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Decomplementation with cobra venom factor prolongs survival of xenografted islets in a rat to mouse model
OBERHOLZER, José, et al.
Abstract
Although the involvement of complement in hyperacute rejection of xenotransplants is well recognized, its role in rejection of devascularized xenografts, such as pancreatic islets, is not completely understood. In this study, we investigated whether complement participates in the immunopathology of xeno-islet transplantation in a concordant rat to mouse model. Rat pancreatic islets were implanted under the kidney capsule of normal and cobra venom factor (CVF)-decomplementized diabetic C57BL/6 mice. Graft survival was monitored by blood glucose levels. Deposition of IgM and C3 on grafted islets in vivo or on isolated islets in vitro (after incubation with normal and decomplementized mouse serum), as well as CD4- and CD8-positive leucocyte infiltration of grafts, was checked by immunohistochemistry. In addition, complement-mediated cytotoxicity on rat islet cells was evaluated by a 3-(4, 5-dimethythiazolyl)-2.5-diphenyl-2H-tetrazolium-bromide (MTT) assay. A significant C3 deposition was found on grafted islets from the first day after transplantation in vivo, as well as on isolated islets after incubation with mouse serum [...]
OBERHOLZER, José, et al . Decomplementation with cobra venom factor prolongs survival of xenografted islets in a rat to mouse model. Immunology , 1999, vol. 97, no. 1, p. 173-80
DOI : 10.1046/j.1365-2567.1999.00742.x PMID : 10447729
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Decomplementation with cobra venom factor prolongs survival of xenografted islets in a rat to mouse model
J. OBERHOLZER, D. YU, F. TRIPONEZ, N. CRETIN, E. ANDEREGGEN, G. MENTHA, D. WHITE,* L. BUEHLER, P. MOREL & J. LOU Division of Surgical Research, Department of Surgery, University Hospital of Geneva, Geneva, Switzerland,
and *Department of Surgery, University of Cambridge, Cambridge, UK
SUMMARY
Although the involvement of complement in hyperacute rejection of xenotransplants is well recognized, its role in rejection of devascularized xenografts, such as pancreatic islets, is not completely understood. In this study, we investigated whether complement participates in the immunopathology of xeno-islet transplantation in a concordant rat to mouse model. Rat pancreatic islets were implanted under the kidney capsule of normal and cobra venom factor (CVF )- decomplementized diabetic C57BL/6 mice. Graft survival was monitored by blood glucose levels.
Deposition of IgM and C3 on grafted isletsin vivoor on isolated isletsin vitro(after incubation with normal and decomplementized mouse serum), as well as CD4- and CD8-positive leucocyte infiltration of grafts, was checked by immunohistochemistry. In addition, complement-mediated cytotoxicity on rat islet cells was evaluated by a 3-(4,5-dimethythiazolyl )-2.5-diphenyl-2H- tetrazolium-bromide (MTT ) assay. A significant C3 deposition was found on grafted islets from the first day after transplantationin vivo, as well as on isolated islets after incubation with mouse serum in vitro. By MTT assay, complement-mediated cytotoxicity for islet cells was found.
Decomplementation by CVF decreased C3 deposition on either isolated or grafted islets, delayed CD4- and CD8-positive leucocyte infiltration, led to significant inhibition of complement-mediated cytotoxicity for islet cells, and prolonged graft survival (mean survival time 21·3 versus 8·5 days;
P<0·01). Our results indicate that decomplementation can prolong the survival time of devascu- larized xenografts across concordant species. The deposition of complement on transplanted islets may contribute to xenograft rejection by direct cytotoxicity and by promoting leucocyte infiltration.
INTRODUCTION that the humoral immune response plays a crucial role in HAR. Xenoreactive natural antibodies and complement pre- Allotransplantation of pancreatic islets is making the step
existing in recipients induce donor endothelial cell activation from an experimental procedure to an accepted alternative to
and damage, which recruit other effector mechanisms, leading pancreas transplantation. While recent results are encourag-
ultimately to graft destruction.4 However, in devascularized ing,1,2 it is evident that this therapeutic approach will be
xenografts, such as pancreatic islets, HAR does not occur and limited by organ shortage. Therefore, xeno-islet transplan-
the term concordant and discordant can not be applied with tation ( XIT ) has been considered as a possible solution.3
the same meaning. Although the T-cell response has been In discordant species combinations, whole organ xenografts
recognized as playing a major role in xeno-islet rejection,5 trigger a hyperacute rejection ( HAR). It has been recognized
XIT in B-cell-deficient mice exhibits a significantly prolonged survival time, suggesting that some humoral factors may also Received 24 September 1998; revised 10 December 1998; accepted
participate in the rejection.6 15 December 1998.
In previous studies, a significant deposition of IgM and Abbreviations: BSA, bovine serum albumin; CVF, cobra venom C3 was found on grafted islets in a rat to mouse model, while factor; DMEM, Dulbecco’s modified Eagle’s medium; FITC, fluor-
this deposition was not seen in islet allotransplantation.7 escein isothiocyanate; HAR, hyperacute rejection; HB-MVEC, human
Although a direct complement-mediated cytotoxicity on xeno- brain microvascular endothelial cells; HBSS, Hanks’ buffered salt
islet cells has been shown in vitro,8 the effect of complement solution; MST, mean survival time; MTT, 3-(4,5-dimethythiazolyl )-
on grafted isletsin vivoremains to be investigated.
2.5-diphenyl-2H-tetrazolium-bromide (=thiazolyl blue); PBS, phos-
phate-buffered saline; XIT, xeno-islet transplantation. In order to evaluate the impact of complement on xeno- islet rejection in a concordant model (as related to organ Correspondence: Dr J. Oberholzer, Division of Surgical Research,
xenotransplantation), rat islets of Langerhans’ were trans- Department of Surgery, University Hospital of Geneva, Rue Micheli-
du-Crest 24, 1211 Geneva, Switzerland. planted into mice decomplementized by cobra venom factor
© 1999 Blackwell Science Ltd
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J. Oberholzer et al.
174
(CVF ) and the survival of grafted islets, as well as the relevant 2.5-diphenyl-2H-tetrazolium-bromide (MTT ) assay as described previously.12 Briefly, fresh normal or decomple- immunohistological changes, were investigated.
Our results indicate that effective decomplementation by mentized mouse serum was diluted in 100ml Dulbecco’s modi- fied Eagle’s medium (DMEM; Gibco BRL, Paisley, UK ) in a CVF significantly prolongs xeno-islet survival in a rat to
mouse model. Complement deposition may participate in the 96-well plate. Heat-inactivated fetal calf serum ( FCS; Gibco) was used as a negative control. HB-MVEC suspension in immunopathology of xeno-islet rejection by direct cytotoxicity
and by promoting T-cell infiltration. 100ml DMEM, containing 2mg/ml actinomycin D (Merck, Darmstadt, Germany) was added to each well (5±104 cells/well ). The final serum concentration was 25%. The cells MATERIALS AND METHODS
were incubated with serum at 37°for 20 hr and then 50ml of 0·5% MTT (Merck) was added to each well. After a 4-hr Animals
Sprague–Dawley male rats (400–500 g) and C57BL/6 male incubation, the plate was centrifuged at 500gfor 10 min and the supernatant discarded. The cells in each well were lysed mice (20–25 g, 6–8 weeks old ) were purchased from BRL
(Basel, Switzerland ) and bred in the animal facilities at the with 100ml/well isopropanol–HCl in a VARI shaker (Dynatech, Zurich, Switzerland ) for 5 min. The optical density University Hospital of Geneva. All animal studies were
approved by the local ethic commission. was measured at 570 nm with an enzyme-linked immuno- sorbent assay ( ELISA) reader. The percentage of cytotoxicity was calculated as follows:
Rat islet isolation
Rat islets were isolated using the intraductal collagenase
digestion technique as described previously.9 Briefly, anaes- % cytotoxicity=OD of control – OD of test OD of control thesia was induced by 5% isofluran (ForeneA; Abbott, Cham,
Switzerland ). After midline incision and exposure of the pancreas, 10 ml Hanks’ balanced saline solution (HBSS ) with
2 mg/ml collagenase type XI (Sigma, St Louis, MO) was Rat islet transplantation
injected into the pancreatic duct. After pancreatectomy, the Diabetes was induced by a single injection of streptozotocin pancreas was digested in a 37°water bath with gentle shaking i.p. (200 mg/kg; Sigma, St Louis, MO). Diabetes was defined for 19 min. The isolated islets were purified further on a Euro- as plasma glucose levels above 20 mmol/l on 3 consecutive Ficoll (Sigma) gradient centrifugation. The purified islets were days. Approximately 250–350 hand-picked, freshly isolated washed three times and resuspended in HBSS solution for islet rat islets were injected beneath the kidney capsule in mice of
transplantation. each group under 1·5% isofluran ( ForeneA) anaesthesia. The
function of grafted islets was evaluated by measurement of
Decomplementation non-fasting plasma glucose levels. Blood samples were taken
C57BL/6 male mice were divided into four groups (control, on the tail vein and glucose was measured in the plasma by CVF1, CVF2 and CVF3). In the CVF1 group, 500mg/kg an automatic analyser (Cobas Mira; Roche Diagnostic, Basel, CVF (Imutran, Cambridge, UK ) was injected intraperitoneally Switzerland ). Values above 11 mmol/l on 3 consecutive days (i.p.) into mice 24 hr prior to islet transplantation. The mice were considered as rejection.
of the CVF2 group received 500mg/kg CVF i.p. 24 hr prior to
and 3 days after islet transplantation. For the CVF3 group a Preparation of rat islet cells
single injection of CVF (500mg/kg) was given to mice on day In order to investigate the complement-mediated cytotoxicity 4 after transplantation. The mice of the control group were for rat islet cells, purified rat islets were digested further using injected with the matching volumes of phosphate-buffered an EDTA-dispase technique as described previously.8Briefly,
saline (PBS ). purified rat islets were incubated in HBSS with 3m EDTA
In order to evaluate the efficiency of decomplementation for 5 min at room temperature. After centrifugation islets were by CVF, serum was collected daily and pooled from four non- incubated in dispase (9 mg/ml in HBSS; Dispase IIA; transplanted mice of the control group, CVF1 and CVF2. Boehringer Mannheim, Mannheim, Germany) for 15 min at The C3 levels in serum were measured by radial immuno- 37°with gentle shaking. The digestion was stopped with cold diffusion using a method as described previously.10Briefly, 1% RPMI (Gibco BRL) containing 10% FCS and the cell suspen- agar was stabilized at 45° in a water bath, and goat anti- sion was passed through a 100-mm mesh screen to discard the mouse C3 (29·5 mg/ml; courtesy of Dr I. Shozo, Geneva, aggregated cells and fibrin. Cells were washed three times with Switzerland ) added at 15150. Gel was poured on a glass plate RPMI, counted, and then resuspended in DMEM containing and circular wells were punched out in the gel. Approximately 10% FCS for the cytotoxicity assay.
7ml of diluted samples and standards, respectively, was added
to the wells. The plates were incubated at room temperature Immunohistochemistry
Kidneys were taken from control and CVF-treated mice on in a moist box for 48 hr, washed in 0·9% NaCl overnight, and
coloured with 1% tannic acid (Sigma, Steinheim, Germany).11 days 1, 4 and 7 after islet transplantation (n=3 for each group and day) and snap-frozen in Tissue Tek (Miles, Elkhart, Ring-shaped precipitates of samples were measured and com-
pared to a standard curve obtained by plotting the square of Indiana, USA). The samples were soaked in liquid nitrogen and then stored at −80° until immunohistological testing.
diameters of standards against dilutions.
The bioactivity of complement in the serum was analysed Tissue sections were incubated with PBS plus 0·5% bovine serum albumen (BSA; Sigma) for 15 min to block non-specific using a cytotoxicity test with human brain microvascular
endothelial cells (HB-MVEC ) by a 3-(4,5-dimethythiazolyl )- binding, and then stained with guinea-pig anti-porcine insulin
© 1999 Blackwell Science Ltd,Immunology,97, 173–180
(Dako, Zug, Switzerland ), goat anti-mouse IgM (Sigma, Buchs, Switzerland ), sheep anti-mouse C3 ( The Binding Site, Birmingham, UK ), rat anti-mouse CD4 and rat anti-mouse CD8 antibody (Serotec, Oxford, UK ), respectively. After a 30-min incubation, the sections were washed and further stained with corresponding fluorescein isothiocyanate ( FITC )- conjugated second antibodies for 45 min. The results were checked under a fluorescence microscope ( Zeiss, Iena, Germany) by three different examiners. Immunostaining was evaluated semi-quantitatively with a scoring system (0,±,+, ++). Each examiner calculated the mean value of his scoring for each treatment group from 3×2 slides per specific staining and corresponding day after transplantation (days 1, 4 and 7).
The freshly isolated and purified rat islets were fixed with
−20°methanol for 10 min, washed twice with PBS, and then Days after CVF administration
Control 1 3 6
% C3 levels
2 4 5 7
100 90 80 70 60 50 40 30 20 10 0 100
90 80 70 60 50 40 30 20 10 0
% cytotoxicity
% C3 levels
Complement-mediated cytotoxicity on HB-MVEC
incubated with PBS containing 0·1% BSA to block non-specific Figure 1. Efficiency of complement depletion by CVF. A single dose of CVF (500mg/kg i.p.) achieved a drop in C3 levels below 10% of binding. Islets were incubated with normal or decomple-
the control for 3 days (measured by radial immunodiffusion).
mentized mouse serum, respectively, for 1 hr at 4°. After three
Complement-mediated cytotoxicity, as measured by a MTT-assay on washings, islets were incubated with a goat anti-mouse IgM
HB-MVEC, was reduced to levels below 20% of the positive control or sheep anti-mouse C3 antibody for 1 hr. Islets were washed
for 4 days. After 1 week C3 levels regained near-normal values.
and further stained by a diluted rabbit anti-goat (Sigma) or
Results are expressed as mean values of triplicates±SD.
rabbit anti-sheep FITC-conjugated antibody (Sigma) for 30 min. The staining was checked under a fluorescence micro-
after the second CVF administration (the CVF2 group) or the scope. The experiment was repeated with three different islet
first CVF administration on day 4 (the CVF3 group) (data isolations.
not shown).
Cytotoxicity assay
Normal or CVF-decomplementized serum was serially diluted Prolonged graft survival in complement-depleted recipients with 100ml DMEM in a 96-well plate. The isolated islet cell
All diabetic mice became normoglycemic within 3 days after suspension with 2mg/ml actinomycin D was added to each
islet transplantation. Compared with control mice, the survival well (104 cells/well ). Rat islet cells were incubated with the
time of grafted islets was significantly prolonged in CVF1 and serum for 20 hr at 37°and then 50ml 0·5% MTT was added
CVF2 mice (19·7 and 21·3 days versus 8·5 days; eachP<0·01).
to each well. After a 4-hr incubation, the plate was centrifuged
However, no difference was found between the decomple- at 500gfor 10 min and the supernatant was discarded. The
mentized groups, CVF1 and CVF2, as shown in Fig. 2. In cells in each well were lysed with 100ml/well isopropanol–HCl
contrast, a single CVF administration at day 4 after islet in a VARI shaker for 5 min. The optical density was measured
transplantation (the CVF3 group) did not prolong the survival at 570 nm with an ELISA reader. The results are expressed as
a percentage of cell survival. The experiment was repeated with three different islet isolations.
RESULTS Evaluation of decomplementation by CVF
After the administration of a single dose of CVF (500mg/kg i.p.), the C3 concentration in the serum had decreased to below 10% of normal levels and persisted for 3 days, as shown in Fig. 1.
A second administration of CVF 4 days after the first could prolong decomplementation for another 3 days (data not shown). The purified CVF, administered at 500mg/kg i.p., did not have any obvious toxic effect on treated animals.
Normal mouse serum at a concentration of 25% induced Days after transplantation
0 5 15 30
% surviving grafts
10 20 25
100 90 80 70 60 50 40 30 20 10 0
Control CVF1 CVF2 CVF3
P < 0.01
71% cytotoxicity on HB-MVEC, compared with heat-
Figure 2. Effect of decomplementation by CVF on xeno-islet graft inactivated FCS. This value was used as a positive control (=
survival in a rat to mouse model. Plasma glucose values above 100% cytotoxicity), as shown in Fig. 1. With a single adminis-
11 mmol/l on 3 consecutive days were considered as a rejection. MST tration of CVF (500mg/kg i.p.) there was a drop in comple- of grafts in the control group (n=6) was 8·5 (7–11) days, while in ment-mediated cytotoxicity to below 20% of the positive prior to transplantation complement-depleted recipients the MST was control. This persisted for 4 days after CVF administration. prolonged significantly to 19·7 (10–26) days for the CVF1 group (n= In addition, the C3 levels were also checked and were 6) and 21·3 (18–30) days for the CVF2 group (n=6) (bothP<0·01).
below 10% of the controls in all grafted mice on the day of There was no significant difference between CVF1 and CVF2 groups or between control and CVF3 groups.
transplantation (the CVF1 and CVF2 groups) and the day
J. Oberholzer et al.
176
time of grafted islets, suggesting an involvement of complement infiltration. The results of immunohistology are summarized in Table 1.
at early, but not late, stages after XIT.
Immunohistological changes after decomplementation Complement and IgM binding on isolated xeno-islets
In order to confirm C3 deposition seen in vivo, isolated rat Immunohistochemistry was performed on kidneys taken on
days 1, 4 and 7 after islet transplantation. islets were incubated with normal or decomplementized mouse serum, and complement and IgM binding was investigated In the control group, IgM deposition was found on grafted
islets from day 1 and persisted weakly until day 7, while a furtherin vitro.
By immunofluorescence staining, IgM binding was found more marked C3 deposition was seen on grafted islets at days
1 and 4, and had decreased on day 7 after transplantation. In only on microvessel-like structures of rat islets ( Fig. 5a). In contrast, C3 binding was homogeneously distributed on the the CVF1 and CVF2 groups, C3 deposition was significantly
reduced ( Fig. 3a–d ) while the IgM deposition was not affected. islet surface (Fig. 5c), suggesting that C3 deposition on xeno- islets might be IgM independent. After incubation of rat islets Moreover, the difference of C3 deposition between control
and CVF1/CVF2 groups became less obvious at day 7 after with serum from CVF-treated mice, C3 binding on islets was significantly decreased while IgM deposition was unchanged transplantation, but the insulin staining showed a different
pattern. Insulin staining in the control group presented a ( Fig. 5b,d ).
scattered pattern, while that in the CVF groups was relatively intact ( Fig. 4a,b). There was no obvious difference in immuno-
Complement-mediated cytotoxicity of mouse serum on rat islet histology between the CVF1 and CVF2 groups, and between
cells the control and CVF3 groups.
Further immunohistological analysis of grafted islets Intact serum of untreated mice exerted a strong complement- mediated cytotoxicity on rat islet cells incubated at higher revealed a CD4- and CD8-positive leucocyte infiltration
beginning at day 4 and becoming more evident on day 7 after serum concentrations, as measured by the MTT assay. This cytotoxicity was serum-concentration dependent. In vivo transplantation in the control group (Fig. 4c,e) as well as in
the CVF3 group. In contrast, CD4- and CD8-positive leuco- decomplementation by CVF could abrogate completely the cytotoxicity of low concentrations of mouse serum (12·5%) cyte infiltration was absent in the CVF1 and CVF2 groups on
day 4 and very weak on day 7 after transplantation ( Fig. 4d,f ), against rat islet cells (97·7±6·8 SD versus 78·7±6·5 SD percentage islet cell survival; P<0·05). Using higher suggesting an involvement of complement in leucocyte
Control CVF
(a) (b)
(c) (d)
Figure 3. Effect of decomplementation on C3 deposition on grafted islets. Immunofluorescence staining on xeno-islets at day 1 after transplantation. Insulin and C3 staining in control (a, c) and CVF-treated recipients (b, d ) Original magnification×150.
© 1999 Blackwell Science Ltd,Immunology,97, 173–180
Control CVF
(a) (b)
(c) (d)
(e) (f)
Figure 4. Effect of decomplementation on leucocyte infiltration. Immunofluorescence staining on xeno-islets at day 7 after transplantation. Staining for insulin, CD4 and CD8 in control (a, c, e) and CVF1 and CVF2 recipients (b, d, f ). Original magnification×150.
Table 1. Summary of the immunohistology of xeno-islet transplantation
Day 1 Day 4 Day 7
Control CVF1+2 Control CVF1+2 Control CVF1+2 CVF3
Parameter (n=3) (n=6) (n=3) (n=6) (n=3) (n=6) (n=3)
IgM + + ± ± ± ± ±
C3 ++ – ++ – + ± ±
CD4 – – + – ++ ± ++
CD8 – – + – ++ ± ++
Symbols: –, negative;±, weak;+, positive;++, strongly positive.
J. Oberholzer et al.
178
Control CVF
(a) (b)
(c) (d)
Figure 5. Effect of decomplementation on IgM and C3 binding on isolated rat isletsin vitro.Immunofluorescence staining for IgM and C3 on freshly isolated rat islets after incubation with intact (a, c) or complement-depleted mouse serum (b, d ). Original magnification×400.
concentrations of mouse serum (50%), in vivo decomple- DISCUSSION mentation by CVF could strongly reduce, but not completely
It is known that activation of complement by xenoreactive block, cytotoxicity of mouse serum on rat islet cells (54·9±3·4
IgM is responsible for HAR of solid organs xenotransplanted SD versus 24·9±1·7 SD percentage islet cell survival;P<0·01).
across discordant species.4However, little is known about the As a control, Sprague–Dawley rat serum, even at higher
role of complement in devascularized tissue xenografts, such concentrations, did not have any cytotoxic effect on rat islet
as pancreatic islets, where HAR does not usually occur. In cells ( Fig. 6).
this study, we show that complement also participates in the rejection of xenografted islets and that decomplementation by CVF prolongs graft survival of xeno-islets in a concordant rat to mouse model.
In our previous and present studies, complement- deposition was found on concordant and discordant xeno- grafted pancreatic islets early after transplantation.7,13 Complement deposition was demonstrated further on isolated rat isletsin vitro, and immunohistological patterns suggested an IgM-independent pathway consistent with previous reports.8In order to evaluate the role of complement in xeno- islet rejection, rat islets were transplanted in complement- depleted mice. Decomplementation was performed by CVF, a constituent of cobra venom with extensive structural homology to mammalian C3. CVF activates complement and leads to
% serum
0 12.5 50
% islet cell survival
25 100
90 80 70 60 50 40 30 20 10 0
Mouse serum Complement- depleted mouse serum (CVF) Rat serum
complement consumption.14Decomplementation by CVF has Figure 6. Complement-mediated cytotoxicity on isolated rat islet cells. been used widely to investigate the role of complement in Isolated rat islet cells were incubated with different concentrations of
HAR.15 fresh serum and the cytotoxicity was measured by an MTT assay.
With a single injection of CVF (500mg/kg i.p.), a reduction Intact homologous rat serum was used as a negative control. Results
of 90% in C3 levels and of 80% in bioactivity was obtained are expressed as mean values of triplicates±SD. *P<0·05, **P<0·01
and persisted for 3–4 days. Immunohistology showed for statistical difference between normal and complement-depleted
mouse serum. that decomplementation with CVF significantly reduced
© 1999 Blackwell Science Ltd,Immunology,97, 173–180
complement deposition on grafted isletsin vivoand on isolated not shown). As no significant difference in survival time or immunohistological pattern was found between the CVF1 and islets in vitro. These results indicate that CVF is efficient for
decomplementation in mice. CVF2 groups, persisting decomplementation could not further delay or finally prevent leucocyte infiltration and rejection.
Complement depletion by CVF the day before transplan-
tation significantly prolonged graft survival, from a mean Due to the redundancy of mechanisms that promote chemo- attraction and activation of cellular responses, it is evident survival time (MST ) of 8·5 days to 20 days. A single dose of
CVF on day 4 did not achieve any prolongation of graft that complement is only one of many factors triggering leuco- cyte infiltration.
survival. Thus, complement seems to have an impact on early
but not late stages of xeno-islet-rejection. The results of the present study demonstrate that comple- ment is also involved in the immunopathology of rejection of By MTT assay, a cytotoxicity of mouse serum on rat
islet cells was demonstrated. This cytotoxicity was serum- non-primary vascularized xenografted tissue. Complement may participate in the rejection of xeno-islets by direct cyto- concentration dependent and complement mediated, as it was
reduced after CVF administration. These results conform with toxicity and by promoting CD4- and CD8-positive leucocyte infiltration. Efficient decomplementation by CVF significantly a previous report by Schaapherderet al. showing that comple-
ment across species is cytotoxic for isolated islet cells.8 prolongs the survival of xenotransplanted islets of Langerhans’
in a concordant rat to mouse model.
Although there was a significant cytotoxic effect of comple- ment on isolated islet cells in vitro, it was not seen on whole
islets eitherin vitro16orin vivo. In fact, in spite of a marked ACKNOWLEDGMENTS complement deposition on grafted islets early after transplan-
This work was supported by grant no. 32-50865.97 from the Swiss tation, insulin staining at days 1 and 4 did not show a different
National Science Foundation (to Ph. Morel J. Philippe and J. Lou).
pattern between control and CVF groups.
We thank Professor Shoso Izui and Professor Ju¨rg A. Schifferli for IgM was only found on microvessels within whole islets,
their valuable scientific suggestions. We also thank Tuzi Radelgruber, while a significant IgM binding was found on all isolated rat
Liliane Fossati, Corinne Sinigaglia, David Matthey-Doret, as well as islets cells after incubation with mouse serum by flow cyto- Raymond Mage for technical assistance.
metric analysis (data not shown). These results may provide an explanation for the difference in complement-mediated
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