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BOR - Papers in Press, published online ahead of print January 19, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.038315
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BIOLOGY OF REPRODUCTION 72, 1224–1231 (2005)
DOI: 10.1095/biolreprod.104.038315
© 2005 by the Society for the Study of Reproduction, Inc.

Neonatal Porcine Sertoli Cells Inhibit Human Natural Antibody-Mediated Lysis1

Jannette M. Dufour 3,4 , Meredith Hamilton 3, Ray V. Rajotte 3,4,5  , and Gregory S. Korbutt 2 3,4,5  

Surgical-Medical Research Institute,3 Department of Surgery,4 Department of Medicine,5 University of Alberta, Edmonton, Alberta, Canada T6J 6J5


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sertoli cells protect cotransplanted cells from allogeneic and xenogeneic rejection. Additionally, neonatal porcine Sertoli cells (NPSCs) survive long-term as xenografts in nonimmunosuppressed rodents. This has led to the hypothesis that NPSCs could be used to prevent cellular rejection in clinical transplantation, thereby eliminating the need for chronic immunosuppression. Prior to transplantation of NPSCs in humans it is necessary to determine whether they are also protected from humoral-mediated xenograft rejection. The presence of Gal{alpha}(1,3)Galß(1,4)GlcNAc-R ({alpha}Gal epitope) as well as binding of human immunoglobulin G (IgG) and IgM to NPSCs was examined by immunocytochemical and fluorescence-activated cell sorter analysis. {alpha}Gal was detected on 88.5% ± 3.0% of NPSCs. Consistent with this, 71.7% ± 1.0% and 65.4% ± 5.2% of NPSCs were bound by IgG and IgM, respectively. When cultured NPSCs underwent an in vitro cytotoxicity assay by incubation with human AB serum plus complement, no increase in cellular lysis was observed, while controls—porcine aorta endothelial cells—were shown to contain >60% dead cells. Finally, activation of the complement cascade was examined by immunohistochemistry. C3 and C4 were deposited on the surface of the NPSC membrane, indicating activation of complement. Although the complement cascade was activated, the membrane attack complex (MAC) was not formed. These data demonstrate that despite expression of {alpha}Gal, binding of xenoreactive antibodies, and the activation of complement, NPSCs survive human antibody and complement-mediated lysis by preventing MAC formation. This suggests that NPSCs may be able to survive humoral-mediated rejection in a clinical situation.

immunology, male reproductive tract, Sertoli cells, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue shortage is a major problem all transplant centers face due to the lack of human organ donors. Therefore, alternate sources of transplantable cells need to be developed. One potential source of tissue is xenogeneic porcine cells. However, before porcine cells can be used successfully, the immunological barriers associated with xenotransplantation, which include the humoral response and cell-mediated rejection, will have to be overcome [1]. Humoral-mediated xenograft rejection is initiated by the binding of preexisting xenoreactive antibodies to surface antigens on the transplanted cells, which leads to activation of the complement cascade and rapid destruction of the tissue [1]. The majority of the xenoreactive antibodies present in human serum are specific for the carbohydrate antigen Gal{alpha}(1,3)Galß(1,4)GlcNAc-R (Gal{alpha}1,3Gal, or {alpha}Gal) [2– 5], which is synthesized by {alpha}1,3-galactosyltransferase and present only in lower mammals, including pigs and New World monkeys [1]. {alpha}Gal is expressed on porcine endothelial cells [6] and has been detected in the testis [7, 8], with weak to no expression on Sertoli cells [7, 8].

Sertoli cells are partially responsible for the immunoprotective environment of the testis [9, 10] and they protect pancreatic islets from allograft [11, 12] and xenograft rejection [13, 14] as well as autoimmune destruction [15, 16]. Furthermore, bovine adrenal chromaffin cells are protected from immune-mediated rejection when cotransplanted with syngeneic Sertoli cells in rats [17]. We have shown previously that neonatal porcine Sertoli cells (NPSCs) are able to survive cell-mediated rejection when transplanted as xenografts in nonimmunosuppressed rodents [18, 19]. This has led to the hypothesis that NPSCs could be used to prevent rejection of islets or other cellular grafts, thereby eliminating the need for chronic immunosuppression. Prior to the use of Sertoli cells in a clinical setting, in addition to demonstrating their safety and efficacy in a large animal model, it is also important to determine whether they can survive humoral-mediated xenograft rejection.

In this study we examined the expression of {alpha}Gal on porcine Sertoli cells and the binding of preformed human xenoreactive antibodies to antigens present on the surface of Sertoli cells. Additionally, we used an in vitro model to study humoral-mediated rejection [20, 21] to assess whether Sertoli cells are susceptible to human antibody/complement-mediated lysis. Finally, components of the complement cascade were analyzed and Sertoli cells were found to survive by inhibiting the formation of the membrane attack complex (MAC).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Isolation

Male Landrace-Yorkshire neonatal pigs (aged 1 to 3 days) were used as Sertoli cell and aorta endothelial cell (i.e., experimental positive controls) donors. Animal experimentation was conducted according to the guidelines of the Canadian Council of Animal Care.

NPSCs were isolated by collagenase and trypsin digestion as described previously [18]. Sertoli cells were cultured in Ham F10 media (supplemented with 10 mM D-glucose, 2 mM L-glutamine, 50 µM isobutylmethylxanthine, 0.5% BSA, 10 mM nicotinamide, 100 U/ml penicillin, 100 µg/ml streptomycin) and 10% heat-inactivated neonatal porcine serum (NPS) for 24 h at 37°C before experimentation. Porcine aortic endothelial cells (PAECs) were isolated from abdominal aortas by cutting them longitudinally and gently rinsing the inner surface with Hanks balanced salt solution (HBSS) supplemented with 0.25% (w/v) BSA (Fraction V; Sigma Chemical Co., St. Louis, MO) to remove red blood cells. Aortas were placed in fresh HBSS and scraped to collect the endothelial cells, which were then transferred to a 50-ml conical tube, resuspended in HBSS, and centrifuged at 1200 x g for 10 min. Cells were washed twice with HBSS and then plated on 6-well tissue culture plates in Dulbecco modified Eagle medium (DMEM; supplemented with 25 U/ml penicillin and 25 µg/ml streptomycin) and 10% fetal calf serum (FCS) at 37°C. The next day, the cells were rinsed and then cultured in fresh media until they reached 80% confluency. At this time, cells were transferred to 100-mm tissue culture-treated plates and cultured in DMEM with 10% FCS. Experiments were performed using cells from passages two to six.

Detection of {alpha}Gal

Tissues analyzed included neonatal porcine testes (n = 5), adult porcine testis (n = 1), freshly isolated NPSCs cultured for 1 day in Ham F10 plus NPS to form cellular aggregates (n = 3), and NPSC grafts removed from SCID mice at 150 days post-transplant (n = 3) [22]. Whole tissues were immersed in Z-fix (Anatech Ltd., Battle Creek, MI), embedded in paraffin, sectioned, and after deparaffinization/rehydration, sections were incubated with 10% hydrogen peroxide, blocked with 20% normal goat serum, and incubated with biotinylated BS-1 isolectin B4 from Bandeiraea simplicifolia BS-1 with specificity for {alpha}-D-galactosyl groups [23, 24] (IB4, 1:25; Sigma) for 30 min. Sections were then incubated with the avidin-biotin complex (ABC)-enzyme complex with diaminobenzidine as chromagen (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin (Zymed Laboratories Inc., South San Francisco, CA). Additionally, NPSCs (n = 5) or PAECs (n = 5) cultured overnight with Ham F10 plus NPS on chamber slides were fixed with 1% paraformaldehyde for 30 min and stained for IB4 as described above. As a control for nonspecific binding, sections and cells were also stained as described above without IB4. To accurately determine the percentage of cells that are positive for {alpha}Gal, 1 x 106 nonfixed single NPSCs or PAECs that had been cultured for 24 h in Ham F10 plus NPS were incubated with fluorescein isothiocyanate (FITC)-conjugated BS-1 isolectin B4 (1:50; Sigma) for 1 h on ice, and analyzed by fluorescence-activated cell sorter (FACS).

Binding of Xenoreactive Immunoglobulin G and M Antibodies Present in Human Serum

To determine the binding of preformed xenoreactive antibodies to NPSCs or PAECs, 1 x 106 nonfixed single cells after culture for 24 h with Ham F10 plus NPS were incubated with heat-inactivated pooled human AB serum (1:16; Nabi BioPharmaceuticals Inc., Boca Raton, FL) for 1 h, followed by FITC-conjugated rabbit anti-human immunoglobulin G (IgG) or IgM (1:10; DAKO Diagnostics, Carpinteria, CA) for 30 min on ice. Negative controls consisted of cells that were incubated with FITC-labeled secondary antibodies alone. The percentage of cells bound by human antibodies was determined by FACS analysis and the Sertoli cell purity was determined on the basis of the proportion of vimentin-positive Sertoli cells using techniques previously described [18]. To assess Sertoli cell purity, a minimum of 500 single cells from each preparation were counted (Table 1). Additionally, NPSCs (n = 3) or PAECs (n = 3) were cultured overnight in Ham F10 plus NPS on chamber slides, incubated with 50% heat-inactivated human AB serum for 1 h, fixed with 1% paraformaldehyde for 30 min, followed by incubation with FITC-conjugated rabbit anti-human IgG (1:20) or IgM (1:20) for 30 min, and examined by fluorescence microscopy. As a control for nonspecific binding, cells were also incubated as above without human serum.


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  		TABLE 1. FACS analysis of {alpha}Gal expression and anti-human IgG and IgM binding.a

In Vitro Cytotoxicity Assay

The in vitro human antibody and complement-mediated cytotoxicity assay was performed similar to that described previously for neonatal porcine islets [21]. Briefly, 2 x 105 NPSCs or PAECs were plated in 24-well tissue culture treated plates and cultured overnight in 1 ml of Ham F10 media (supplemented as above) and NPS. The next morning, 0.5 ml of media was removed and cells were incubated at 37°C in one of four groups. Cells in group 1 (50% human serum plus complement) were incubated for 1 h with 0.5 ml of heat-inactivated pooled human AB serum and 0.5 ml of Ham F10 media (supplemented as above) plus NPS. For cells in group 1, 200 µl of media was then removed and replaced with 200 µl of rabbit complement from 3- to 4-wk-old rabbits (Pel-Freeze, Brown Deer, WI). Cells were incubated with complement for an additional 30 min. Cells in group 2 (media alone; i.e., no human serum or complement was added) were incubated for 1.5 h with 0.5 ml of fresh Ham F10 media (supplemented as above) and 0.5 ml of Ham F10 media (supplemented as above) plus NPS. Cells in group 3 (human serum alone; i.e., no complement was added) were incubated for 1.5 h with 0.5 ml of heat-inactivated pooled human AB serum and 0.5 ml of Ham F10 media (supplemented as above) plus NPS. Cells in group 4 (complement alone; i.e., no human serum was added) were incubated for 1 h with 0.5 ml of fresh Ham F10 media (supplemented as above) and 0.5 ml of Ham F10 media (supplemented as above) plus NPS. For cells in group 4, 200 µl of media was then removed and replaced with 200 µl of rabbit complement from 3- to 4-wk-old rabbits. Cells were incubated with complement for an additional 30 min. Rabbit complement from 3- to 4-wk-old rabbits was used because it has been demonstrated previously that it does not contain xenoreactive antibodies to porcine cells [21]. At the end of the cytotoxicity assay, media was removed and cell survival was analyzed using the LIVE/ DEAD Viablility/Cytotoxicity Assay (Molecular Probes, Eugene, OR) or MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (R&D Systems, Minneapolis, MN) as described in the protocol supplied by the manufacturer.

For the LIVE/DEAD assay, cells were washed with PBS and incubated for 45 min with 4 µM ethidium homodimer-1 and 2 µM calcein AM in PBS. Live cells were detected by green fluorescence due to the uptake of Calcein AM, while dead cells were detected by red fluorescence due to the uptake of ethidium homodimer-1. Identical fields were examined with an inverted microscope using a separate filter to detect green or red fluorescence, and cell survival was quantified by counting the number of green (live) and red (dead) cells. A minimum of 500 cells were counted in each group. For the MTT assay, cells were incubated with 200 µl of modified Eagle medium and 25 µl of MTT for 5 h at 37°C, followed by the addition of 200 µl of detergent to each well and further incubation overnight at 37°C. The absorbance of the supernatant was measured at 570 nm using a spectrophotometer with a reference wavelength of 650 nm. All assays were performed in triplicate with data expressed as mean absorbance. As a negative control for the MTT assay, a group of cells were lysed by incubating the cells for 1.5 h in 1% Triton X-100.

Detection of C4, Factor B, C3, and the Membrane Attack Complex on NPSCs and PAECs

NPSCs and PAECs (1 x 105 cells/well) were cultured overnight on chamber slides in 400 µl of Ham F10 media (supplemented as above) with NPS (n ≥ 3 per antibody). The next morning, cells were treated in one of four groups (human serum plus complement, media alone, human serum alone, or complement alone) as described above (In Vitro Cytotoxicity Assay) except the human serum was not heat-inactivated, and a total 0.4 ml instead of 1 ml of media was used. At the end of the cytotoxicity assay, cells were fixed with 1% paraformaldehyde for 30 min and immunostained for human C4, factor B, C3, or the MAC. Slides were incubated with 10% hydrogen peroxide, blocked with 20% normal goat serum or 20% normal rabbit serum, and incubated with goat anti-human C4 (1:4000; Calbiochem, San Diego, CA), goat anti-human factor B (1:2000; Calbiochem), goat anti-human C3 (1:5000; Calbiochem), or rabbit anti-human C5b-9 (MAC, 1:2000; Calbiochem) primary antibodies for 30 min, followed by the appropriate biotinylated secondary antibody (1:200; Vector Laboratories). Sections were then incubated with the ABC-enzyme complex with diaminobenzidine as chromagen and counterstained with hematoxylin.

Statistical Analysis

Data are expressed as means ± SEM of n independent experiments. Statistical significance of difference between multiple comparisons was calculated by one-way analysis of variance (ANOVA). A Scheffé F-test was used to determine specific differences between means when determined as significant by ANOVA. A P value of < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of {alpha}Gal on Sertoli Cells

The expression of {alpha}Gal on NPSCs was first examined in various testicular tissue sections. Consistent with previous reports [7, 8], Sertoli cells within intact neonatal porcine testis (Fig. 1A) and adult testis (Fig. 1D) were negative for {alpha}Gal. Similarly, tissue sections of Sertoli cell aggregates isolated from neonatal porcine testes (Fig. 1B) as well as Sertoli cell aggregates that had been matured by transplantation for 150 days under the kidney capsule of SCID mice (Fig. 1C) were also negative for {alpha}Gal. As previously shown, {alpha}Gal was present on vascular endothelial cells within the testis [8] (Fig. 1A) and in Sertoli cell grafts (Fig. 1C). Interstitial Leydig cells were also positive for {alpha}Gal (Fig. 1, A and D).



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  		FIG. 1. Immunohistochemistry for the detection of {alpha}Gal expression. Paraffin-embedded sections (A–D) and cells cultured as a monolayer on chamber slides and fixed for 30 min with 1% paraformaldehyde (E and F) were immunostained for IB4 lectin (brown). Samples included neonatal porcine testis (A), NPSCs isolated from neonatal porcine testis and cultured overnight to form cellular aggregates (B), NPSC aggregates that had been transplanted for 150 days underneath the kidney capsule of a SCID mouse (C), adult porcine testis (D), PAECs (E), and NPSCs (F). Sections were counterstained with hematoxylin (blue). Arrow, Sertoli cell; arrowhead, blood vessel. Bar in (D) = 50 µm (A–D). Bar in (F) = 100 µm (E and F)

In contrast, when NPSCs cultured on chamber slides and fixed for 30 min with paraformaldehyde (i.e., nonparaffin-embedded) were examined, the Sertoli cells were found to express {alpha}Gal (Fig. 1F), although the staining intensity was much lower than that observed on control PAECs (Fig. 1, compare E and F). All control sections and cells stained without IB4 were negative. This suggests the {alpha}Gal antigen is lost or masked on Sertoli cells during processing of the paraffin-embedded tissue or that the humoral milieu present in tissue alters the expression of {alpha}Gal on Sertoli cells. To further assess this expression, cells were also analyzed for {alpha}Gal expression by FACS analysis (Fig. 2), and 88.5% ± 3.0% of the NPSCs were {alpha}Gal positive (Table 1). The cellular composition of the Sertoli cell preparations used for FACS analysis consisted of approximately 87% Sertoli cells, suggesting nearly all of the Sertoli cells express {alpha}Gal. Even though a large percentage of the NPSCs were positive for {alpha}Gal by FACS analysis, the mean fluorescence intensity was significantly lower than on PAECs (Fig. 2, compare A and B).



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  		FIG. 2. Detection of {alpha}Gal on Sertoli cells and PAECs. FACS analysis of nonfixed PAECs (A) and NPSCs (B) stained with a FITC-conjugated IB4 lectin (1:50) for 1 h to detect {alpha}Gal (solid line). Negative controls were cells not stained with IB4 (dotted line)

Binding of Human Preformed Xenoreactive Antibodies to Sertoli Cells

The expression of {alpha}Gal on the cell surface of NPSCs suggests they may be recognized by antibodies present in human serum. Therefore, we examined the deposition of human IgG and IgM on Sertoli cells by immunohistochemistry (Fig. 3) and FACS analysis (Table 1). When NPSCs were incubated with 50% heat-inactivated human AB serum followed by anti-human IgG or IgM, the Sertoli cells were clearly positive (Fig. 3, F and H) and exhibited a staining intensity similar to that observed with PAECs (Fig. 3, compare F and H with B and D). In contrast, only background levels of fluorescence were detected when cells were incubated in the absence of human serum (Fig. 3, A, C, E, and G). By FACS analysis, 71.7% ± 1.0% and 65.4% ± 5.2% of the NPSCs reacted with anti-human IgG and IgM antibodies, respectively (Table 1). Because these preparations contained greater than 86% Sertoli cells, this suggests that the majority of the cells express xenoantigens reactive with human serum. Control PAECs were 91.9% ± 3.5% and 92.2% ± 0.7% positive for human IgG and IgM binding, respectively.



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  		FIG. 3. Binding of human xenoreactive antibodies to NPSCs and PAECs. PAECs (A–D) and NPSCs (E–H) were cultured on chamber slides in media alone (A, C, E, and G) or with 50% heat-inactivated human AB serum (B, D, F, and H). Cells were then examined for the presence of anti-human IgG (A, B, E, and F) or IgM (C, D, G, and H) binding. Bar in (H) = 100 µm (A–H)

Sertoli Cells Survive Human NaturalAntibody-Mediated Lysis

Because NPSCs were bound by human xenoreactive antibodies, we wanted to investigate whether they are susceptible to human natural antibody-mediated lysis. The ability of NPSCs and PAECs to survive human antibody/rabbit complement-mediated cellular lysis was assessed by the LIVE/DEAD assay (n = 4) after incubation with heat-inactivated human AB serum plus rabbit complement. When cells were cultured in media alone, 89.3% ± 1.8% of the NPSCs and 88.4% ± 3.1% of the PAECs were viable (i.e., negative for ethidium homodimer-1, a marker for cell death). After exposure to human serum plus complement the viability of the NPSCs did not change, with 87.8% ± 1.4% of the cells alive, whereas the proportion of PAECs lysed after exposure to human serum and complement increased, with only 37.8% ± 13.6% viable cells remaining.

To further verify these results, cell survival was also measured with the MTT assay (Fig. 4). NPSCs and PAECs were incubated with 50% heat-inactivated human AB serum for 1 h followed by 30 min with the addition of 20% rabbit complement. Controls included cells that were incubated with media alone, 50% heat-inactivated human AB serum alone, 20% rabbit complement alone, or Triton X-100 alone. After exposure to human serum plus complement there was a significant increase in NPSC survival (163.7% ± 4.7% survival; Fig. 4) compared to the media control. In contrast, there was a significant decrease in viable PAECs (29.7% ± 9.0% survival; Fig. 4) after incubation with human serum plus complement. There was also a significant increase in NPSC survival when they were incubated with human serum alone (171.0% ± 22.7% survival; Fig. 4), while there was no significant change in PAEC survival (136.5% ± 10.3% survival; Fig. 4) compared with those in media alone. The percentage of viable NPSCs and PAECs was also not statistically different after exposure to rabbit complement alone when compared to media alone (Fig. 4). Incubation with Triton X-100 led to greater than 95% cellular lysis (Fig. 4).



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  		FIG. 4. Susceptibility of NPSCs and PAECs to human natural antibody-mediated lysis as assessed by the MTT assay. NPSCs and PAECs were incubated with media alone, heat-inactivated human serum plus rabbit complement, heat-inactivated human serum alone, rabbit complement alone, or Triton X-100 alone as described in Materials and Methods. Negative control values (media alone) for NPSCs and PAECs were arbitrarily set equal to 100%, and the relative percentage of cell viability was calculated for each condition. The data are presented as the mean + SEM of four individual experiments in triplicate. Means bearing different superscript letters are significantly different at P < 0.05

Sertoli Cells Inhibit Formation of the MembraneAttack Complex

To understand a possible mechanism for NPSCs resistance to antibody/complement-mediated lysis it is important to examine the role of the complement cascade. We analyzed components of the complement system to first determine whether complement is activated, and if so, which step in the cascade is inhibited by NPSCs to allow their survival. To examine activation of the classical versus the alternative pathway, immunohistochemistry was performed for C4 (classical pathway) and factor B (alternative pathway). C4 was detected on the surface of NPSCs and PAECs after incubation with human serum and human serum plus rabbit complement (Fig. 5, A–D), indicating activation of the classical pathway. The majority of the NPSCs were negative for factor B, however, occasional patches of positive cells were observed (Fig. 5, G and H). The deposition of factor B on PAECs was variable with cells that ranged from negative to highly positive (Fig. 5, E and F).



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  		FIG. 5. Immunohistochemical analysis of C4, factor B (fB), C3, and MAC deposition on NPSCs and PAECs after exposure to human serum alone or human serum plus complement. PAECs (A, B, E, F, I, J, M, and N) and NPSCs (C, D, G, H, K, L, O, and P) cultured overnight on chamber slides in media with NPS were incubated for either 1.5 h with 50% human AB serum alone (A, C, E, G, I, K, M, and O) or for 1 h in 50% human AB serum followed by 30 min with the addition of 20% rabbit complement (B, D, F, H, J, L, N, and P). Following incubation the cells were fixed for 30 min in 1% paraformaldehyde and immunostained for C4 (A–D), fB (E–H), C3 (I–L) and MAC (M–P). Bar in (P) = 50 µm (A–P)

To identify which step in the complement cascade is inhibited by NPSCs, cells were examined for the presence of C3 and MAC, two factors activated downstream in the complement cascade. Consistent with previous reports that indicate PAECs are lysed by human serum and complement [25, 26], PAECs were positive for C3 and MAC (Fig. 5, I, J, M, and N). Of interest, NPSCs were positive for C3 (Fig. 5, K and L), but negative or very low for MAC (Fig. 5, O and P). This suggests that NPSCs survive complement activation by inhibiting formation of the MAC. There was no reactivity with any of the antibodies when cells were incubated with media alone or rabbit complement alone (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrate that NPSCs are resistant to human natural antibody-mediated cellular lysis in vitro, while PAECs are killed. Previously, we have shown that NPSCs are able to survive long-term as discordant xenografts in nonimmunosuppressed Lewis rats and BALB/c mice [18, 19]. Because rodents have preformed anti-porcine antibodies, these data taken together suggest NPSCs are able to survive humoral-mediated xenograft rejection both in vitro and in vivo, although it remains to be determined whether NPSCs can survive in humans.

Not only did NPSCs inhibit human antibody and complement-mediated cellular lysis, but the survival of NPSCs increased after exposure to 50% heat-inactivated human serum or 50% heat-inactivated human serum followed by rabbit complement. This increase is most likely due to the trophic effect of growth factors present in the serum or to up-regulation of growth factors produced by Sertoli cells (or both). Sertoli cells are known to produce many growth factors, including the insulin-like growth factor, transforming growth factors alpha and beta, basic fibroblast growth factor, neurotropin-3, and nerve growth factor [27, 28]. These growth factors can have both autocrine and paracrine effects in the developing testis [28], and therefore may function in our cultures to stimulate proliferation of Sertoli cells. It has also been shown previously that 10% calf serum can increase the growth of testicular cells isolated from newborn rats [28–30]. It is probable that factors present in human serum combined with factors secreted by the Sertoli cells act synergistically to increase the Sertoli cell survival.

Sertoli cells also exhibit remarkable immunoprotective properties. Not only can they protect themselves from cell-mediated rejection, when transplanted as allografts [11, 31] and xenografts [13, 18, 19, 32], but they are also able to prevent rejection of other cells. In the native testis, Sertoli cells are responsible for creating a suitable microenvironment to support the developing germ cells. This includes creation of the blood-testis barrier to physically separate the germ cells from immunological surveillance [33, 34] and production of immunoprotective factors [35]. This is necessary because the advanced germ cells express unique surface antigens that can elicit an immune response [36, 37]. Additionally, Sertoli cells cotransplanted with islets are able to protect islets from allogeneic rejection [11, 12] and autoimmune destruction [15, 16], and to prevent rejection of cotransplanted xenogeneic islets [13, 14] or neuronal cells [17]. The current study supports the concept that Sertoli cells are immunoprotective by demonstrating that they also survive natural human antibody-mediated cytolysis.

This study also demonstrated that NPSCs do not express {alpha}Gal when examined on paraffin-embedded sections, although they were positive for {alpha}Gal when analyzed by FACS or fixed with 1% paraformaldehyde on chamber slides. Two previous studies have examined {alpha}Gal expression on Sertoli cells [7, 8]. Dor et al. reported that Sertoli cells in paraffin-embedded testis sections from 1- and 22-mo-old pigs were negative for {alpha}Gal [8], while McKenzie et al. reported that Sertoli cells were weakly positive [7]. A similar contradiction has been observed previously in connective tissue [6], for which Oriol et al. reported that connective tissue was weakly positive for {alpha}Gal in frozen tissue sections but negative in paraffin-embedded tissue. This apparent discrepancy may be explained by tissue processing. The low level of {alpha}Gal expression observed on Sertoli cells may have been masked during processing of the paraffin-embedded tissue. Additionally, {alpha}Gal can be localized to glycoproteins and glycolipids [38]. {alpha}Gal expressed on lipids could be extracted by organic solvents during tissue processing and paraffin-embedding, and thereby would not be detected. For FACS analysis and staining on chamber slides, the cells were not exposed to these organic solvents and would therefore retain {alpha}Gal on glycolipids. This suggests that NPSCs may express {alpha}Gal on glycolipids, although further analysis is required for verification. The positive reaction for {alpha}Gal on the blood vessels of the testis and Sertoli cells grafts in paraffin-embedded sections can be explained because endothelial cells are known to express {alpha}Gal on a triad of glycoproteins [39], which would be stable during the processing of paraffin-embedded tissue. Another possible explanation for the variable {alpha}Gal expression is that the local environment present in the testis may repress {alpha}Gal on Sertoli cells. Sertoli cells in vivo are exposed to hormones and growth factors, and interact with other cells such as Leydig cells, germ cells, and peritubular myoid cells, which have all been demonstrated to regulate Sertoli cell products [40, 41]. The effect of these factors and interactions on {alpha}Gal expression requires further investigation.

Humoral-mediated xenograft rejection occurs when human serum antibodies bind xenoantigens located on the surface of target cells [1]. The resulting antigen-antibody complex activates an enzymatic cascade via the classical pathway leading to formation of the MAC and cellular lysis [1]. The alternative pathway also terminates in formation of the MAC and cellular lysis; however, it can be activated by several mechanisms including spontaneous activation and amplification after the classical pathway has been activated [42]. The ability of Sertoli cells to survive human antibody/ complement-mediated lysis could be either an active process due to the production of immunoprotective factors by Sertoli cells or a passive process in which Sertoli cells do not express xenoantigens, and thus are not recognized by xenoreactive antibodies. In this study, we observed that NPSCs express {alpha}Gal and are also bound by human IgG and IgM. Moreover, complement was activated via the classical pathway as indicated by the deposition of the complement proteins C4 and C3. Complement may have also been activated via the alternative pathway because patches of factor B were also detected. This would suggest that Sertoli cells are able to prevent cellular lysis associated with complement activation by producing one or more immunoprotective factors that may be able to inhibit formation of MAC because there was little to no deposition of the MAC on the surface of NPSCs. Consistent with this observation, Sertoli cells are also known to produce high levels of clusterin [43], which has been shown to inhibit formation of the MAC in vitro [44]. However, a more recent study suggests that the physiological concentration of clusterin in human serum is not high enough to protect cells from complement lysis [45]. It has been reported that the concentration of clusterin in seminal plasma is at least 10-fold higher than in blood [46, 47], and therefore, the local concentration of clusterin secreted by Sertoli cells may be high enough to provide their protection.

An alternative mechanism is that Sertoli cells survive complement-mediated cytolysis by producing a combination of complement inhibitors. In addition to clusterin, Sertoli cells have been shown to produce many immunoprotective factors [35] including mRNA for complement inhibitors such as the membrane cofactor protein (MCP) [48]. Additionally, CD59 and decay-accelerating factor (DAF) are expressed in the testis, although it is not known whether Sertoli cells produce these factors [49–51]. Moreover, spermatozoa are coated with the complement inhibitors clusterin, DAF, MCP, and CD59, all of which are hypothesized to be needed to protect sperm against complement lysis in the female reproductive tract [47, 52]. It is possible that one of the many functions of Sertoli cells in the testis is to secrete complement inhibitors, which coat the spermatozoa and protect them from complement lysis.

Previously, it was speculated that complement inhibitors are species specific, and therefore, porcine complement inhibitors expressed by porcine tissues may be unable to inhibit human complement cytolysis. However, more recent studies indicate that porcine MCP, CD59, and DAF are all effective in inhibiting human complement at least in vitro [49–51, 53, 54]. Our data support the ability of porcine cells to survive human complement, although the specific factors involved are unknown. Current studies are underway to identify the factors important for Sertoli cell survival.

In summary, NPSCs are not susceptible to human antibody and complement-mediated lysis. NPSCs survive despite expression of {alpha}Gal, binding of natural xenoreactive antibodies found in human serum, and activation of complement. It is likely that NPSCs inhibit complement-mediated cell lysis by producing one or more factors that inhibit formation of the MAC. Further study of the immunoprotective factors produced by Sertoli cells may identify novel proteins that are important for survival of transplant rejection.


   ACKNOWLEDGMENTS
 
We acknowledge the technical assistance of Alana Eshpeter, Crystal Harris, Dawne Colwell, and James Lyon. We also thank Dr. Kwan Hee Kim (Washington State University) and Karen Seeberger (University of Alberta) for critically reading the manuscript.


   FOOTNOTES
 
1 Supported in part by the Alberta Heritage Foundation for Medical Research (AHFMR), Canadian Institutes of Health Research (CIHR) grant FRN-8030, and the Juvenile Diabetes Research Foundation (JDRF). G.S.K. is a senior scholar of the AHFMR, and recipient of a Career Development Award from the JDRF. J.M.D. is a recipient of a Postdoctoral Fellowship from AHFMR and CIHR. Back

2 Correspondence: Gregory S. Korbutt, Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2N8. FAX: 780 492 1627; korbutt{at}ualberta.ca Back

Received: 29 November 2004.

First decision: 22 December 2004.

Accepted: 18 January 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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