TRANSPLANTATION OF ALGINATE MICROCAPSULES: Generation of... : Transplantation (original) (raw)
Transplantation of microencapsulated islets of Langerhans has been proposed as a treatment of type 1 diabetes, which ideally should make immune suppression therapy unnecessary. The scarcity of human islet tissue for implantation, however, poses a limitation to this approach. Thus, it is important to develop immunoisolation systems for xenotransplantation of islets of Langerhans. One device, which could reverse diabetes in at least allograft transplantations of pancreatic islets, is the alginate poly-L-lysine (PLL*) capsule, consisting of a core of calcium alginate gel, a PLL membrane, and an outer coating of alginate (1). The PLL/alginate membrane is permeable to low molecular weight substances such as oxygen, insulin, and interleukin (IL-1), but the membrane is supposed to be impermeable to immune cells, immunoglobulins, and tumor necrosis factor (TNF) (2). Although the capsules can protect islet cells from cell-mediated destruction, antigens can be released from the capsules, which may subsequently activate host immune cells and lead to pathological reactions in the recipient.
We have previously demonstrated that alginates with a high content of mannuronic acid (high M) evoke an inflammatory response by stimulating monocytes to produce proinflammatory cytokines, such as TNF, IL-1, and IL-6 (3). Alginates with a high content of guluronic acid (high G), however, do not stimulate monocytes to produce proinflammatory cytokines (4). Despite the fact that high G-alginates stimulate cytokine production to a very low extent (4), little is known about their antigenicity. Thus, the generation of antibodies against alginate, which bind to the capsule surface, may result in a localized immune response against the capsules. Likewise, antibody production may occur if islet antigens are released from the encapsulated islets. We therefore considered it important to study both the antigenicity of empty alginate capsules and also responses to encapsulated porcine islet-like cell clusters (ICC) transplanted to mice and rats.
MATERIALS AND METHODS
Alginates. Alginates from three sources were used in this study. A highly purified alginate with a high content of guluronic acid (high G-alginate) was obtained from Pronova Biopolymer (Drammen, Norway). The polymer was extracted from stipes of Laminaria hyperborea and had a guluronic acid content, FG, of 68% and an average number of guluronic residues in the G-blocks (NG>1) of 14 as determined by 1H-NMR-spectroscopy (5). The alginate had a molecular weight of 250,000, which was estimated from intrinsic viscosity measurements using the Mark-Howink-Sacurada equation (6). The second type of deacetylated alginate (Poly-M) highly enriched in mannuronic acid (96% mannuronic acid and molecular weight >300,000) was isolated from Pseudomonas aeruginosa grown on agar plates at low temperatures as described previously (7). The third alginate type was made from Macrocystic pyrifera (high M-alginate) and had a mannuronic acid content of 68% and a molecular weight of 210,000. The material was purified by a repeated combination of alkali treatment (0.2 M NaOH) at 45°C, precipitation with ethanol, and extraction of the precipitate by ethanol and chloroform. The material was finally dissolved in pyrogen-free water, filtered through a 0.22-mm membrane filter (Millipore, Bedford, MA), and lyophilized. Lipopolysaccharide contamination in the alginates was measured by the LAL assay (KABI Vitrum, Chromogenix, Mölndal, Sweden). The level of endotoxin was <10 ng/mg in poly-M, <5 ng/mg in the high M-alginate, and <5 ng/mg in the high G-alginate material. The alginates (poly-M, high G-alginate, and high M-alginate) were tested for protein contamination by using the Bio-Rad Protein Assay (Bio-Rad Laboratories GmbH, München, Germany), but no proteins were detected in either the poly-M, the high M-alginate, or the high G-alginate, indicating a protein concentration of less than 0.1% in the alginate solutions.
Preparation and injection of empty alginate capsules, encapsulated ICC, and bovine serum albumin (BSA)-Dynabeads. For antibody studies against alginates, female Balb/c mice (20 g) and Wistar rats (200 g) (Mogbals, Ry, Denmark) were used as recipients. Different types of capsules (see below) were injected twice intraperitoneally (i.p.) with an interval of 2 months between each injection. Each group of mice or rats consisted of five to six animals. Two types of empty capsules were made: one type had high M-alginate as a core and outer coating (high M-alginate-PLL-high M-alginate), and a second type had high G-alginate as core and coating (high G-alginate-PLL-high G-alginate). Mice and rats were injected with 1 ml and 5 ml of alginate capsules, respectively. Control animals received the same volumes of 0.9% NaCl. Serum samples were harvested by vein puncture before the start of the experiments (preimmune serum) and 2 weeks after the second injection of alginate capsules.
For antibody studies against ICC antigens, Wistar rats were used as recipients. ICC were obtained from fetuses of pregnant sows (Department of Medical Cell Biology, Uppsala, Sweden). The procedure for isolation and culture of fetal porcine ICC has been described in detail elsewhere (8). ICC were suspended at a concentration of 2000 ICC/ml in a sterile filtered 1.8% high G-alginate solution. Inhomogeneous alginate beads were made by letting droplets of the ICC/alginate solution fall into an aqueous solution containing Ca++ ions (0.8% CaCl2·2H2O) in the presence of mannitol as osmolythe. The size of the alginate beads was controlled by a coaxial air stream, and the average diameter was 0.7±0.05 mm (9). The alginate beads were collected from the Ca++ solution and washed three times in 0.9% NaCl. The alginate PLL membrane was added by suspending the alginate beads in a solution of 0.1% PLL with molecular weight of 21 kDa (Sigma Chemical Co., St. Louis, MO) in 0.9% NaCl for 10 min. Finally, the alginate capsules were coated with 0.1% high G-alginate. The duration of the alginate coating step was 10 min. Each capsule contained one to two ICC. All capsules with or without ICC and also free-floating ICC were hand-picked to transplant approximately the same cell mass to each animal. Only ICC with intact morphology and without signs of necrosis were used. Free or encapsulated ICC were washed three times in 0.9% NaCl before being injected into the peritoneal cavity of rats. Some rats received an i.p. injection of macrocapsules (diameter 4-5 mm) that was made by suspending encapsulated ICC (as described above) in a suspension of 1.8% high G-alginate solution. Droplets of this mixture were gelled in a 0.8% CaCl2 solution in the presence of mannitol as osmolythe.
In some experiments BSA, either in a solution of 0.9% NaCl or bound to Dynabeads, was used as antigen. Dynabeads with reactive epoxy groups (Dynal, Oslo, Norway) were coated with BSA (Sigma) by incubating 150 mg of Dynabeads with 300 μg of BSA in phosphate-buffered saline (PBS) buffer for 48 hr at 20°C. To prevent the release of unbound BSA, the particles were washed extensively in PBS five times during a period of 48 hr. This procedure yielded Dynabeads with 2.6 μg of BSA/mg. The BSA and BSA-Dynabeads were injected i.p. into rats (BSA=37.3 μg/animal) either free or encapsulated in alginate. To study whether G-alginate could act as an adjuvant, rats were injected i.p. with BSA (37.3 μg/animal) mixed with high G-alginate gel fragments (1 ml, slurry of gelled alginate), soluble high G-alginate (1 ml), or added together with empty high G-alginate capsules (1 ml). Injections of BSA mixed with 0.3 ml of complete Freund's adjuvant (Sigma) served as a positive control. The antibody response against BSA was measured in serum samples harvested via puncture of the great saphenous vein 2 weeks after the last injection.
Detection of antibodies in serum samples. Serum samples were tested for antibodies against alginates, PLL, or BSA by a method described by Jahr et al. (10). Briefly, immunoplates (Nunc, Roskilde, Denmark) were coated with 50 μl of alginate (100 μg/ml), PLL (100 μg/ml), or BSA (2 μg/ml) diluted in PBS and incubated overnight at 4°C. For detection of antibodies against ICC, immunowells were coated with 50 μl of sonicated ICC (100 ICC/200 μl PBS) diluted 1/100 in PBS. Antibodies bound to the immunowells coated with alginates were detected with peroxidase-labeled antibodies against rat or mouse immunoglobulins (Zymed Laboratories, San Francisco, CA). Peroxidase-labeled antibodies were detected with o-phenylenediamine as a substrate (Dakopatts, Glostrup, Denmark). Antibodies bound to immunowells coated with BSA or ICC were detected by using biotinylated monoclonal antibodies against rat immunoglobulins (Zymed). Bound biotinylated antibodies were detected with a streptavidin peroxidase complex (DAKO). The absorbance at 490 nm was measured with a microplate reader (Bio-Rad Laboratories). The results have been presented either as absorbance (OD 490) or as ELISA units. In the latter case, 1 unit is the dilution of serum where the absorbance is 50% of the maximum absorbance.
Detection and quantification of rat IgE. The ELISA was performed according to the procedure provided by the manufacturer (LO-IMEX, Experimental Immunology Unit, University of Louvin, Brussels, Belgium).
In vitro immunization and sheep red blood cell (SRBC) plaque-forming assay. Generation of an anti-SRBC primary antibody response in vitro was performed according to the method of Faxvaag et al.(11). Briefly, freshly prepared spleen lymphoid cells from female NMRI mice (Bomholt Gaard, Denmark) (0.5×106 cells/100 μl medium/well), peritoneal feeder cells (2.5×103/50 μl medium/well), and SRBC (1×106/30 μl medium/well) were used. These cells were cocultured with poly-M, high G-alginate, or high M-alginate in different concentrations in 96-well culture plates (Costar, Cambridge, MA) for 5 days at 37°C in a gas phase consisting of 5% CO2 in humidified air. The cells were washed three times and resuspended in 200 μl of medium. The number of B cells producing anti-SRBC IgM antibodies per culture was estimated by the Cunningham Technique (12). Briefly, 50 μl of the lymphocyte suspension, 25 μl of guinea pig complement, 50 μl of 25% SRBC, and culture medium to 210 μl were mixed, administered in standard Cunningham slides, and incubated for 2 hr at 37°C in a gas phase consisting of 5% CO2 in humidified air. Plaque-forming cells were counted with an inverted microscope at low magnification.
Histology and immunocytochemistry. Implants and tissue from lungs, pancreas, kidneys, and intestine with peritoneal cells were fixed in 10% formalin, dehydrated in ethanol, and embedded in paraffin. Four-micrometer thick sections were prepared and stained with hematoxylin, erythrocin, and saffron. Kidneys were frozen in liquid nitrogen and prepared for freeze sectioning. Four-micrometer thick sections were placed on PLL (Sigma)-coated glass slides, air dried, and fixed in acetone for 10 min at room temperature. The sections were thereafter stained with fluorescein isothiocyanate-labeled antibodies (goat anti-rat IgG H+ L chain and goat anti-rat IgM (Biodesign International, Kennebunk, ME).
RESULTS
Antibody responses against alginates in mice and rats. In the first set of experiments, mice were injected with high M-alginate (64% M) alginate or high G-alginate (68% G) capsules i.p. Mice given high M capsules developed antibodies against the capsule material(Fig. 1A). However, mice receiving high G capsules did not develop antibodies against the capsule material (Fig. 1B). We also tested sera from mice injected with high M-alginate capsules for antibody activity against high G-alginate and mice injected with high G-alginate capsules for antibody activity against high M-alginate, but no antibody activities were detected in any of these groups (data not shown). When Wistar rats were used as recipients, no alginate antibody responses were detected in animals receiving high M or high G capsules (data not shown). These results indicate that antibody responses against alginate are dependent on both the alginate type and the recipient species. Mouse and rat serum samples were also examined for antibodies against PLL after injection of alginate-PLL-alginate capsules, however, no PLL antibodies were detected (data not shown).
(A) Antibodies against high M-alginate in sera from mice that received two injections of high M capsules. (B) Antibodies against high G-alginate in sera from mice that received two injections of high G capsules. Preimmune sera (□) were harvested before injection, and immune sera (
) were obtained 14 days after the second injection of capsules. ***P<0.001 versus preimmune sera, using Student's unpaired t test. Results are presented as mean ± SEM, n=5 mice.
Generation of antibodies against ICC. We then tested the ability of alginate microcapsules to protect against an antibody response against encapsulated xenogeneic tissue. Rats received 50 nonencapsulated or 50 encapsulated ICC in high G-alginate-PLL capsules. Antibodies against ICC antigens were detected after 6 weeks in sera from rats injected with either free or encapsulated ICC (Fig. 2). There was a further increase of the antibody titer 2 weeks after a second injection of free or encapsulated ICC (Fig. 2). However, 8 weeks after the second ICC injection, the amount of ICC antibodies had started to decrease.
Antibodies against ICC in sera from rats that received empty capsules (□), free ICC (), or encapsulated ICC (▪). Parallel groups of rats were treated with 15 mg/kg day of CsA (CyA). Control rats received the same volume of 0.9% NaCl (▦). Tx I, first transplantation; Tx II, second transplantation. Results are presented as mean ± SEM, n=5-6 rats.
Having observed that encapsulated ICC evoke a profound antibody response, we tested whether cyclosporine (CsA) could inhibit this response. When CsA at a dose of 15 mg/kg and day was injected subcutaneously, the humoral response against these xenoantigens in rats was completely abolished (Fig. 2).
In a separate series of experiments, the antibody production tended to be higher in rats receiving encapsulated ICC compared with rats injected with free ICC. This was probably not due to tolerance induction because a dose-related increase in the ICC antibody response was found in groups of rats receiving from 1 to 100 free ICC per rat (Table 1). Another possibility is that encapsulation of ICC may result in protruding islets or slow release of antigens, which continuously stimulate the antibody response leading to higher amounts of antibodies in serum. To test this hypothesis, 50 ICC were injected i.p. encapsulated in high G-alginate-PLL capsules or in macrocapsules. The ICC antibody response was highest in rats injected with ICC in macrocapsules (Fig. 3).
Generation of antibodies against nonencapsulated porcine ICC in rats
Antibodies against ICC in sera from rats that received 50 encapsulated ICC (□) or 50 encapsulated ICC in macrocapsules (▪). Control rats received the same volume of 0.9% NaCl (▦). The rats received two transplantations (see Materials and Methods), and sera were obtained 14 days after the second transplantation. Results are presented as mean ± SEM, n=5 rats.
Stability of microcapsules in vivo. When encapsulated islets are injected in vivo, the possibility exists that some of the capsules may break and release their content. If this occurs, an antibody response against encapsulated ICC is likely to occur. The mechanical stability of high G microcapsules in vivo was therefore studied by encapsulating BSA, which was immobilized on Dynabeads. The BSA-Dynabeads were injected i.p. in rats either as free beads or encapsulated in high G-alginate-PLL-alginate capsules. It was found that free BSA-Dynabeads stimulated the generation of BSA antibodies in rats (Fig. 4). However, by encapsulating the BSA-Dynabeads before injection, there was a complete prevention of the antibody response.
Antibodies against BSA in rats immunized with BSA, empty alginate capsules, BSA-Dynabeads, encapsulated BSA-Dynabeads, BSA-complete Freund's adjuvant, BSA-alginate gel fragments, BSA-alginate solution, and BSA-alginate capsules. Results are presented as mean ± SEM, n=5 rats.
Effects of alginates as adjuvants. Polysaccharides of different types may act as an adjuvant by increasing the immune response against antigens (3). Because it was found that rats developed high titers of ICC antibodies when they were injected with encapsulated ICC, the possibility exists that alginate may act as an adjuvant by increasing the ICC antibody response. BSA has previously been used to evaluate the adjuvant effect of silicone gels in rats (13). Thus, rats were injected with BSA mixed with a high G-alginate gel slurry, soluble high G-alginate, or added together with empty high G-alginate capsules. Injection of BSA mixed with complete Freund's adjuvant served as a positive control. There was no increase in the BSA antibody response in rats receiving BSA in combination with any of the different alginate combinations (Fig. 4).
In an additional experiment, it was tested whether alginates affected the generation of antigen-specific B cells. In vitro immunization with SRBC was performed in the absence and presence of different alginate types, and the number of SRBC-specific B cells was determined. The results from this experiment implicate that high G-alginate did not increase the number of SRBC-specific B cells compared with untreated controls. In contrast to this, a higher adjuvant activity was observed when poly-M or lipopolysaccharide was added (Fig. 5).
Formation of SRBC antibodies in vitro in the presence of 40 μg/ml Poly-M (▨), 8 μg/ml Poly-M (▦), 1.6 μg/ml Poly-M (▧), 1.6 μg/ml lipopolysaccharide (LPS) (▪), 40 μg/ml high M-alginate (▩), 40 μg/ml high G-alginate (▦), and control medium (□). The number of B cells producing SRBC IgM antibodies was quantified by a plaque-forming assay as described in Materials and Methods. Results are presented as mean of 4-6 replicated cultures. SD<15%.
In vivo findings. There were no symptoms or signs of serum sickness or any cutaneous, respiratory, or gastrointestinal manifestations that were characteristic of the disease of immediate-type hypersensitivity reactions in any of the mice and rats used in this study. Moreover, all mice and rats appeared healthy, and gained weight and behaved normally. Urine from all rats was analyzed for protein, blood, and cylinders. We found small traces of protein in all groups including controls, but no signs of blood and cylinders. When the abdominal cavities of rats were macroscopically investigated, we could not see any signs of inflammation in the surroundings of the injected capsules. In all mice and rats, some capsules were adhered to the abdominal wall and intestines, while other capsules were free floating. With regard to these in vivo findings, there were no obvious differences between the two types of capsules used.
Histological findings. Kidneys, pancreases, and intestines with peritoneal cells were examined at the end of the study (14-30 days after the second injection). There were no pathological changes in any of the groups studied. Moreover, there were no signs of peritoneal inflammation or vasculitis. When the tissues were examined after immunofluorescent processing using antiserum against IgM and IgG, there were no differences compared with the age-matched control animals.
DISCUSSION
Transplantation of insulin-producing allo- or xenografts that are immune protected by microencapsulation may be a future treatment of type 1 diabetes patients. It is therefore of importance to know whether the capsule material and the encapsulated graft are able to sensitize the host to antibody generation. Production of antibodies may result in unwanted immune responses against the capsule and the graft, and may cause immune complex disorders in the recipient.
There has been a discrepancy in the literature between the properties of high M-alginate and high G-alginate as capsule material. This is, in our opinion, basically due to the different ways of preparing capsules. In the traditional Lim and Sun technique, the calcium gel core is dissolved by sequestering the calcium ions after alginate polycation has been formed, leaving capsules with a liquid core surrounded by a thin polyanion polycation membrane (14). This type of capsule benefits from an alginate with a relatively low content of guluronic acid (high M-alginate), mainly because gels made from this material dissolve more rapidly, but also because there is a somewhat better binding between PLL and diequatorially linked mannuronic acid residues. The major obstacle with liquid core capsules is their mechanical weakness, because their integrity relies only on the outer membrane and a high internal osmotic pressure. More stable capsules can be formed by omitting the de-gelling step and thus keeping a calcium alginate gel surrounded by PLL. It is essential for this type of capsule to minimize the osmotic swelling of the gel core, which is obtained by using a material with a high content of G-blocks (high G-alginate) and consequently a strong cooperative binding of the cross-linking calcium ions. The difference in stability between high G solid core capsules and liquid core capsules has been addressed earlier (15). In essence, this is the background for our choice of G-rich materials for our experiments.
In this study, we found that empty high M-alginate capsules implanted into the peritoneal cavity of mice produced a detectable antibody response. However, this response was not seen when empty high G-alginate capsules were transplanted. Furthermore, alginate with more than 90% mannuronic acid increased the number of antibody-producing B cells. This was not detected for high G-alginate. Previous studies have shown that highly purified high M-alginate, but not high G-alginate, stimulates monocytes to produce TNF, IL-1, and IL-6 (3, 16) and that this response is due to a CD14-dependent process (3). Taken together these results show that high M-alginate has higher immune-stimulating activities than high G-alginate, both in terms of antibody and cytokine induction. Our data also indicate that empty high G-alginate-PLL capsules do not contain or leak enough antigenic alginate to stimulate antibody production when injected i.p. into mice and rats.
The present study also shows that xenotransplantation in rats of ICC either as free nonencapsulated grafts or after encapsulation in a PLL-alginate capsule evoked a humoral immune response. The highest antibody response was found in animals transplanted with encapsulated ICC, however, the antibody titers declined with time. Proteins released from encapsulated ICC may bind (nonspecifically) to the outer capsule surface and trigger complement activation and subsequently a local inflammation. Weber et al. (17) have reported that CTLA4-Ig is able to prolong survival of microencapsulated neonatal porcine islet xenografts in nonobese diabetic mice. CTLA4-Ig is a fusion protein, which inhibits T-helper cell activation and cellular immune responses in mice by binding to CD80 and CD86 on the antigen-presenting cells (18). By this intervention they were also able to prevent a pericapsular cellular response to capsules transplanted to nonobese diabetic mice (17). Moreover, humoral responses to xenografts have previously been reported in rats transplanted with porcine and canine islets in biohybrid diffusion chambers (19). Transplantation of encapsulated human islets to human recipients did not lead to humoral or cellular responses unless the patient was already sensitized (20). However, in the present study, microencapsulation of the ICC xenografts did not prevent the humoral and cellular responses in the recipients. Despite this, studies by other groups (21, 22) have demonstrated survival of xenogeneic transplanted microencapsulated islets for long periods in recipients without immunosuppression. The reasons for these discrepancies remain to be found.
We also demonstrate herein that the humoral response raised against the encapsulated ICC was prevented by treating the animals with a low dose of CsA from the day of transplantation. This conforms to data of previous studies indicating that CsA inhibits antibody production only if the treatment starts before the sensitization; but CsA treatment after sensitization has no effect (23). It is noteworthy that xenotransplantation of islets to rats using a combination of uncoated alginate beads and CsA did not produce any evidence of a humoral response at 103-176 days after transplantation, even though CsA treatment was discontinued in some of the recipients (24).
Immunization of animals may lead to the appearance of circulating immune complexes. The appearance and fate of immune complexes depend on the amount and nature of the antigens and antibodies involved. Species type, genetic dispositions, and physical status of the host are also involved (25). Immune complexes are eliminated by >99% by phagocytes, predominantly by the liver's Kupffer cells. Overload of this system may cause diseases provocated by precipitating immune complexes (26). We examined glomerular mesangium in the kidneys for immune complexes containing IgG and IgM, but all of these examinations were negative. This indicates that the amount of antigens and antibodies in these transplanted animals did not precipitate any disorder. However, it has to be taken into account that low numbers of ICC and capsules were transplanted in our experiments. These results are in accordance with experiences from experiments with biohybrid diffusion chambers, in which xenoantibodies against islets appeared, but no hypersensitivity or diseases caused by immune complexes were observed (19).
In summary, this study demonstrates that high G-alginate alone does not give rise to an antibody response. Also, high G-alginate does not act as an adjuvant by increasing the antibody response to other antigens. However, high G-alginate capsules do not prevent the release of xenoantigens from the capsules, because xenoantibodies are produced in rats implanted with encapsulated porcine ICC. This humoral immune response can be prevented by treatment with CsA, which may indicate that immunosuppression should be considered in the transplantation of encapsulated xenografts.
Acknowledgments. The authors thank Tove Mette Sætrum, Line Jensen, and Margareta Engkvist for expert technical assistance. We also thank Harald Aarset and his staff in the Department of Pathology, University Hospital of Trondheim, for histological examination of the kidneys.
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* Abbreviations: BSA, bovine serum albumin; CsA, cyclosporine; G, guluronic acid; high G, alginates with a high content of guluronic acid; high M, alginates with a high content of mannuronic acid; ICC, islet-like cell clusters; IL, interleukin; i.p., intraperitoneally; M, mannuronic acid; PBS, phosphate-buffered saline; PLL, poly-L-lysine; SRBC, sheep red blood cells; TNF, tumor necrosis factor.
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