*Abbreviations: C6D, C6-deficient; CsA, cyclosporine; DSG, deoxyspergualin; H&E, hematoxylin and eosin; IL, interleukin; MAC, membrane attack complex of complement; MMF, mycophenolate mofetil; PAP, peroxidase-antiperoxidase; RAPA, rapamycin.
Insulin-dependent mellitus continues to present a therapeutic challenge. According to the Diabetes Control and Complications Research Trial, strict metabolic control of serum glucose reduced the risk of secondary complications of diabetes by as much as 76% (1). However, it has also been shown that long-term normalization of hemoglobin A1c can be achieved only in about 5% of diabetes. Successful whole organ pancreas transplantation invariably induces euglycemia in 100% of patients, but surgical risk, complications associated with the exocrine portion of the pancreas, and organ availability limit transplants to a minority of patients. Islet cell transplantation could significantly reduce risk and morbidity, but xenoislet transplantation could further address the problem of organ shortage.
In most models of discordant pancreas xenoislet transplantation other than those with mouse recipients, the islets fail to restore a normoglycemic state, as these islets are immediately nonfunctional. Although primary nonfunction of islet allografts has been shown to be macrophage-dependent(2), the mechanism of primary nonfunction of islet xenografts is unknown. In immediately vascularized solid organ xenografts, hyperacute rejection occurs in minutes to hours (3,4). In these models, preformed natural antibodies bind to antigens on donor vascular endothelium and activate complement, which initiates tissue injury and graft destruction. In certain species combinations, complement activation may be induced directly by the xenogeneic endothelium through the alternative pathway (5). However, current evidence suggests that, in primarily avascularized xenografts, primary non-function may occur but there is no hyperacute rejection(6-8), as immunosuppression that does not decrease preexisting antibody levels and does not inhibit complement reduces the incidence of primary nonfunction. At present, it is unknown whether complement is involved in primary nonfunction of islet xenografts. However, primary nonfunction could occur through a number of mechanisms involving complement. C1q and C3b can contribute to an inflammatory response(9,10) and C3a, C4, and C5a may recruit a number of inflammatory cells, including macrophages, which can affect post-transplant islet cell function. The membrane attack complex (MAC*), composed of C5b-9, in sublytic concentrations may induce several proinflammatory processes, including the release of interleukin (IL)-8 and monocyte chemoattractant protein-1 (11).
In the work reported herein, we tested whether the MAC plays a role early dysfunction of islet xenografts. We used a C6-deficient (C6D) strain of PVG RT1C rats (12) as recipients in a dog-to-rat xenoislet transplant model. The lack of C6 prevents formation of the MAC, the final common effector for both complement pathways. We found that nonimmunosuppressed C6D rats, similar to normocomplementemic rats, uniformly develop primary nonfunction. In immunosuppressed recipients, C6D rats exhibited a slight reduction in primary nonfunction.
MATERIALS AND METHODS
Animals. PVG RT1C normocomplementemic rats were purchased from Harlan-Sprague Dawley Inc. (Indianapolis, IN) and PVG RT1C C6D rats were purchased from Bantin & Kingman Laboratories(Fremont, CA). All rats were received at a weight of approximately 250 g, and during the course of the study they were maintained on standard rodent chow(Teklad 4% Rat/Mouse Diet; Harlan Teklad Co., Madison, WI) and given water ad libitum. Mongrel canine donors, 15-20 kg, were obtained from Twin Valley Kennels, Spring Green, WI. For all experiments, the Principles of Laboratory Animal Care (NIH Publication 86-23, revised 1985) and the regulations of the University of Minnesota Animal Care Committee were followed.
Complement activity. The integrity of the complement pathway in PVG rats was tested by a total serum hemolytic complement activity assay(CH50 assay) based on a published procedure (13) and was carried out in 96-well flat-bottom microtiter plates (Corning, Corning, NY). Samples were serially diluted 2-fold from undiluted C6D rat serum of from an initial 1:10 dilution of serum from normocomplementemic pr C6D rats reconstituted with C6. Fifty microliters of diluted serum were mixed with 50µl of sensitized sheep red cells at 108 cells/ml in Veronal buffer. One hundred microliters of Veronal buffer were added, and the plates were incubated at 37°C with occasional mixing. Degree of hemolysis was measured by turbidity at 650 nm in a Vmax kinetic plate reader (Molecular Devices, Sunnyvale, CA). CH50 is expressed as the serum dilution yielding 50% reduction in turbidity. Serum from normal Lewis rats was used to determine baseline rat CH50 activity. Sera from untreated PVG rats were stored at-80°C before use for CH50 determinations. Highly purified human C6(Quidel, San Diego, CA) was used to reconstitute C6D serum at 100-250µg/ml. Normocomplementemic PVG rats had serum complement levels similar to those of Lewis rats. Serum from C6D rats had undetectable hemolytic complement. The CH50 activity of the C6D rats could be restored to normal levels of addition of C6, confirming that these rats lack C6 but have normal activity of all other complement components.
Experimental groups and immunosuppression. Animals were divided into seven groups (Table 1). Group I consisted of normocomplementemic PVG rats, and group II consisted of C6D rats received 10,000 islet equivalents and no immunosuppression. Groups III, IV, and V consisted of C6D rats that received 5,000, 10,000, and 20,000 islet equivalents, respectively. Their immunosuppression protocol is listed inTable 1. In groups VI and VII, normocomplementemic rats and C6D rats, respectively, received 10,000 islet equivalents each. Their immunosuppression protocol is also listed in Table 1. Animals received immunosuppression in the following doses: deoxyspergualin(DSG) 2.5 mg/kg i.p., mycophenolate mofetil (MMF) 30 mg/kg p.o., cyclosporine(CsA) 10 mg/kg i.p., and rapamycin (RAPA) 1.5 mg/kg p.o. The MMF was suspended in a carboxymethylcellulose/polysorbate/ethanol vehicle, the DSG in distilled water, and the CsA in an ethanol/45% intralipid solution. The RAPA was a king gift of Dr. S. Sehgal (Wyeth-Ayerst Research, Princeton, NJ). It should be noted that, in the doses administered, these regimens are nondiabetogenic. Immunosuppression was started 3 days before transplantation and stopped when the animal had 3 days of hyperglycemia, defined as peripheral blood glucose greater than 200 mg/dl.
Diabetes induction. Rats received 45 mg/kg streptozotocin intravenously (Upjohn, Kalamazoo, MI) and were screened for the presence of diabetes by measuring tail vein blood sugars starting 2 days after injection. All animals demonstrated blood glucose greater than 325 mg/dl on 2 consecutive days, including the day of the transplant, as measured by a glucose oxidase assay in a Beckman glucose analyzer 2(14).
Islet isolation. All dogs were fasted the night before surgery, and then a duodenum-preserving pancreatectomy was performed according to a published protocol (15) under general inhaled anesthesia. The pancreas was digested and islets isolated by a modified automated collagenase digestion method of Recordi (16). Briefly, the pancreas was weighed and distended with 250 ml of a 2.5 mg/ml solution of collagenase (lot 13125, Crescent Chemical Co., Haupauge, NY). It was then placed in a filter-screen chamber and agitated until islets, stained with dithizone (Fisher Chemical, Fair Lawn, NJ), were observed separating from the acinar parenchymal tissue under phase contrast microscopy. Digestion was stopped at this point by diluting the solution with a large volume of cold Hanks' balanced salt solution. Dispersed pancreatic tissue was washed several times. The washed digest was purified on discontinuous Euro-Ficoll gradients with a COBE 2991 cell separator (16). The islet-containing layer was collected into small conical tubes and washed several times with Hanks' balanced salt solution. At this time, samples of 10,000 islet equivalents were prepared for transplantation. An islet equivalent is defined as the amount of tissue contained in islets with sections that have a diameter of at least 150 µm.
Anesthesia and transplantation. All rats were anesthetized with ketamine hydrochloride (10 mg/kg i.m.) and Nembutal (30 mg/kg s.c.). They were also given 0.03 ml of heparin and 0.07 ml of atropine subcutaneously to prevent portal vein clotting and aspiration, respectively. A midline incision was made, and the small bowel was laid out on the anterior abdominal wall. A small tributary of the superior mesenteric vein was identified and cannulated with a 25-gauge butterfly needle. The islets, in a total volume of 3 ml, were then injected into the vein, and they subsequently drained into the liver parenchyma via the portal vein. The abdomen was closed in two layers with a running 5-0 vicryl suture. The animal was allowed to recover in its cage, which had been supplied with clean bedding.
Postoperative monitoring and immunohistochemistry. Animals were bled from the tail vein at 6 hr after transplant and then once each on every postoperative day until permanent nonfunction was certain (3 consecutive days of blood glucose greater than 200 mg/dl). The first of these 3 days was designated the day of nonfunction. The animals were then euthanized with ether. The livers were harvested, sectioned into small pieces, placed in embedding medium, quick-frozen in prechilled 2-methylbutan, and stored in-70°C in preparation for immunohistologic studies. Sections were also fixed in neutral-buffered formalin, embedded in paraffin, and sectioned for light microscopy.
Cryostat sections were fixed in acetone and stained by a three-layer peroxidase-antiperoxidase (PAP) method or by direct immunofluorescence. Briefly, sections were incubated overnight with primary antibodies at 4°C, followed by incubations at room temperature with bridging antibodies, PAP complexes, and the substrate diaminobenzidine. Sections were slightly counterstained with hematoxylin. For negative controls, the primary antibodies were replaced with normal serum. Tissue sections known to express the relevant antigens served as positive controls. Primary anti-rat antibodies were directed against insulin, CD3 cells (Dako, Carpinteria, CA), leukocyte common antigen (OX-1), monocytes and macrophages (ED-1), CD4 T cells/macrophages (W3/25), CD8 T cells (OX-8) (Serotec, Oxford, England), and polymorphonuclear neutrophils (Inter-Cell Technologies, Hopewell, NJ). Developing reagents consisted of rabbit anti-mouse and sheep anti-rabbit antibodies with the appropriate PAP complex. Fluorescein isothiocyanate-conjugated, affinity-isolated goat antibodies were specific for rat IgM and IgG (Jackson ImmunoResearch Labs, Inc., West Grove, PA) and C3 (Organon Teknika/Cappel, Durham, NC).
Histologic analysis was performed in rats from groups I, II, VI, and VII. Semiquantitative assessment of cell infiltrates was carried out in nondiabetic, nonimmunosuppressed animals on formalin-fixed, hematoxylin and eosin (H&E)-stained liver sections. In each animal the number of inflammatory foci per 20 medium power fields was counted. For both groups at 6 hr after transplant, 10 islets per animal were assessed semiquantitatively in each of three animals per group for both peri-insular as well as intra-insular leukocyte infiltrates according to the following scale: 0(absent), 1 (a few cells), 2 (moderate infiltration), 3 (severe infiltration). At 48 hr after transplant, the largest cross-diameter and its perpendicular of 10 inflammatory infiltrates per animal in each of three animals per group were measured in centimeters on a television screen. Immunostained sections from animals of each group were evaluated semiquantitatively for the presence of various leukocytes.
Statistics. A one-tailed t test was used for statistical analysis of unpaired data, and a log-rank analysis was used to compare actuarial graft survival in experimental groups. The Fisher exact test was used to compare survival rates at posttransplant day 4 in groups VI and VII. Comparison of the size of the inflammatory foci was performed using a two-way analysis of variance with repeated measures on one factor.
Posttransplant islet cell function. We initially investigated whether the absence of C6 in nonimmunosuppressed rats decreased the incidence of primary nonfunction in a canine islet xenograft model. Results showed that all rats in groups I and II (normal complement levels and C6D, respectively) experienced primary nonfunction (Table 1), as evidenced by hyperglycemia and intact xenoislet insulin staining at 6 hr (see below). We then modified our protocol by adding immunosuppression and varying the amount of islet equivalents transplanted. Our initial results with a two- or three-drug immunosuppressive regimen excluding CsA met with limited success(Table 1). The incidence of primary nonfunction was 100% in group III, which received MMF and DSG and was transplanted with 5,000 islet equivalents; 50% in group IV, which received these two drugs plus RAPA and 10,000 islet equivalents; and 82% in group V, which received this same three-drug protocol and 20,000 islet equivalents. The mean functional islet survival times for groups III, IV, and V were 0, 1, and 0.27 days, respectively. Therefore, we added CsA to the regimen. With this four-drug protocol, the incidence of primary nonfunction was 33% in normocomplementemic (group VI) and 10% in C6D rats (group VII). As the C6D rats are unable to assemble the MAC of complement, these results suggested that the MAC may play a minor role in the mechanism of primary nonfunction.
It was of interest of compare the posttransplant functional survival in rats receiving the four-drug immunosuppressive regimen. Normocomplementemic rats (group VI) had a range of posttransplant survival from 0 to 4 days with a mean of 1.57±0.33 days. On the other hand, C6D rats (group VII) had a posttransplant survival from 0 to 5 days with a mean of 2.70±0.67 days (P=0.038, Table 1). Not only was the average length of posttransplant islet cell function longer in the C6D rats, but the percentage of recipients in group VII with posttransplant islet function was larger than in group VI on all posttransplant days(Fig. 1), reaching significance on day 4(P<0.05). On day 4, 50% of C6D animals demonstrated functional islets, while only 14% of normocomplementemic animals retained islet function. On day 5, there was not a single normocomplementemic recipient that had functioning canine islet cells, while 20% of C6D recipients demonstrated adequate islet cell function to maintain serum glucose levels under 200 mg/dl. These results suggest that rats that are unable to form the MAC maintain canine islet xenografts somewhat longer than control rats.
Immunohistochemistry and histology. To ascertain whether the MAC plays a role in the response to islet xenografts, we compared canine islets in nonimmunosuppressed C6D and normocomplementemic rats at 6 and 48 hr after transplantation. Islets were found embolized in small branches of the portal vein, partially surrounded by fibrin thrombi. At 6 hr after transplant, islets discernible on H&E sections were composed of cells with typical fine eosinophilic granulation of the cytoplasm(Fig. 2, A and B). The full spectrum from intact islets without inflammatory cells to small, dense, heterogeneous infiltrates without islet cells was seen in both groups. Leukocytes were found mainly in the direct vicinity of the islets, but also within individual grafts. At 48 hr after transplant, endocrine cells were very difficult to recognize on H&E sections, secondary to ongoing massive cellular rejection (Fig. 2, C and D). Infiltrates increased in size considerably between 6 and 48 hr after transplant. The few remaining islet cells within infiltrates occasionally showed an acidophilic cytoplasm and a condensed nucleus.
No difference was found regarding the number, intensity, and size of infiltrates in normocomplementemic and C6D rats. At 6 hr after transplant, the score (mean±SEM) for cell infiltration was 2.1±0.2 for the normocomplementemic rats and 2.0±0.2 for the C6D rats. Cell infiltration scores at 48 hr were 3.0 in all specimens. Size of inflammatory foci could not be measured at 6 hr because the cell infiltrates were not well delineated. At 48 hr after transplant, the largest diameter of the inflammatory foci and its perpendicular (mean±SEM) were 7.4±0.7 by 4.1±0.4 for the normocomplementemic rats and 7.2±1.0 by 3.4±0.3 for the C6D rats. The differences were not statistically significant.
Immunohistologically, islet from nonimmunosuppressed animals stained positively for insulin at 6 and 48 hr after transplant. At 6 hr after transplant, there was no difference between normocomplementemic and C6D rats with regard to discernible islet number or size of intact islets(Fig. 2, E and F). At 48 hr, insulin positivity was reduced in both groups to very few foci of a few cells within dense inflammatory infiltrates (Fig. 2, G and H). At 6 hr after transplant, the majority of inflammatory cells were neutrophils, but a small number of macrophages, as well as a few T cells, were found in the infiltrates. At 48 hr after transplant, macrophages represented the vast majority of leukocytes. There was also a moderate number of neutrophils and T cells. There was no apparent difference in the composition of the inflammatory infiltrates between normocomplementemic and C6D rats.
In rats immunosuppressed with the four-drug protocol, insulin-positive cells were discerned at 6 and 48 hr after transplant, but there was no staining at 96 hr. No differences were observed in the intensity and composition of the cell infiltrates in normocomplementemic and C6D rats. Most early infiltrating cells were neutrophils, but there were also macrophages and T cells. At 48 hr after transplant, there was a preponderance of macrophages with a moderate number neutrophils and T cells.
Deposition of IgM, IgG, and C3 was similar on islets and surrounding tissue from all groups.
In these studies, we investigated the potential role of the MAC of complement in the pathogenesis of primary nonfunction of canine islets transplanted into rats. We compared the function and histology of transplanted islets in rats with a normal complement system with those that were C6D and thus unable to assemble the MAC. It is known that islet xenografts transplanted into recipients other than mouse never function in the absence of extensive immunosuppression, a process designated primary nonfunction. Primary nonfunction occurs in pig-to-rat(17) and canine-to-rat models (6,18,19). Though certain mediators that are operative in primary nonfunction of islet allografts may be similar to those found in islet xenografts, there is no conclusive evidence for a single dominant mechanism. A number of mechanisms have been proposed to explain this phenomenon(6,19-24). Infiltration with inflammatory cells, especially macrophages, is considered a major mechanism (20,22,24), and deposition of IgG may mediate adhesion of polymorphonuclear leukocytes(6). Release of cytokines from infiltrating cells may induce nitric oxide production by β cells, which in turn could lead to islet cell dysfunction (6,21). Furthermore, a protective role for IL-10 and transforming growth factor-β has been proposed (23). Finally, complement participation mediated by immunoglobulin deposition or by direct complement activation may also play a role in primary nonfunction (6,19).
Complement-mediated hyperacute rejection is the first major immunological hurdle in immediately vascularized discordant xenografts(5). As others have hypothesized, however, complement may play a role in primary nonfunction of islet xenografts as well. Although human preformed antibodies may bind to porcine islet cells and induce complement-mediated cytotoxicity (25,26), the function of intact pig islets is maintained in the presence of human serum(27). However, the MAC in sublytic concentrations might promote the release of IL-8 and MCP-1 (11), as well as prostaglandins, leukotrienes, and oxygen radicals (10) and thus contribute to inflammation that may be part of the mechanism through which the islets suffer primary nonfunction. In addition, C1q is an inflammatory mediator (3,10), and C3a and C5a can act as strong chemoattractants and anaphylatoxins and might play a role in primary nonfunction.
The current study was undertaken to elucidate a potential participation of the MAC in primary nonfunction of canine islet xenografts in the rat. A slight but significant difference was noted in the incidence of primary nonfunction in heavily immunosuppressed normocomplementemic PVG rats compared with C6D rats: 33% and 10%, respectively. Furthermore, islet cell function was prolonged somewhat in C6D rats. However, the role of the MAC in primary nonfunction appears to be a minor one because islet xenograft function was also prolonged in all groups immunosuppressed with three or four drugs when compared with the nonimmunosuppressed groups. Moreover, there was no difference in the incidence of primary nonfunction or functional islet survival between normocomplementemic and C6D rats when the recipients were not immunosuppressed. All recipients in these groups failed to show any islet function, and the incidence of primary nonfunction was 100%. Although, histologically, there was no tissue destruction observed at 6 hr after transplant, the animals remained hyperglycemic, further supporting the phenomenon of primary nonfunction. We carefully compared the intensity and composition of the cellular infiltrates in nonimmunosuppressed normocomplementemic and C6D rats and found no differences. These observations suggest that the MAC may play only a minor role in increasing the incidence of primary nonfunction and decreasing functional islet survival time, which becomes evident only with the addition of heavy immunosuppression.
It is clear that the four-drug regimen the rats received played a major role in supporting posttransplant islet cell function. Each additional drug decreased primary nonfunction and increased functional survival time. The results were better with the four-drug regimen that included CsA than with the two- or three-drug protocols. The situation is analogous to that in islet allograft, where the incidence of primary nonfunction fell from 37% to 7% with the addition of CsA (2). In our model, the need for a quadruple-drug regimen makes it difficult to draw conclusions regarding the mechanism of primary nonfunction. The drugs used act on multiple effector mechanisms that may potentially participate in primary nonfunction as they inhibit lymphocyte DNA synthesis, cytokine production including IL-1β and IL-2, as well as macrophage activity and leukocyte adherence(21,28-30).
In conclusion, our studies indicate that the absence of the MAC does not preclude the uniform development of primary nonfunction of canine islets transplanted into nonimmunosuppressed rats. Thus these studies exclude the MAC as a major pathophysiologic mediator of primary nonfunction. However, our results suggest that, in heavily immunosuppressed rats, an intact MAC may be one of several minor factors that impair complete abrogation of primary nonfunction in this model.
Acknowledgements. The authors acknowledge the excellent technical assistance of Ann Gaare, Tom Gilmore, and Jan Shivers in the development of this work, as well the assistance of Dr. Angelika Gruessner in the statistical analysis.
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