolation had taken place. A rating of 1 meant that up to a quarter of the villous epithelial cells contained vacuoles. Ratings of 2 and 3 meant that between a quarter and a half or between a half and three quarters of the cells contained vacuoles. A rating of 4 meant that virtually all the villous epithelial cells contained vacuoles. Materials Colostrum was collected from ten sows over a period of 3 weeks, individual samples being stored at - 20° C. All samples were later pooled and the concentration of porcine IgG present determined by quantitative electro-immunodiffusion. The concentration of porcine IgG was found to be 9-77 % (wlv). This has been approximated to 10 % (w/v) in the text. Purified porcine IgG, Cohn fraction II, came from KochLight Laboratories Ltd., Colnbrook, Bucks. Human IgG, Cohn fraction II, came from Sigma Chemical Company, St Louis, Mo. 63178. Both proteins were stored at - 20° C. All other reagents were of A.R. grade. RESULTS
Absorption of human IgG by the new-born pig intestine Accelerator effect It has already been shown that the absorption of bovine IgG by the new-born pig is critically dependent upon the amount of colostrum fed to the animal (Pierce & Smith, 1967a). In those experiments both bovine IgG and putative accelerator substances had been presented to the pig gut in different dilutions, no attempt being made to separate these two variables. It was decided in the present work to examine these variables separately to determine which of these factors was responsible for the previously observed results. Human IgG (2 % w/v) was placed in the duodenum of new-born pigs in the presence and absence of 10 ml. sow colostrum. The subsequent appearance of human IgG in venous blood, monitored over the ensuing 9 hr, is shown in Fig. 1. Human IgG administered in Krebs-bicarbonate saline failed to reach the blood in any significant quantity. The low figures indicate a complete inability to detect human IgG in the plasma of one or more of the six animals used in these experiments. The substitution of sow colostrum for the balanced saline medium increased the subsequent absorption of human IgG by several orders of magnitude.
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 477 In a third set of experiments 10 % (w/v) porcine IgG, the same concentration as that found in sow colostrum, was administered together with 2 % (w/v) human IgG in Krebs-bicarbonate medium. The rate at which human IgG appeared in the blood might have been fractionally slower
0-8
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6 8 10 Time (hr) Fig. 1. Time course for appearance of human IgG in new-born pig plasma following intra-duodenal administration. Human IgG was administered as a 2% (wlv) solution in Krebs-Henseleit medium with no additional protein present (-0-); with 10% (w/v) porcine IgG present (-A\-); or with 10% (w/v) porcine IgG present in sow colostrum (-El-). The interrupted line shows the mean absorption curve obtained for conditions where porcine IgG was present. Individual values give means + s.E. of ten determinations. Points with arrows show where one or more samples failed to give a detectable precipitin line at assay (< 0-0125 mg human IgG ml.-' plasma).
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than when administered in colostrum (Fig. 1), but the over-all shape of the curve and the final plateau concentrations were identical. The final mean concentration of human IgG in plasma under these conditions was 0 55 mg ml-'. The period for most rapid absorption occurred 3-5 hr after administration of the protein solution. The first conclusion to be reached from these experiments is that Krebs-Henseleit medium has no inherent inhibiting effect on the absorption of human IgG. Absorption from this solution proceeds well provided 10 % (w/v) porcine IgG is present. The second conclusion is that, although these experiments show facilitation of human IgG absorption by sow
K. A. BURTON AND M. W. SMITH 478 colostrum, they show nothing that cannot be reproduced by the presence of porcine IgG alone. Specific binding of porcine IgG to membrane receptors would be expected to inhibit the absorption of a related molecule (human IgG). The fact that the opposite occurred strongly suggests that facilitation takes place through some other, non-specific process.
Vacuole formation The most obvious event occurring in the pig intestine following the ingestion of colostrum is the formation of large numbers of protein-filled
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Fig. 2. Endocytotic response of new-born pig small intestine to administered solutions of immunoglobulins. Human IgG (2 %, w/v) was given in KrebsHenseleit medium with (-A-) or without (-0-) 10% w/v porcine IgG. Human IgG (2 %, w/v) was also presented in sow colostrum containing 10% (w/v) porcine IgG(-LI-). The experiments were those described in Fig. 1. The broken line gives the mean endocytotic index produced by solutions containing porcine as well as human IgG. Values give means + S.E. for Fig. 1. as
vacuoles. Histological sections of intestine, taken from piglets used for the previous experiments, were therefore stained for protein and the degree of vacuolation calculated. The results obtained are shown in Fig. 2. An endocytotic index of 4 indicates that between 75 and 1 00 % of the villous
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 479 cells contained protein-filled vacuoles. Values probably give underestimates since small inclusions, beyond the resolving power of the light microscope, will obviously escape detection. Virtually no protein-filled vacuoles were detected in intestines following administration of 2 % (w/v) human IgG in Krebs-Henseleit medium. The small amount of vacuolation which did occur was confined to the distal region, showing that the administered solution had travelled a considerable distance down the tract. The addition of 10% (w/v) porcine IgG to 2 % (w/v) human IgG in Krebs-Henseleit medium stimulated endocytosis. Maximal effects were seen in the mid-intestine in a region seen previously to be most active in transporting bovine IgG under in vitro conditions (Pierce & Smith, 1967b). Solutions of sow colostrum containing 2 % (w/v) human IgG induced endocytosis to a similar extent to that produced by the mixed proteins administered in saline. The over-all curve was slightly lower but the difference was not statistically significant. The mean curve drawn from these two groups of animals gave a maximal mean endocytotic index of 2*7 in segment 4 (equivalent to nearly 70 % of all cells being vacuolated). The rise in endocytotic index seen on going down the intestine from segment 1 to 4 represents an increasing ability of the tissue to endocytose protein, but the fall that succeeds it from segment 6 to 10 probably arises because protein fails to reach these areas of the intestine. Analyses to prove this point were not, in fact, carried out in these experiments. These results show a strong correlation between the ability to form protein-filled vacuoles and the ability to transport human IgG (Fig. 1). If it is endocytosis alone which determines whether human IgG shall be transported then one would predict that increasing the concentration of human IgG in saline would, by itself, be sufficient to stimulate transport and that this would eliminate the effect of porcine IgG on the absorption of human IgG. This was tested for in a further series of experiments.
Absorption of human IgJ at high concentration The design of these experiments was identical to that described in the previous section. The rate at which human IgG, administered as a 10 % (w/v) solution in Krebs-Henseleit medium, appeared in the plasma is compared with that for a similar solution containing an additional 10% (w/v) porcine IgG in Fig. 3. Human IgG was first detected in the plasma 2-5 hr after instillation into the duodenum, but the concentration was then already twice that seen following the administration of 2 % (w/v) human IgG (Fig. 1). The plasma concentration then rose rapidly reaching stable levels 3-4 hr later. The final concentration reached appeared lower when the administered solution contained porcine as well as human IgG, but the variability in results made this difference statistically insignificant.
480 K. A. BURTON AND M. W. SMITH The time course for human IgG absorption following administration in sow colostrum was very similar to that found for the other two groups of animals. The final concentration in plasma appeared slightly higher, but again the variability between animals made this difference statistically
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Time (hr) Fig. 3. Time course for appearance of human IgG in plasma of new-born pigs following intra-duodenal administration. Human IgG was given as a 10% (w/v) solution in Krebs-Henseleit medium (-O-); together with 10 % (w/v) porcine IgG in Krebs-Henseleit medium (-A-) or together with 10 % (w/v) porcine IgG in sow colostrum (-El-). Each point derives from determinations carried out on samples obtained from six animals. The interrupted line gives the mean absorption curve constructed from values pooled from all three experimental situations.
insignificant. A mean absorption curve, incorporating all experimental results, gave a final mean plasma concentration for human IgG of 1-5 mg ml.-1, this concentration being reached after a period of 6-5 hr. This equilibrium concentration is less than would be predicted were the amount
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 481 of human IgG absorbed to be directly proportional to the concentration administered (0.55 mg ml.-' for a 2 % (w/v) solution would then be equivalent to 2-75 mg ml.-' for a 10 % (w/v) solution). Saturation of the absorptive process through the administration of large amounts of protein has been reported previously (Pierce & Smith, 1967 a). It is interesting to find in the present work that any facilitating effect colostrum might have on human IgG absorption disappears under these conditions. Vacuole formation following administration of high concentrations of human
Igo The degree of endocytosis correlated well with the amount of human IgG transported when the concentration of administered protein was low (Figs. 1 and 2). The administration of larger amounts of protein probably leads to some degree of saturation of transport. The degree of endocytosis encountered under these conditions was next measured to see whether the previously determined correlation still applied. The experimental material consisted of segments of intestines taken from animals at the end of experiments, the results of which are summarized in Fig. 3. Sectioning, staining and estimation of endocytotic index were as described previously. The results obtained are shown in Fig. 4. The general pattern of endocytosis, low in proximal and distal intestine, maximal in mid-intestine, was similar to that seen previously. Values for endocytotic index in both proximal and distal segments were similar for all three administered solutions. Endocytosis in the mid-intestine was, however, higher in the presence of added protein, whether that protein was present in the form of porcine IgG or sow colostrum. The mean index from experiments involving administration of 10 % (w/v) human IgG in colostrum or with 10 % (w/v) porcine IgG added to human IgG-containing Krebs-Henseleit solution was about 2-75 (segments 3-6). This compared with an index of 145 for 10% (w/v) human IgG administered alone. The correlation between transport of human IgG and the degree of endocytosis is thus seen to break down when the total concentration of administered protein becomes high enough to saturate the transport process. The small intestine will, of course, absorb porcine as well as human IgG and much of this transport probably involves a common mechanism. Under these conditions it is the total amount of protein transported (porcine plus human IgG) which should be considered in relation to endocytosis. The transport of porcine IgG was therefore measured and added to that previously determined for human IgG. Fig. 5 relates total protein transport to the protein concentration of administered solutions and to the degree of endocytosis which these solutions produce. Cumulative endocytotic indices have been calculated by summation of indices obtained
482 K. A. BURTON AND M. W. SMITH from individual intestinal segments (see Figs. 2 and 4) to give a measure of total endocytotic response. Increasing the concentration of administered protein above 15 % (w/v) (total protein load greater than 1P5 g) had little or no effect on protein transport. This verified previous similar work using
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Fig. 4. Endocytotic response of new-born pig intestine to immunoglobulincontaining media. The experiments were those described in Fig. 3. The ad. ministered solutions consisted of 10% (w/v) human IgG in KrebsHenseleit medium (-0-); 10% (w/v) human IgG +10% (w/v) porcine IgG in Krebs-Henseleit medium (- A-) or 10 % (w/v) human IgG + 10 % (wlv) porcine IgG in sow colostrum (-EI-). The interrupted line shows the mean endocytotic response in the mid-intestine following the intra-duodenal administration of solutions containing human plus porcine IgG. Individual values give means + s.E. of estimations carried out on plasma obtained from twelve animals.
bovine IgG in the pig (Pierce & Smith, 1967 a). Endocytosis also seemed to have reached a maximum over this range of protein concentration. Both endocytosis and the amount of total protein transported showed a linear relation to protein concentration over the range 2-12 % (w/v). The correlation between total protein transport and the degree of endocytosis therefore held good for all concentrations of protein tested. It is possible
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 483 that the bulk of porcine and human IgG molecules share a common route of transport, through the vacuolar system of the anterior and midintestine. 4
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Fig. 5. Relation between cumulative endocytotic index and total protein transport by new-born pig intestine. Plasma concentrations of immunoglobulins, human or human plus porcine, are plotted against the cumulative endocytotic index obtained by integration of curves shown in Figs. 2 and 4. The numbers refer to the following solutions: 1 and 2: 2 and 10% (w/v) human IgG in Krebs-Henseleit medium; 3 and 4: 10% (w/v) porcine IgG in Krebs-Henseleit medium containing 2 and 10% (w/v) human IgGrespectively; 5 and 6: 10% (w/v) porcine IgG in sow colostrum containing other colostral proteins (10% w/v) plus 2 and 10% (w/v) human IgG respectively.
Absorption of porcine IgG by the new-born pig intestine Selectivity All the evidence presented so far suggests that the pig intestine is likely to absorb 7S immunoglobulins from different species with equal facility. Yet it is also true to say that the animal variation encountered is itself
K. A. BURTON AND M. W. SMITH large enough to make small differences in transport rate statistically insignificant. One way round this problem is to measure the transport of two or more proteins in the same animal. This was done in the next series of experiments. The rate at which porcine IgG, presented in sow colostrum, reached the plasma of new-born pigs is compared with that for human IgG, dissolved in the same medium, in Fig. 6A. The time course for absorption was similar but the plasma concentration of porcine IgG was consistently higher than that for human IgG. A straight comparison of the pooled 484
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Fig. 6. Selective transport of porcine IgG by new-born pig intestine. a, plasma concentrations of porcine (-e-) and human (-O-) IgG following the intra-duodenal administration of both at concentrations of 10 % (wlv) in sow colostrum. b, Difference between porcine and human IgG transport, porcine IgG being presented either as sow colostrum (-J-) or as a soluble commercial preparation (-C-). Individual differences give means + S.E. of determinations carried out on six animals.
values in Fig. 6A shows this difference to be not significant, the actual plasma concentration of porcine and human IgG varying by a factor of 3 to 4 between different animals and litters. This variation was nevertheless
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 485 consistent, the transport of both human and porcine IgG being high or low according to the animal used. For this reason it was considered valid to represent these results as differences in transport between porcine and human IgG, all differences being determined in the same sample of plasma taken from the same animal (Fig. 6B). Values greater than 0 indicate porcine IgG to be transported more readily than human IgG. These differences were significant for times of absorption greater than 6-5 hr. Porcine IgG was present as sow colostrum while the human IgG had been prepared commercially. It could be that purification of the human IgG caused some denaturation and that this was why transport was slightly lower. Experiments were therefore repeated using a mixed protein solution containing 10 % (w/v) human and 10 % (w/v) porcine IgG, both proteins being obtained commercially. The time course for absorption, not shown here, was similar to that shown previously (Fig. 6A). The difference in transport between porcine and human IgG, plotted in Fig. 6B, was also similar. It is concluded from this that the pig intestine can show a real ability to preferentially transport porcine IgG, but that this ability is small in relation to the large amount of non-specific transport taking place. DISCUSSION
The belief that the ability of an intestine to transport immunoglobulins could be judged solely by the presence or absence of protein-filled vacuoles in the mucosa has, in the past, proved a particularly nasty trap for the unwary. The original finding that vacuoles disappeared from the rat mucosa at a time when macromolecular transport ceased (Clarke & Hardy, 1 969a, b) appeared, at first sight, to support the Brambell hypothesis. This support proved, however, to be short-lived once it became clear that macromolecular-induced vacuolation could also occur in species showing no trans-cellular movement of proteins (Clarke & Hardy, 1970). The pig falls into an intermediate class in that it absorbs immunoglobulins at birth and the intestine becomes vacuolated, but these vacuoles persist for up to 2 weeks though the transport of immunoglobulins ceases within a few hours (Clarke & Hardy, 1971). This unsatisfactory state of affairs was partly resolved by the subsequent discovery that the major route for immunoglobulin transport in the rat was through a system. of coated vesicles confined to the more proximal parts of the small intestine (Rodewald, 1973). The fact that the rat intestine transported immunoglobulins when vacuoles were present thus proved to be merely coincidental. It is not surprising, in these circumstances, to find all the more recent work on immunoglobulin absorption assuming that virtually all immunoglobulin transport takes place via these coated vesicles. There are, however, reasons
486 K. A. BURTON AND M. W. SMITH to doubt whether this latest assumption is any more valid than the earlier ones. A distinction must be made here between species such as the rat and mouse, where homologous proteins are absorbed more readily than heterologous ones, and species such as the pig or cow, where it is difficult if not impossible to show this sort of discrimination. There is now ample evidence proving the involvement of coated vesicles in the selective transport of immunoglobulins in the rat intestine (Rodewald, 1976), and one must assume here that the vacuolar system is only important for protein degradation. Present results suggest that this does not apply to the pig, however, where the vacuolar system is thought to transport most of the absorbed immunoglobulins. In the present work, a good correlation was always seen to exist between the ability of different concentrations of different immunoglobulins to induce endocytosis and the rate at which they were absorbed. It is suggested that this correspondence does not reflect mere coincidence and that a major route for the non-selective transport of immunoglobulins in the pig intestine lies through the vacuolar system at the time of its formation. It is also possible that some porcine IgG crosses the piglet intestine by selective means. This suggestion, made originally from an analysis of serum levels of immunoglobulins following oral administration (Payne quoted by Wilson, 1962; Pierce & Smith, 1967a), was verified in the present work, where proteins were administered intraduodenally. Quite a large fraction of orally-administered immunoglobulin enters the blood in degraded form, some of this degradation taking place in the stomach (Pierce & Smith, 1967a; Hardy, 1969). Injecting immunoglobulins directly into the duodenum will reduce, but probably not eliminate, such proteolysis. The possibility that human IgG is degraded more extensively than porcine IgG and that this is why porcine IgG is transported more readily, cannot be definitely excluded. No attempt was made to identify possible products of immunoglobulin break-down in the present work, but the assay system should have shown more than one precipitin line if they had been present in more than trace amounts. The fact that only one line was detected provides some support for the view that the intestine can, to a limited extent, transport porcine in preference to human IgG. The postulated route for such transport would be through the coated vesicles, shown by Hardy, Hockaday & Tapp (1971) to be widely distributed within the pig small intestine at birth. It has already been stated that sow colostrum, like that of the cow, contains factors capable of stimulating the absorption of different immunoglobulins (Hardy, 1970). Part of this effect could arise from the trypsin inhibitor activity of colostrum or from the action of unknown factors on cellular metabolism. It is probably no coincidence, though, that
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 487 these accelerator effects have, up till now, only been seen in species where the post-natal transport of immunoglobulins is transient and mainly nonselective. These are situations where the initiation of endocytosis probably plays a crucial part in determining the over-all efficiency of immunoglobulin transport. Results obtained in the present work show sow colostrum and purified porcine IgG to be indistinguishable in their effects on the trans-cellular transport of human IgG. They also initiate endocytosis to a similar extent. It seems likely that the action of colostrum as an accelerator of immunoglobulin absorption in this species derives mainly from its ability to initiate endocytosis. We wish to express our thanks to Mr R. W. Ash and his staff for providing us with a plentiful supply of new-born, unsuckled pigs during the time when this work was carried out. REFERENCES BALFouR, W. E. & COMLINE, R. S. (1962). Acceleration of the absorption of unchanged globulin in the new-born calf by factors in colostrum. J. Physiol. 160, 234-257. BRAMBELL, F. W. R. (1966). The transmission of immunity from mother to young and the catabolism of immunoglobulins. Lancet ii, 1087-1093. CLARKE, R. M. & HARDY, R. N. (1969a). The use of [12.5]polyvinyl pyrrolidone K60 in the quantitative assessment of the uptake of macromolecular substances by the intestine of the young rat. J. Physiol. 204, 113-125. CLARKE, R. M. & HARDY, R. N. (1969b). An analysis of the mechanism of cessation of uptake of macromolecular substances by the intestine of the young rat ('closure'). J. Physiol. 204, 127-134. CLAiRE, R. M. & HARDY, R. N. (1970). Structural changes in the small intestine associated with the uptake of polyvinyl pyrrolidone by the young ferret, rabbit, guinea-pig, cat and chicken. J. Physiol. 209, 669-687. CLARKE, R. M. & HARDY, R. N. (1971). Histological changes in the small intestine of the young pig and their relation to macromolecular uptake. J. Anat. 108, 63-77. HARDY, R. N. (1969). The break-down of [1311]globulin in the digestive tract of the new-born pig. J. Physiol. 205, 435-451. HARDY, R. N. (1970). Absorption of macromolecules from the intestine of the newborn animal. In Physiology of Digestion and Metabolism in the Ruminant, ed. Phillipson, A. T., pp. 150-165. Newcastle upon Tyne: Oriel. HARDY, R. N., HOCKADAY, A. R. & TAPP, R. L. (1971). Observations on the structure of the small intestine in foetal, neo-natal and suckling pigs. Phil. Trans. R. Soc. B 259, 517-531. JoNEs, E. A. & WALDMANN, T. A. (1972). The mechanism of intestinal uptake and transeellular transport of IgG in the neonatal rat. J. clin. Invest. 51, 2916-2927. KREBs, H. A. & HENSELEIT, K. (1932). Untersuchungen fiber die Harnstoffbildung im Tierkorper. Hoppe-Seyler's Z. physiol. Chem. 210, 33-66. LAURELL, C.-B. (1966). Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analyt. Biochem. 15, 45-52. MORRIS, B. & MoRRIs, R. (1974). The absorption of 125I-labelled immunoglobulin G by different regions of the gut in young rats. J. Physiol. 241, 761-770. PAYNE, L. C. (1962). Unpublished data cited by T. H. Wilson in Intestinal Absorption. Philadelphia: W. B. Saunders.
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PIERCE, A. E. & SMITH, M. W. (1967a). The intestinal absorption of pig and bovine immune lactoglobulin and human serum albumin by the new-born pig. J. Phy8iol. 190, 1-18. PIERCE, A. E. & SMITH, M. W. (1967b). The in vitro transfer of bovine immune lactoglobulin across the intestine of new-born pigs. J. Physiol 190, 19-34. RODEWALD, R. (1973). Intestinal transport of antibodies in the newborn rat. J. cell Biol. 58, 189-211. RODEWALD, R. (1976). Intestinal transport of peroxidase-conjugated IgG fragments in the neonatal rat. In Maternofoetal Transmission of Immunoglobulin8 ed. HEMMINGS, W. A., pp. 137-153. Cambridge: University Press.
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ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT ACROSS THE SMALL INTESTINE OF THE NEW-BORN PIG BY K. A. BURTON AND M. W. SMITH From the Agricudltural Research Council Institute of Animal Physiology, Babraham, Cambridge CB2 4AT
(Received 3 January 1977) SUMMARY
1. Measurements of porcine and human IgG transport across the small intestine of the new-born pig have been made in vivo and related to the amount of endocytosis taking place. 2. The amount of immunoglobulins transported, following intraduodenal administration to conscious animals, is directly related to the degree of endocytosis which these proteins produce. Administration of protein in amounts sufficient to cause a maximal endocytotic response causes saturation of the protein transporting process. 3. Absorption of small amounts of human IgG from the small intestine can be accelerated by the addition of sow colostrum or porcine IgG in quantities sufficient to stimulate endocytosis. These effects disappear when the amount of human IgG administered is itself sufficient to fully stimulate endocytosis. 4. Preferential transport of porcine over human IgG can be demonstrated when both are given as a single solution to individual pigs. The degree of preference is, however, small in relation to the total amount of immunoglobulin transported. 5. The initial formation of eindocytotic vacuoles in the pig intestine seems, unlike the situation in rats and mice, to provide a major route for the trans-cellular movement of macromolecules including immunoglobulins. Present evidence suggests that, though sow colostrum can on occasion stimulate such transport, it does so merely by acting as a proteincontaining solution to increase the amount of endocytosis taking place. INTRODUCTION
The route by which immunoglobulins (IgG) cross the intestinal epithelium of new-born animals has recently been questioned (Jones &
K. A. BURTON AND M. W. SMITH Waldmann, 1972; Rodewald, 1973). It was originally thought that the large protein-filled vacuoles, formed in the mucosa following the ingestion of colostrum, represented part of a single pathway for the transcellular transport of proteins. Endocytosis was assumed to be a non-selective process, selectivity in transport being achieved subsequently through the specific binding of homologous immunoglobulins to receptors on the surface of the vacuolar membrane and the proteolytic digestion of unbound protein (Brambell, 1966). The need to make these multiple assumptions disappeared once it became established that immunoglobulins could enter the mucosa by more than one route. It was this second route of transport, involving initial binding to the microvillar membrane followed by packaging and transport within small coated vesicles, which accounted for the specific transport of homologous proteins. Uptake of immunoglobulins into the vacuolar system was shown, on the other hand, to be nonselective and there was no subsequent transport of protein from these vacuoles into the intercellular spaces (Rodewald, 1973). The idea that immunoglobulin entry into vacuoles was a terminal event was borne out by more recent experimental findings showing negligible amounts of homologous immunoglobulins to be absorbed across the distal ileum of the neo-natal rat (Morris & Morris, 1974). Although this newer hypothesis proved satisfactory for rats and mice, it failed to account for the non-selective transport of immunoglobulins seen to take place in pigs and other animals. It could be that coated vesicles are absent from the mucosa of these animals or that, if present, they lack the ability to preferentially transport homologous proteins. It could also be that coated vesicles are present and functional, but that their effect is lost through the simultaneous transport of large amounts of immunoglobulins and other proteins through an open-ended, nonselective, vacuolar system. The present work attempts to distinguish between some of these possibilities by measuring the in vivo transport of immunoglobulins across a restricted area of the new-born pig intestine under different conditions. The amount of vacuole formation at the end of these experiments was also estimated and related to the overall efficiency of protein transport in this tissue. Both pig and cow colostrum contain factors capable of increasing the rate at which immunoglobulins cross the intestine of new-born animals (Balfour & Comline, 1962; Hardy, 1970). A secondary aim of the present work was to see whether sow colostrum could facilitate the absorption of human immunoglobulin across the pig intestine. Facilitation did occur, the amount correlating well with the ability of the added compound to stimulate endocytosis. The implication of these findings to the action of accelerator substances in general is discussed. 474
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 475 METHODS Animal8 Piglets were removed immediately after birth. They were not allowed to suck the sow. Parturition was induced on day 112 of gestation by the previous I.M. injection of prostaglandin analogues (ICI 80996 or 79939). Normal gestation period in the pig is 114 days. All piglets used came from the Babraham herd of Large Whites. They were operated on within 1 hr of collection and kept warm subsequently through the use of a thermostatically controlled electric blanket. Intravenous glucose was given both before and during the experiment (see below). Operative procedure Each animal was anaesthetized with Fluothane (Imperial Chemical Industries Ltd, Macclesfield, Cheshire) for cannulation of its jugular vein (FG nylon i.v. cannula, outside diameter 1-34 mm, Portex Ltd, Hythe, Kent). The cannula was positioned finally so as to lie in or adjacent to the heart. A small mid line incision was next made immediately distal to the sternum to expose the stomach. The stomach was moved with blunt forceps to reveal the duodenum which was then cannulated with non-toxic polyvinyl tubing (Portex Ltd, Hythe, Kent). Both i.V. and intraduodenal cannulae were then strapped to the back of the animal. Heparin (25 i.u.) and glucose (2 ml. 50 % (w/v) solution) were injected i.v. and the animals left to recover consciousness. Solutions of immunoglobulins, made up in sow colostrum or in balanced saline (Krebs & Henseleit, 1932) at 370 C, were finally injected into the duodenum over a 5 min period. The volume of solution injected was 10 ml. in all cases. Piglets used in these experiments had a mean body weight of 1-29 + 0 04 kg (mean + s.E. for thirty-two animals). Nine 1 ml. samples of blood were removed during the experiment, an equal volume of 50 % (wlv) glucose being added on each occasion to keep the fluid volume constant. Experiments took either 8-5 or 9*5 hr to complete. The duodenal cannula was covered with adhesive plaster following administration of protein solution to avoid any possibility that the cannula might catch or move relative to the animal. The amount of jugular cannula left free was also kept to a minimum for a similar reason. In fact the piglets spent the whole of the experimental period either asleep or lying quietly on the electrically heated blankets. It was often possible to obtain blood samples without waking the animal. The whole procedure was tolerated without obvious signs of discomfort. Determination of immunoglobulin Blood taken for analysis was spun at top speed on a micro-centrifuge (Quickfit Instrumentation, Stone, Staffs), for 6 min. The separated plasma was stored at 40 C and analyses completed within the next two days. Determination of immunoglobulins was by electrophoresis through agarose containing specific antibodies to the proteins (Laurell, 1966), immunoglobulins in the samples being first coupled to albumin with glutaraldehyde to confer a net negative charge on the molecule. Part of the assay system came as a kit from ICL Scientific, Euclid, California, 92708, but the specific antisera to porcine and human IgG were prepared in sheep locally using conventional means. These antisera were absorbed against pooled samples of serum taken from unsuckled, new-born piglets before being incorporated into 1 % (w/v) agarose-buffer medium. The final concentration of antiserum in agarose was kept as low as possible to increase the sensitivity of the assay. Unknown samples were run alongside standards and the height of precipitin rockets compared directly. The height of precipitin rocket obtained was reproducible for any given standard, the
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error over the concentration range 25-800 /zg ml.-' varying from 2-7 to 5-5 % (means + s.E. of nineteen and eleven observations respectively).
Quantification of endocyto8i8 The small intestine, dissected out at the end of each experiment, was divided into ten segments, the middle piece from each segment being fixed in formal saline (4 % (v/v) formaldehyde in isotonic saline). Sections cut at 4 ,um were then triple-stained for protein using Heidenhain's Azan reagent and the degree of vacuolation assessed by counting the number of vacuolated cells present per section of villus. It was decided to adopt a rating system 0-4 to represent the degree of endocytosis taking place. A rating of 0 meant that no vacuolation had taken place. A rating of 1 meant that up to a quarter of the villous epithelial cells contained vacuoles. Ratings of 2 and 3 meant that between a quarter and a half or between a half and three quarters of the cells contained vacuoles. A rating of 4 meant that virtually all the villous epithelial cells contained vacuoles. Materials Colostrum was collected from ten sows over a period of 3 weeks, individual samples being stored at - 20° C. All samples were later pooled and the concentration of porcine IgG present determined by quantitative electro-immunodiffusion. The concentration of porcine IgG was found to be 9-77 % (wlv). This has been approximated to 10 % (w/v) in the text. Purified porcine IgG, Cohn fraction II, came from KochLight Laboratories Ltd., Colnbrook, Bucks. Human IgG, Cohn fraction II, came from Sigma Chemical Company, St Louis, Mo. 63178. Both proteins were stored at - 20° C. All other reagents were of A.R. grade. RESULTS
Absorption of human IgG by the new-born pig intestine Accelerator effect It has already been shown that the absorption of bovine IgG by the new-born pig is critically dependent upon the amount of colostrum fed to the animal (Pierce & Smith, 1967a). In those experiments both bovine IgG and putative accelerator substances had been presented to the pig gut in different dilutions, no attempt being made to separate these two variables. It was decided in the present work to examine these variables separately to determine which of these factors was responsible for the previously observed results. Human IgG (2 % w/v) was placed in the duodenum of new-born pigs in the presence and absence of 10 ml. sow colostrum. The subsequent appearance of human IgG in venous blood, monitored over the ensuing 9 hr, is shown in Fig. 1. Human IgG administered in Krebs-bicarbonate saline failed to reach the blood in any significant quantity. The low figures indicate a complete inability to detect human IgG in the plasma of one or more of the six animals used in these experiments. The substitution of sow colostrum for the balanced saline medium increased the subsequent absorption of human IgG by several orders of magnitude.
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 477 In a third set of experiments 10 % (w/v) porcine IgG, the same concentration as that found in sow colostrum, was administered together with 2 % (w/v) human IgG in Krebs-bicarbonate medium. The rate at which human IgG appeared in the blood might have been fractionally slower
0-8
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6 8 10 Time (hr) Fig. 1. Time course for appearance of human IgG in new-born pig plasma following intra-duodenal administration. Human IgG was administered as a 2% (wlv) solution in Krebs-Henseleit medium with no additional protein present (-0-); with 10% (w/v) porcine IgG present (-A\-); or with 10% (w/v) porcine IgG present in sow colostrum (-El-). The interrupted line shows the mean absorption curve obtained for conditions where porcine IgG was present. Individual values give means + s.E. of ten determinations. Points with arrows show where one or more samples failed to give a detectable precipitin line at assay (< 0-0125 mg human IgG ml.-' plasma).
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than when administered in colostrum (Fig. 1), but the over-all shape of the curve and the final plateau concentrations were identical. The final mean concentration of human IgG in plasma under these conditions was 0 55 mg ml-'. The period for most rapid absorption occurred 3-5 hr after administration of the protein solution. The first conclusion to be reached from these experiments is that Krebs-Henseleit medium has no inherent inhibiting effect on the absorption of human IgG. Absorption from this solution proceeds well provided 10 % (w/v) porcine IgG is present. The second conclusion is that, although these experiments show facilitation of human IgG absorption by sow
K. A. BURTON AND M. W. SMITH 478 colostrum, they show nothing that cannot be reproduced by the presence of porcine IgG alone. Specific binding of porcine IgG to membrane receptors would be expected to inhibit the absorption of a related molecule (human IgG). The fact that the opposite occurred strongly suggests that facilitation takes place through some other, non-specific process.
Vacuole formation The most obvious event occurring in the pig intestine following the ingestion of colostrum is the formation of large numbers of protein-filled
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Fig. 2. Endocytotic response of new-born pig small intestine to administered solutions of immunoglobulins. Human IgG (2 %, w/v) was given in KrebsHenseleit medium with (-A-) or without (-0-) 10% w/v porcine IgG. Human IgG (2 %, w/v) was also presented in sow colostrum containing 10% (w/v) porcine IgG(-LI-). The experiments were those described in Fig. 1. The broken line gives the mean endocytotic index produced by solutions containing porcine as well as human IgG. Values give means + S.E. for Fig. 1. as
vacuoles. Histological sections of intestine, taken from piglets used for the previous experiments, were therefore stained for protein and the degree of vacuolation calculated. The results obtained are shown in Fig. 2. An endocytotic index of 4 indicates that between 75 and 1 00 % of the villous
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 479 cells contained protein-filled vacuoles. Values probably give underestimates since small inclusions, beyond the resolving power of the light microscope, will obviously escape detection. Virtually no protein-filled vacuoles were detected in intestines following administration of 2 % (w/v) human IgG in Krebs-Henseleit medium. The small amount of vacuolation which did occur was confined to the distal region, showing that the administered solution had travelled a considerable distance down the tract. The addition of 10% (w/v) porcine IgG to 2 % (w/v) human IgG in Krebs-Henseleit medium stimulated endocytosis. Maximal effects were seen in the mid-intestine in a region seen previously to be most active in transporting bovine IgG under in vitro conditions (Pierce & Smith, 1967b). Solutions of sow colostrum containing 2 % (w/v) human IgG induced endocytosis to a similar extent to that produced by the mixed proteins administered in saline. The over-all curve was slightly lower but the difference was not statistically significant. The mean curve drawn from these two groups of animals gave a maximal mean endocytotic index of 2*7 in segment 4 (equivalent to nearly 70 % of all cells being vacuolated). The rise in endocytotic index seen on going down the intestine from segment 1 to 4 represents an increasing ability of the tissue to endocytose protein, but the fall that succeeds it from segment 6 to 10 probably arises because protein fails to reach these areas of the intestine. Analyses to prove this point were not, in fact, carried out in these experiments. These results show a strong correlation between the ability to form protein-filled vacuoles and the ability to transport human IgG (Fig. 1). If it is endocytosis alone which determines whether human IgG shall be transported then one would predict that increasing the concentration of human IgG in saline would, by itself, be sufficient to stimulate transport and that this would eliminate the effect of porcine IgG on the absorption of human IgG. This was tested for in a further series of experiments.
Absorption of human IgJ at high concentration The design of these experiments was identical to that described in the previous section. The rate at which human IgG, administered as a 10 % (w/v) solution in Krebs-Henseleit medium, appeared in the plasma is compared with that for a similar solution containing an additional 10% (w/v) porcine IgG in Fig. 3. Human IgG was first detected in the plasma 2-5 hr after instillation into the duodenum, but the concentration was then already twice that seen following the administration of 2 % (w/v) human IgG (Fig. 1). The plasma concentration then rose rapidly reaching stable levels 3-4 hr later. The final concentration reached appeared lower when the administered solution contained porcine as well as human IgG, but the variability in results made this difference statistically insignificant.
480 K. A. BURTON AND M. W. SMITH The time course for human IgG absorption following administration in sow colostrum was very similar to that found for the other two groups of animals. The final concentration in plasma appeared slightly higher, but again the variability between animals made this difference statistically
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Time (hr) Fig. 3. Time course for appearance of human IgG in plasma of new-born pigs following intra-duodenal administration. Human IgG was given as a 10% (w/v) solution in Krebs-Henseleit medium (-O-); together with 10 % (w/v) porcine IgG in Krebs-Henseleit medium (-A-) or together with 10 % (w/v) porcine IgG in sow colostrum (-El-). Each point derives from determinations carried out on samples obtained from six animals. The interrupted line gives the mean absorption curve constructed from values pooled from all three experimental situations.
insignificant. A mean absorption curve, incorporating all experimental results, gave a final mean plasma concentration for human IgG of 1-5 mg ml.-1, this concentration being reached after a period of 6-5 hr. This equilibrium concentration is less than would be predicted were the amount
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 481 of human IgG absorbed to be directly proportional to the concentration administered (0.55 mg ml.-' for a 2 % (w/v) solution would then be equivalent to 2-75 mg ml.-' for a 10 % (w/v) solution). Saturation of the absorptive process through the administration of large amounts of protein has been reported previously (Pierce & Smith, 1967 a). It is interesting to find in the present work that any facilitating effect colostrum might have on human IgG absorption disappears under these conditions. Vacuole formation following administration of high concentrations of human
Igo The degree of endocytosis correlated well with the amount of human IgG transported when the concentration of administered protein was low (Figs. 1 and 2). The administration of larger amounts of protein probably leads to some degree of saturation of transport. The degree of endocytosis encountered under these conditions was next measured to see whether the previously determined correlation still applied. The experimental material consisted of segments of intestines taken from animals at the end of experiments, the results of which are summarized in Fig. 3. Sectioning, staining and estimation of endocytotic index were as described previously. The results obtained are shown in Fig. 4. The general pattern of endocytosis, low in proximal and distal intestine, maximal in mid-intestine, was similar to that seen previously. Values for endocytotic index in both proximal and distal segments were similar for all three administered solutions. Endocytosis in the mid-intestine was, however, higher in the presence of added protein, whether that protein was present in the form of porcine IgG or sow colostrum. The mean index from experiments involving administration of 10 % (w/v) human IgG in colostrum or with 10 % (w/v) porcine IgG added to human IgG-containing Krebs-Henseleit solution was about 2-75 (segments 3-6). This compared with an index of 145 for 10% (w/v) human IgG administered alone. The correlation between transport of human IgG and the degree of endocytosis is thus seen to break down when the total concentration of administered protein becomes high enough to saturate the transport process. The small intestine will, of course, absorb porcine as well as human IgG and much of this transport probably involves a common mechanism. Under these conditions it is the total amount of protein transported (porcine plus human IgG) which should be considered in relation to endocytosis. The transport of porcine IgG was therefore measured and added to that previously determined for human IgG. Fig. 5 relates total protein transport to the protein concentration of administered solutions and to the degree of endocytosis which these solutions produce. Cumulative endocytotic indices have been calculated by summation of indices obtained
482 K. A. BURTON AND M. W. SMITH from individual intestinal segments (see Figs. 2 and 4) to give a measure of total endocytotic response. Increasing the concentration of administered protein above 15 % (w/v) (total protein load greater than 1P5 g) had little or no effect on protein transport. This verified previous similar work using
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Fig. 4. Endocytotic response of new-born pig intestine to immunoglobulincontaining media. The experiments were those described in Fig. 3. The ad. ministered solutions consisted of 10% (w/v) human IgG in KrebsHenseleit medium (-0-); 10% (w/v) human IgG +10% (w/v) porcine IgG in Krebs-Henseleit medium (- A-) or 10 % (w/v) human IgG + 10 % (wlv) porcine IgG in sow colostrum (-EI-). The interrupted line shows the mean endocytotic response in the mid-intestine following the intra-duodenal administration of solutions containing human plus porcine IgG. Individual values give means + s.E. of estimations carried out on plasma obtained from twelve animals.
bovine IgG in the pig (Pierce & Smith, 1967 a). Endocytosis also seemed to have reached a maximum over this range of protein concentration. Both endocytosis and the amount of total protein transported showed a linear relation to protein concentration over the range 2-12 % (w/v). The correlation between total protein transport and the degree of endocytosis therefore held good for all concentrations of protein tested. It is possible
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 483 that the bulk of porcine and human IgG molecules share a common route of transport, through the vacuolar system of the anterior and midintestine. 4
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Fig. 5. Relation between cumulative endocytotic index and total protein transport by new-born pig intestine. Plasma concentrations of immunoglobulins, human or human plus porcine, are plotted against the cumulative endocytotic index obtained by integration of curves shown in Figs. 2 and 4. The numbers refer to the following solutions: 1 and 2: 2 and 10% (w/v) human IgG in Krebs-Henseleit medium; 3 and 4: 10% (w/v) porcine IgG in Krebs-Henseleit medium containing 2 and 10% (w/v) human IgGrespectively; 5 and 6: 10% (w/v) porcine IgG in sow colostrum containing other colostral proteins (10% w/v) plus 2 and 10% (w/v) human IgG respectively.
Absorption of porcine IgG by the new-born pig intestine Selectivity All the evidence presented so far suggests that the pig intestine is likely to absorb 7S immunoglobulins from different species with equal facility. Yet it is also true to say that the animal variation encountered is itself
K. A. BURTON AND M. W. SMITH large enough to make small differences in transport rate statistically insignificant. One way round this problem is to measure the transport of two or more proteins in the same animal. This was done in the next series of experiments. The rate at which porcine IgG, presented in sow colostrum, reached the plasma of new-born pigs is compared with that for human IgG, dissolved in the same medium, in Fig. 6A. The time course for absorption was similar but the plasma concentration of porcine IgG was consistently higher than that for human IgG. A straight comparison of the pooled 484
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Fig. 6. Selective transport of porcine IgG by new-born pig intestine. a, plasma concentrations of porcine (-e-) and human (-O-) IgG following the intra-duodenal administration of both at concentrations of 10 % (wlv) in sow colostrum. b, Difference between porcine and human IgG transport, porcine IgG being presented either as sow colostrum (-J-) or as a soluble commercial preparation (-C-). Individual differences give means + S.E. of determinations carried out on six animals.
values in Fig. 6A shows this difference to be not significant, the actual plasma concentration of porcine and human IgG varying by a factor of 3 to 4 between different animals and litters. This variation was nevertheless
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 485 consistent, the transport of both human and porcine IgG being high or low according to the animal used. For this reason it was considered valid to represent these results as differences in transport between porcine and human IgG, all differences being determined in the same sample of plasma taken from the same animal (Fig. 6B). Values greater than 0 indicate porcine IgG to be transported more readily than human IgG. These differences were significant for times of absorption greater than 6-5 hr. Porcine IgG was present as sow colostrum while the human IgG had been prepared commercially. It could be that purification of the human IgG caused some denaturation and that this was why transport was slightly lower. Experiments were therefore repeated using a mixed protein solution containing 10 % (w/v) human and 10 % (w/v) porcine IgG, both proteins being obtained commercially. The time course for absorption, not shown here, was similar to that shown previously (Fig. 6A). The difference in transport between porcine and human IgG, plotted in Fig. 6B, was also similar. It is concluded from this that the pig intestine can show a real ability to preferentially transport porcine IgG, but that this ability is small in relation to the large amount of non-specific transport taking place. DISCUSSION
The belief that the ability of an intestine to transport immunoglobulins could be judged solely by the presence or absence of protein-filled vacuoles in the mucosa has, in the past, proved a particularly nasty trap for the unwary. The original finding that vacuoles disappeared from the rat mucosa at a time when macromolecular transport ceased (Clarke & Hardy, 1 969a, b) appeared, at first sight, to support the Brambell hypothesis. This support proved, however, to be short-lived once it became clear that macromolecular-induced vacuolation could also occur in species showing no trans-cellular movement of proteins (Clarke & Hardy, 1970). The pig falls into an intermediate class in that it absorbs immunoglobulins at birth and the intestine becomes vacuolated, but these vacuoles persist for up to 2 weeks though the transport of immunoglobulins ceases within a few hours (Clarke & Hardy, 1971). This unsatisfactory state of affairs was partly resolved by the subsequent discovery that the major route for immunoglobulin transport in the rat was through a system. of coated vesicles confined to the more proximal parts of the small intestine (Rodewald, 1973). The fact that the rat intestine transported immunoglobulins when vacuoles were present thus proved to be merely coincidental. It is not surprising, in these circumstances, to find all the more recent work on immunoglobulin absorption assuming that virtually all immunoglobulin transport takes place via these coated vesicles. There are, however, reasons
486 K. A. BURTON AND M. W. SMITH to doubt whether this latest assumption is any more valid than the earlier ones. A distinction must be made here between species such as the rat and mouse, where homologous proteins are absorbed more readily than heterologous ones, and species such as the pig or cow, where it is difficult if not impossible to show this sort of discrimination. There is now ample evidence proving the involvement of coated vesicles in the selective transport of immunoglobulins in the rat intestine (Rodewald, 1976), and one must assume here that the vacuolar system is only important for protein degradation. Present results suggest that this does not apply to the pig, however, where the vacuolar system is thought to transport most of the absorbed immunoglobulins. In the present work, a good correlation was always seen to exist between the ability of different concentrations of different immunoglobulins to induce endocytosis and the rate at which they were absorbed. It is suggested that this correspondence does not reflect mere coincidence and that a major route for the non-selective transport of immunoglobulins in the pig intestine lies through the vacuolar system at the time of its formation. It is also possible that some porcine IgG crosses the piglet intestine by selective means. This suggestion, made originally from an analysis of serum levels of immunoglobulins following oral administration (Payne quoted by Wilson, 1962; Pierce & Smith, 1967a), was verified in the present work, where proteins were administered intraduodenally. Quite a large fraction of orally-administered immunoglobulin enters the blood in degraded form, some of this degradation taking place in the stomach (Pierce & Smith, 1967a; Hardy, 1969). Injecting immunoglobulins directly into the duodenum will reduce, but probably not eliminate, such proteolysis. The possibility that human IgG is degraded more extensively than porcine IgG and that this is why porcine IgG is transported more readily, cannot be definitely excluded. No attempt was made to identify possible products of immunoglobulin break-down in the present work, but the assay system should have shown more than one precipitin line if they had been present in more than trace amounts. The fact that only one line was detected provides some support for the view that the intestine can, to a limited extent, transport porcine in preference to human IgG. The postulated route for such transport would be through the coated vesicles, shown by Hardy, Hockaday & Tapp (1971) to be widely distributed within the pig small intestine at birth. It has already been stated that sow colostrum, like that of the cow, contains factors capable of stimulating the absorption of different immunoglobulins (Hardy, 1970). Part of this effect could arise from the trypsin inhibitor activity of colostrum or from the action of unknown factors on cellular metabolism. It is probably no coincidence, though, that
ENDOCYTOSIS AND IMMUNOGLOBULIN TRANSPORT 487 these accelerator effects have, up till now, only been seen in species where the post-natal transport of immunoglobulins is transient and mainly nonselective. These are situations where the initiation of endocytosis probably plays a crucial part in determining the over-all efficiency of immunoglobulin transport. Results obtained in the present work show sow colostrum and purified porcine IgG to be indistinguishable in their effects on the trans-cellular transport of human IgG. They also initiate endocytosis to a similar extent. It seems likely that the action of colostrum as an accelerator of immunoglobulin absorption in this species derives mainly from its ability to initiate endocytosis. We wish to express our thanks to Mr R. W. Ash and his staff for providing us with a plentiful supply of new-born, unsuckled pigs during the time when this work was carried out. REFERENCES BALFouR, W. E. & COMLINE, R. S. (1962). Acceleration of the absorption of unchanged globulin in the new-born calf by factors in colostrum. J. Physiol. 160, 234-257. BRAMBELL, F. W. R. (1966). The transmission of immunity from mother to young and the catabolism of immunoglobulins. Lancet ii, 1087-1093. CLARKE, R. M. & HARDY, R. N. (1969a). The use of [12.5]polyvinyl pyrrolidone K60 in the quantitative assessment of the uptake of macromolecular substances by the intestine of the young rat. J. Physiol. 204, 113-125. CLARKE, R. M. & HARDY, R. N. (1969b). An analysis of the mechanism of cessation of uptake of macromolecular substances by the intestine of the young rat ('closure'). J. Physiol. 204, 127-134. CLAiRE, R. M. & HARDY, R. N. (1970). Structural changes in the small intestine associated with the uptake of polyvinyl pyrrolidone by the young ferret, rabbit, guinea-pig, cat and chicken. J. Physiol. 209, 669-687. CLARKE, R. M. & HARDY, R. N. (1971). Histological changes in the small intestine of the young pig and their relation to macromolecular uptake. J. Anat. 108, 63-77. HARDY, R. N. (1969). The break-down of [1311]globulin in the digestive tract of the new-born pig. J. Physiol. 205, 435-451. HARDY, R. N. (1970). Absorption of macromolecules from the intestine of the newborn animal. In Physiology of Digestion and Metabolism in the Ruminant, ed. Phillipson, A. T., pp. 150-165. Newcastle upon Tyne: Oriel. HARDY, R. N., HOCKADAY, A. R. & TAPP, R. L. (1971). Observations on the structure of the small intestine in foetal, neo-natal and suckling pigs. Phil. Trans. R. Soc. B 259, 517-531. JoNEs, E. A. & WALDMANN, T. A. (1972). The mechanism of intestinal uptake and transeellular transport of IgG in the neonatal rat. J. clin. Invest. 51, 2916-2927. KREBs, H. A. & HENSELEIT, K. (1932). Untersuchungen fiber die Harnstoffbildung im Tierkorper. Hoppe-Seyler's Z. physiol. Chem. 210, 33-66. LAURELL, C.-B. (1966). Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analyt. Biochem. 15, 45-52. MORRIS, B. & MoRRIs, R. (1974). The absorption of 125I-labelled immunoglobulin G by different regions of the gut in young rats. J. Physiol. 241, 761-770. PAYNE, L. C. (1962). Unpublished data cited by T. H. Wilson in Intestinal Absorption. Philadelphia: W. B. Saunders.
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