The Grafted Fetal Rat Pancreas

The Grafted Fetal Rat Pancreas

Features ofDevelopment and Rejection

P. R. Millard, MD, MRC Path, J. F. W. Garvey, BSc, MB, BS, RACS,Elena L. Jeffery, AIMLS, and P. J. Morris, PhD, FRCS

Pancreata from 17-18-day gestational rat embryos have been transplanted between re-lated and unrelated adult rats rendered diabetic with streptozotocin. In separate experi-ments aliograft recipients were given the immunosuppressive agent cyclosporin A. Themorphologic changes in most of these grafts were followed during the first 2 weeksafter transplantation. The endocrine, but not the exocrine, tissue developed fully in theisografts and did so at a rate corresponding to that reported for in vitro cultures. Withinthe allografted pancreata there was no maturation of the exocrine components, and endo-crine tissue only reached full maturity if immunosuppression was used. Allograft rejec-tion was similar morphologically to that of other tissues, involving cellular and humoralfactors. The cells within the graft evoking rejection were not identified. These studies in-dicate that if rejection could be adequately suppressed, the endocrine parts of the fetalpancreas would survive and develop. (Am J Pathol 1980, 100:209-224)

THE COMPLICATIONS encountered by many of today's patientswith diabetes mellitus highlight the urgency for finding improved meth-ods of treating this potentially disabling and life-threatening disorder.Theoretically, replacing the pancreas should cure the disorder and pre-vent its- protean complications. The unsatisfactory results of whole organpancreatic allotransplantation in man have stimulated laboratory researchinto the alternatives of isolated islet transplantation and of whole fetalpancreas transplantation.'

Transplantation of fetal pancreas in the rat by the use of the techniqueestablished by Brown et al 2 has been investigated recently within our lab-oratories in both syngeneic and allogeneic combinations. Suppression ofthe allograft reaction has been attempted with the immunosuppressive re-agent cyclosporin A.3 Brown et al 2 have made similar studies and have re-corded the histologic appearances in isografts at the first week and at 2-3months after transplantation, but not the changes seen during these peri-ods or the histologic changes in allografts following rejection.4 Morpho-logic studies of fetal pancreatic cultures have clearly demonstrated thatthere are different developmental patterns for the endocrine and exocrine

From the Department of Pathology and the Nuffield Department of Surgery, University of Oxfordand John Radcliffe Hospital, Headington, Oxford.

Supported by a grant from the Medical Research Council (UK) and the British Diabetic Associa-tion.

Accepted for publication February 18, 1980.Address reprint requests to P. R. Millard, MD, MRCPath, Consultant Pathologist, Department of

Histopathology, John Radcliffe Hospital, Headington, Oxford OX3 9DU, England.

0002-9440/80/0709-0209$01.00 209©D American Association of Pathologists

210 MILLARD ET AL American Journalof Pathology

components of the pancreas.56 These studies also suggest that if trans-plantation is contemplated, an optimum period exists for obtaining a max-imum yield of islet tissue. We have examined sequential biopsy specimensof isografted fetal pancreata in adult diabetic rats to determine whetherdevelopment of the immature pancreas proceeded normally followingisotransplantation in the rat. We also examined a similar group of allo-graft biopsy specimens to determine the pattern of the immune responseto the graft and its effect upon the development of the fetal pancreas. Theeffect of cyclosporin A on pancreatic development and on the rejectionprocesses has also been compared with the histologic appearance of thefetal pancreatic allografts.

Materials and Methods

Animals

Inbred LEW (RT-1c), DA(RT-la) and (DA X LEW) Fl hybrid rats from Bantin andKingman, Aldborough, Hull were used. Isografts, LEW to LEW, and allografts, (DA xLEW) Fl to LEW, were undertaken with time-mated 17-18-day embryos of either sex.The recipients were rendered diabetic with a single intravenous dose of 65 mg/kg strepto-zotocin,7 a pancreatic f8-cell toxin.

TransplantationOne to three weeks after streptozotocin 6-8 pancreata were implanted in equal num-

bers under each renal capsule by the technique of Brown et al.2 This process included theadministration of 0.5-3 units of Burroughs Wellcome Ultralente insulin, based on themorning's serum glucose level, for the first 8 days to allow maturation of the grafts in anormoglycemic environment. Cyclosporin A in olive oil was given to LEW recipients ofDA fetal pancreas by gavage in doses ranging from 10 to 40 mg/kg/day.3

HistologyAt various times, generally Days 3, 6, 8, 10, 14, and 21, laparotomy was performed and

one grafted pancreas was removed for histologic examination. In the cyclosporin-A-treated group similar specimens were obtained either at the time of death or at sacrifice.The tissues were fixed in a formol-saline solution and processed to paraffin wax. All sec-

tions were stained with Bullard's acid hematoxylin and eosin and with aldehyde fuchsin toidentify ,8 granules in islet cells. Selected sections were stained by Perls' method to dem-onstrate ferric iron and by the periodic-acid Schiff reaction (PAS) for zymogen granules.Positive PAS staining occurred in the acini of the control adult rat pancreas.The grafts were examined for evidence of maturation and rejection, and to allow com-

parison between grafts some features were scored 0-4+. As described below, mature isletcells developed from undifferentiated basophilic epithelial cells, through immature or pre-cursor eosinophilic cells. These cells in the early grafts formed ducts with the later appear-ance of acini and finally mature islets. Each of these pancreatic cell types and formedstructures, as well as the infiltrates of lymphoid cells, macrophages, and polymorph cells,were scored (Tables 1 and 2).

Vol. 100, No. 1 GRAFTED FETAL RAT PANCREAS 211July 1980

Results

lsografts

The whole fetal pancreatic isografts were removed from under the kid-ney capsule between 1 and 21 days after transplantation (Table 1). Duringthis interval pancreatic tissue developed and differentiated. Differenti-ation, however, was confined to the endocrine component, and no matureexocrine parts were recognized at any time.

Days 1-3

In these biopsies pancreatic tissue included two cell types (Figure 1).The predominant cell was cubical, with a large basophilic nucleus andpale staining basophilic cytoplasm, which was amorphous and included no,B or zymogen granules. A few of these cells were in division, and theywere regarded as uncommitted, or undifferentiated, cells. The cells werearranged in clumps, some of which were penetrated by spaces, and theclumps were closely apposed to one another, often with apparent sharingof cells between the clumps. Randomly scattered among these cells wereothers differentiated by partly granular eosinophilic cytoplasm. The gran-ules were fine and gave the cytoplasm a ground-glass appearance. By the3rd day after transplant more of these eosinophilic cell types were evidentand the granules had become evenly dispersed throughout their cyto-plasm. The aldehyde fuchsin preparations revealed a few fine minutestrands of poorly staining material in most of these cells, but there was noPAS-positive material, and they were therefore interpreted as immature,or precursor, endocrine cells. Their nuclei at this time were less roundedand more uniformly and more deeply basophilic than on Day 1 and hadindistinct nuclear membranes. They were pleomorphic, and the overallappearance was ill-defined and smudged. None of these cells was in divi-sion. The cells were aggregated in small isolated acini and were some-times budding either as solid cell collections or as incipient acini fromducts. The ducts were small, mildly dilated, and lined by low-set cubicalcells of the undifferentiated type, although occasional eosinophilic cellsand degenerate cells were dispersed among them.The stroma of the graft by the 2nd day of transplantation had loosely

packed plump cells with spindle-shaped nuclei and indistinct pale eo-sinophilic cytoplasm. Between these cells there was edema and in manygrafts, scattered erythrocytes. The occasional small mononuclear cell and,in some grafts, macrophages with foamy cytoplasm sometimes in consid-erable numbers lay between some of these stromal cells. Neutrophil poly-

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morphs were only evident adjacent to degenerate pancreatic cells. Capil-laries, frequently engorged, as well as venules and arterioles, were presentin the Day 2 and Day 3 specimens.

Days 6-10

Pancreatic tissue had developed between the 6th and 8th post-transplant days to the extent that islet cells with distinct aldehyde-fuch-sin-positive granules had formed (Figure 2). The cytoplasm of the cellswas less granular and less eosinophilic than in the precursor cells and wasalso more plentiful. The islet cells were larger than their precursors, andtheir nuclei were paler but more rounded. Islet cells lay in small groups of2-4 cells, as well as randomly and isolated, but were not evident in all ofthe biopsy specimens. Precursor and undifferentiated cells had the sameappearance as in the early biopsies but did not include many dividingcells. In the occasional graft the pancreatic tissue was assuming a lobulatepattern, while in others it was either entirely necrotic or necrotic withinthe central parts. In both of these necrotic areas there was usually a sub-stantial mixed cellular infiltrate. Other stromal features were identical tothose in the early biopsies but with additional hemosiderin- and erythro-cyte-laden macrophages in slight-to-moderate numbers.

Days 14-21

Throughout the 2nd and 3rd week after transplantation islets becamemore numerous and larger, and some included capillaries separating theislet cells (Figure 3). Isolated random islet cells were still seen. Precursorand undifferentiated cells persisted, but more of the latter were degener-ate, and the ducts they lined were more widely dilated and less numerousthan in the early biopsies. Even so, occasional mitoses were found, and thebudding of the precursor cells from some of these ducts was still recogniz-able. Precursor cells in small numbers were still present in the 21-daybiopsy specimens. The stroma included the same spectrum of cellular andvascular findings as was evident during the 1st week after transplantation,but there were also distinct thin fibrous bands embracing parts of the pan-creatic tissue, and macrophages filled with hemosiderin were often scat-tered throughout the stroma. From Day 14, adipose tissue formed a mod-erate component of some of the grafts' parenchyma.

Allografts-Untreated Recipients

Allografts of entire fetal pancreata were examined between 1 and 21days after transplantation (Table 2), and they included the same morpho-logic spectrum as that within the isografted pancreata. However, pancre-


atic tissue did not reach the same degree of maturity. In addition, therewas more necrosis and hemorrhage, and mononuclear cell infiltrates weremore conspicuous. Vascular fibrinoid necrosis was a unique feature ofsome of these grafts.

Endocrine precursor- cells were recognized in the 1- and 3-day biopsyspecimens (Figure 4), and, as in the isograft specimens, islet cells wereseen by Day 6. However, islet cells were only recognized in one 6-dayspecimen and in none of the other 4 similar samples or samples from anyexperiments in which subsequent biopsies were done (Table 2). None ofthe allograft biopsy specimens at any time included formed islets. Hemor-rhage and necrosis, often confined to the central portions of the grafts, wasapparent within many grafts from the 3rd day after transplant and affectedboth pancreatic and stromal compartments. Infiltrating mononuclear cellswere recognized in small numbers from Day 3, when they included largelymphocytes and macrophages with foamy cytoplasm and some containingengulfed red cells or filled with hemosiderin. Variable numbers of neutro-phil polymorphs were also present. By Day 6 (Figure 5) large and smalllymphocytes were found as well as dividing cells and macrophages similarto those in the earlier grafts. Plasma cells were only rarely identified. Allcells were in substantial numbers and were uniformly dispersed through-out the graft. This cellular infiltrate persisted during the second week, butby the third week, when the grafts were largely fibrotic, fewer cells wereevident. Fibrinoid necrosis of arterioles and capillaries was recognized atDay 10 in two expermients and at Day 14 in a further one and affectedseveral vessels within the substance of these grafts (Figure 6). The changewas patchy and focal, and there were neutrophil polymorphs within boththe affected vessel wall and the adjacent graft stroma. All vessels werepatent.

Recipients Given Cyclosporin A

Fifty-six allografts from 14 rats receiving differing doses of cyclosporinA were examined between 1 and 26 days after grafting.3 Overall in ap-pearance they were the same as those in the unimmunosuppressed allo-grafts, but islets with aldehyde-fuchsin-staining granules were evident inone biopsy specimen by Day 9 and in 14 others between Days 10 and 14(Figure 7). The cellular infiltrate included the same range of cells as oc-curred in the control allografts, but the cells appeared fewer in numberand were more in evidence at the periphery of the graft rather than uni-formly dispersed. Fibrinoid necrosis was present in three grafts, occurringat 14 and 26 days after transplantation.


DiscussionBoth tissue culture and insulin and enzyme assay studies have estab-

lished that only the endocrine portions of the fetal pancreas reach matu-rity in vitro.56 Isografts of fetal pancreas to privileged sites have resultedin similar findings. Our observations with fetal pancreatic isografts 8 con-firm the published findings of Brown et al 2 that these grafts can com-pletely correct the streptozotocin-induced diabetes in the rat and indeedproduce functional results superior to those achieved with adult islets. In12 of 16 consecutive rats with isografts the diabetic state was completelyameliorated and, in some recipients, remains so at beyond 9 months.Half of these 12 rats were normoglycemic from the day after insulin wasstopped, ie, Day 9, and in 5 others at 1 month after transplantation. Asingle rat took 121 days to establish permanent normoglycemia. Histologi-cally, the endocrine portion of these pancreata developed at a rate com-parable to that reported both in vivo and in vitro.5'f Mature islets wereobserved at birth and on Day 22 after coitus, which is equivalent to the 6thposttransplant day, and then proceeded, as in vivo, to enlarge. However,in marked contrast, but just as in vitro, the exocrine component failed tomature. No mature exocrine tissue was seen at any time after transplanta-tion, a phenomenon that at present is unexplained but that might be due tothe associated denervation or to the absence of the secretagogues thatwould normally stimulate the exocrine tissue of the intact pancreas. Themature isografts were remarkable for the absence of any significant fibrosisand for the adipose tissue that enveloped them, neither of which has beensatisfactorily explained. The fat may possibly impede stimuli invoking in-sulin release and/or the transport of insulin to sites of action and could bethe response to the lipotrophic stimulating effect of the insulin-secretingcells.

Fetal pancreatic allografts in nonimmunosuppressed recipients fail tocorrect diabetes because immune destruction of the grafted tissue occursbefore the endocrine parts are functionally effective. Comparison of theisografts that included islets and the allografts of the same time, Day 6 on-ward after transplantation (Tables 1 and 2), supports this contention, sincein the latter there was no endocrine tissue (Figure 5). The rejection proc-ess was predominantly cellular but also, as is demonstrated by the vascu-lar lesions within some grafts, involved humoral components (Table 2 andFigure 6). Specific studies have confirmed that a humoral component is in-volved and that specific antibody is present at this time in high titers.9 Thelymphocytes within the allografts were interpreted as a part of the cellu-lar immune response even though similar but fewer cells were evident inthe isografts (Tables 1 and 2). Lymphocytes in both grafts were small and


often difficult to distinguish from the nuclei of the fibrous tissue, andtherefore when doubt existed, a score of + was given, a practice whichmay have overemphasized their presence. Even so, from Day 6, signifi-cantly greater numbers of lymphocytes were found in the allografts than inthe isografts, an observation that implies an immunologic basis for theirpresence (Figures 2 and 5). Large cells containing hemosiderin or en-gulfed red cells and having a foamy cytoplasm were designated as macro-phages, and many were found in both types of grafts. More of these cellsmay have been present, but in the absence of engulfed material and spe-cific marker studies they were not categorized as such. Overall, some allo-grafts could have harbored very considerable numbers of macrophagesand, while they could be part of an immune response, it is unusual to seesuch numbers. Finding these cells in both types of grafts suggests that tis-sue destruction from an ischemic response rather than a primary immuno-logic response is partially responsible for their presence. A similar ex-planation is also more tenable for the neutrophil polymorphs seen in bothtypes of graft.The rejection process was evident morphologically within 3 days after

transplantation and established by 6 days. The pattern, timing, and devel-opment of this rejection process mirrors that of many other tissues and or-gans 10 and is comparable to that reported with allotransplants of free is-lets between adult rats or mice implanted in the peritoneum," beneaththe kidney capsule,'2 or in the liver,'3"14 as well as to that in whole pan-creata between adult dogs.'5 Islet tissue and whole pancreas can thus initi-ate rejection regardless of its maturity or species of origin; and these, andour observations, do not, at least morphologically, support the claim thatislet tissue stimulates a more aggressive immune response than thatmounted to other transplanted tissue.'6

Franklin et al 14 found that rejection of islet transplants occurred mor-phologically at least 48 hours before an elevation of serum glucose. How-ever, Morris et al 17 have suggested that the apparent aggressive rejectionof islets may be in part due to the use of a more sensitive method (ie,serum glucose) for monitoring rejection than exists for the detection ofrenal, cardiac, or other forms of organ rejection. Even so, it is conceivablethat the response is real and that at least in part it is stimulated or abro-gated by the released insulin. From our observations immune rejectioncan be induced by immature pancreatic tissue, for on Day 3 (Table 2; Fig-ure 4) there were no mature islet cells but only uncommitted and pre-cursor endocrine cells within the allografts, and yet mononuclear cells hadappeared. Exactly which cells are responsible for the induction of the im-mune response is not at all clear, but undifferentiated pancreatic cells


could be involved. Recent findings of Parr 18 support this conclusion. Us-ing immunoferritin labeling of mouse H-2 antigens, Parr failed to demon-strate these antigens on ,8 cells, although they were evident on pancreaticduct, acinar, and endothelial cells. However, it was not clear againstwhich specificity of the H-2 complex the alloantisera were directed, and itmay be that only Ia or only SD antigens were absent from islets. If both Iaand SD antigens were absent from islet cells, their presence on anothercellular component of the pancreatic islet tissue, for example, vascular en-dothelium, might be responsible for the induction of the immune responseto the islet cells as suggested by Parr.'8The use of cyclosporin A confirmed that with immunosuppression of

the recipient islet development progresses in a fashion similar to that inthe fetal pancreatic isograft (Figure 7), and studies from these laboratoriesshow that such tissue will function effectively for up to 15 days after trans-plantation (7 days after the last treatment with exogenous insulin).3These findings corroborate that it is rejection that is destroying the isletcells in unsuppressed recipients and not a failure of development. Fetalpancreatic allografts with adequate immunosuppression can therefore beexpected to provide sources of insulin both experimentally and clinically.

In functional studies cyclosporin A was less effective in suppressingrejection of fetal pancreas allografts than it was in suppressing kidneyallografts.'9 The morphologic features of rejection in the control and thecyclosporin-A-treated allografted pancreata were similar, althoughpreservation of intact islets was apparent at the low dose of 10 mg/kg/day (Figure 7). In the light of the very considerable immunosuppressantproperties of cyclosporin A,20 the present findings are a little disappoint-ing, since this agent did not appear to impair islet maturation and there-fore might be a useful immunosuppressive drug for use with fetal pancre-atic allografts. If an effective immunosuppressive regimen could beevolved, fetal pancreatic allografts, and possibly xenografts, would pro-vide the ideal therapy for the patient with diabetes mellitus, relieving himof many of the inadequacies and complications associated with present-day management of the disease.

References

1. Karl RC, Scharp DW, Ballinger WF, Lacy PE: Progress report: Transplantation ofinsulin-secreting tissues. Gut 1977, 18:1062-1072

2. Brown JB, Clark WR, Molnar IG, Mullen YS: Fetal pancreas transplantation for re-versal of streptozotocin-induced diabetes in rats. Diabetes 1976, 25:56-64

3. Garvey JFW, McShane P, Millard PR, Morris PJ: The effect of Cyclosporin A onexperimental pancreas allografts. Transplant Proc (In press)


4. Mullen Y: Survival times of fetal pancreas allografts, Pancreas transplantation fordiabetes mellitus. Ann Intern Med 1978, 89:951-965

5. Hegre OD, McEvoy RC, Bachelder V and Lazarow A: Fetal rat pancreas: Differen-tiation of the islet cell component in vivo and in vitro. Diabetes 1973, 22:577-583

6. McEvoy RC, Hegre OD, Leonard RJ, Lazarow A: Fetal rat pancreas: Differenti-ation of the acinar cell component in vivo and in vitro. Diabetes 1973, 22:584-589

7. Rakieten N, Rakieten ML, Nadkarni MV: Studies on the diabetogenic action ofstreptozotocin (NSC-37917). Cancer Chem Rep 1963, 29:91-98

8. Garvey JFW, Morris PJ, Millard PR: Early rejection of allogeneic foetal rat pan-creas. Transplantation 1979, 27:342-344

9. Garvey JFW, Winearls CG, Millard PR, Morris PJ: (In press)10. Herbertson BM: The morphology of allograft reactions, Immunological Aspects of

Transplantation Surgery. Edited by RY Calne. Medical and Technical Publishing Co.Ltd., 1973, pp 4-38

11. Weber C, Zatriqi A, Weil R, McIntosh R, Hardy MA, Reemtsma K: Pancreatic isletisografts, allografts and xenografts: Comparison of morphology and function. Surgery1976, 79:144-151

12. Bowen KM, Andrus L, Lafferty KJ: Successful allotransplantation of mouse pancre-atic islets to nonimmunosuppressed recipients. Diabetes (In press)

13. Slater D, Mangnall Y, Smythe A, Fox M: The rejection of islets of Langerhans fol-lowing intrahepatic transplantation in the rat. Acta Endocrinol [Suppl] (Copenk)1976, 205, 83:295-302

14. Franklin WA, Schulak JA, Reckard CR: The fate of transplanted pancreatic isletsin the rat. Am J Pathol 1979, 94:85-96

15. Acosta JM, Nardi GL, Reeves G, Pinn V, Buceta JC, Sologa JC: Histopathologicchanges in the allografted canine pancreas. Arch Surg 1973, 106:844-847

16. Matas AJ, Sutherland DER, Steffes MW, Najarian JS: A collective review: Islettransplantation. Surg Gynecol Obstet 1977, 145:757-772

17. Morris PJ, Finch DR, Garvey JF, Poole MD, Millard PR: Suppression of rejectionof allogeneic islet tissue in the rat. Diabetes (In press)

18. Parr EL: The absence of H-2 antigens from mouse pancreatic ,8-cells demonstratedby immunoferritin labelling. J Exp Med 1979, 150:1-9

19. Homan WP, Fabre JW, Williams KA, Millard PR, Morris PJ: Studies on the immu-nosuppressive properties of Cyclosporin A in rats receiving renal allografts. Trans-plantation (In press)

20. Calne RY, Rolles K, White DJG, Thiru S, Evans DB, McMaster P, Dunn DC, Crad-dock GN, Henderson RG, Aziz S, Lewis P: Cyclosporin A initially as the only im-munosuppressant in 34 recipients of cadaveric organs: 32 kidneys, 2 pancreas, and 2livers. Lancet 1979, 2:1033-1036

AcknowledgmentsWe thank Mrs. R. Hunt for typing the manuscript and Mr. R. Holton for help with the photogra-

phy.


[Illustrationsfollow]

1

2

Figure 1-lsograft, F97, at Day 3. Precursor endocrine cells with positive aldehyde fuchsin granules(inset) and undifferentiated pancreatic cells forming acini and ductules. The stroma of the graft isloose, with a few mononuclear cells. (H&E, X300; inset, aldehyde fuchsin, x816) Figure 2-Isograft, F97, at Day 6. Precursor endocrine cells are more numerous than at Day 3 (Figure 1) andislets are beginning to form (center). Many cells include aldehyde-fuchsin-staining granules (inset).(H&E, x300; inset, aldehyde fuchsin, x81 6)

i

3

4

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. .

Figure 3-Isograft, F97, at Day 21. Many formed islets with a few acini and ductules. There are afew mononuclear cells, and fat surrounds the grafted tissue. (H&E, x300) Figure 4-Allograft,F65, at Day 3. Appearances similar to those in the Day 3 isograft (Figure 1). Aldehyde-fuchsin-posi-tive granules are in some endocrine precursor cells (inset). (H&E, x300; inset, aldehyde fuchsin,x816)

l

5

i

6

Figure 5-Allograft, F65, at Day 6. The stroma is diffusely infiltrated by mononuclear cells, and thepancreatic tissue is degenerate. The appearances contrast markedly with those of the isograft atthis time (Figure 2). (H&E, x420) Figure 6-Allograft, F76, at Day 10. Fibrinoid necrosis of anartery with mononuclear cells in the surrounding tissue, which includes no viable pancreatic cells.(H&E, x420)

Figure 7-Allograft, E78, at Day 14. The recipient received 10 mg/kg/day of cyclosporin A for 14days and was normoglycemic for 13 days. In contrast to Figure 6, islets are present, and there arefew mononuclear cells. (H&E, x420)

The Grafted Fetal Rat Pancreas

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