Background

The pig is the donor animal of choice for human xenotransplantation. In the most relevant pig-to-baboon model, pig organs transplanted into baboons are hyperacutely rejected by natural xenoantibodies, which mainly bind to -galactosyl (Gal) epitopes expressed at the surface of endothelial cells. Recent advances in controlling hyperacute rejection have led to improved survival of these xenografts, and it is now important to identify Gal binding sites in other cells and tissues that may be subject to immunologic attack. To this end, we have studied whether Gal antibodies bind to glycated proteins of the extracellular matrix in the kidney and other organs most likely to be used for human xenotransplantation.

Methods

High-titer anti-Gal antibodies, similar to human natural xenoantibodies, were prepared in baboons, and their reactivity with components of pig extracellular matrix was tested by serology and immunohistology.

Results

The antibodies recognized epitopes of immobilized murine, bovine or porcine thyroglobulin, laminin, heparan sulfate proteoglycans, and fibronectin. In sections of pig tissue, the antibodies bound to endothelial and certain epithelial cells, as shown in previous studies, and also to mesenchymal cells, basement membranes, and extracellular matrices, in which they colocalized with matrix glycoproteins, especially laminin and heparan sulfate proteoglycans.

Conclusions

These results suggest that when pig xenografts can be made to survive for prolonged periods, the reactivity of Gal antibody with matrix molecules can induce basement membrane and matrix lesions similar to those induced in laboratory animals by antilaminin and antiheparan sulfate proteoglycans antibodies.

METHODS

Animals

Baboons (Papio anubis; 25 to 31 kg body wt) were obtained from the Biomedical Research Foundation (Houston, TX, USA). One two-day-old pig and 15 miniature pigs were purchased from Harlan Sprague-Dawley (Sinclair Research, Inc., Columbia, MO, USA). Tissues from four outbred adult pigs were obtained from a local abattoir. The protocol for animal studies was formally approved by the Columbia University Review Board.

Antigen preparation and immunization

Four baboons were immunized with 1 mg Gal conjugated to bovine serum albumin (BSA; 14-atom spacer; Dextra Laboratories, Ltd., Reading, UK) in equal volume of incomplete Freund's adjuvant (Sigma, St. Louis, MO, USA) at multiple subcutaneous and intradermal sites. After three weeks, a subcutaneous booster injection of 500 mg of Gal/BSA in incomplete Freund's adjuvant was given. After the first bleeding, the baboons received boost injections every four weeks and were bled every six weeks (<10% of blood volume). The same protocol was used to immunize a sheep with BSA. Preimmune baboon sera were used as comparison with the baboon immune sera, and Gal antibody-negative sera from normal rabbit and pigs were used as controls. The -globulin fractions were obtained by precipitation in 50% ammonium sulfate. IgG was isolated using ImmunoPure Immobilized protein A column (Pierce, Rockford, IL, USA). The flow-through, devoid of IgG, was used as preparation containing mainly IgM. The total protein concentration was measured by a standard Bradford protein assay (Bio-Rad, Hercules, CA, USA). The concentration of -globulins, IgG, and IgM was determined by radial immunodiffusion⁴. Gal antibodies were affinity purified from baboon anti-Gal serum on a 0.2 mL Gal-conjugated silica beads column (Synsorb 115; Chembiomed, Edmonton, Alberta, Canada), as previously described⁵. Titers of adsorbed and subsequently eluted antibodies and of the flow-through were determined by enzyme-linked immunosorbent assay (ELISA) on Gal/BSA, mouse laminin, and porcine endothelial cells. Silica beads conjugated with Gal1-4GlcNac-R epitopes were used as a specificity control. Other baboon anti-Gal aliquots were depleted of Gal reactivity by extensive absorption with Gal-rich rabbit erythrocytes⁶ and were also tested by ELISA.

Other antibodies, lectins, components of the extracellular matrix and chemicals

Antimouse laminin antibody was purchased from Sigma Chemical Co. and antimouse perlecan and antirat fibronectin antibodies from Chemicon Int. Inc. (Temecula, CA, USA). Antibodies to rat heparan sulfate proteoglycans⁷ and to podocallyxin⁸ were gifts from Dr. M.G. Farquhar and R.A. Orlando. All secondary, conjugated, and in a few instances, unconjugated affinity-purified antibodies and lectins were purchased from Sigma. Biocoat matrigel thin-layer plates were purchased from Becton Dickinson (Frankin Lakes, NJ, USA). Human plasma fibronectin, bovine plasma fibronectin, porcine thyroglobulin, mouse laminin from mouse Englebert-Holm-Swarm (EHS) tumor, laminin from human placenta, heparan sulfate proteoglycans from mouse EHS tumor, bovine chondroitin sulfate A, bovine hyaluronate, rat type I collagen, and chicken type II collagen were obtained from Sigma, and bovine type III collagen and chicken Tenascin were from Chemicon International, Inc. The 54 kd rabbit tubular basement membrane nephritogenic antigen, a gift from Drs. Butkowshi and Charonis, was prepare as previously described⁹.

Agarose antihuman IgM (-chain specific), biotin (long arm) NHS (Blanks) N-hydroxysuccinidyl-6-(biotinamide) hexanoate, BNHS, o-phenyledediamine dihydrochloride, 3-3'-dimethoxybenzidine, ovalbumin, pig albumin, BSA, protease inhibitors cocktail, and p-nitrophenylphosphate, heparinase III, -galactosidase, and -galactosidase were obtained from Sigma. Other reagents were Micro-Ouchterlony Kit (ICN, Costa Mesa, CA, USA) and nitrocellulose membranes, gels, and Tris-glycine (Bio-Rad).

Enzyme-linked immunosorbent assay

Gal reactivity on immobilized Gal/BSA or Gal epitope-rich pig thyroglobulin and mouse laminin and on single components of the extracellular matrix was studied with the method of Engvall and Perlman¹⁰. The reactivity of the baboon anti-Gal with murine extracellular matrix was determined using plates of murine basement membrane-like matrix of EHS tumor (Matrigel; Benton Dickinson) and that with cultured porcine endothelial cells with the method described by Platt et al¹¹. Controls were normal rabbit and pig serum (which do not contain Gal antibody), baboon anti-Gal flow-through of Gal immunoadsorption column, baboon anti-Gal absorbed with Gal/BSA or Gal-rich rabbit erythrocytes, and sheep anti-BSA.

Western blot analysis

Components of the extracellular matrix (5 mg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions on 4 to 15% gradient gels¹². The proteins were transferred to nitrocellulose membranes, incubated with 10 mg/mL of primary antibodies, followed by secondary peroxidase-conjugated antibody. Prior to electrophoresis, heparan sulfate proteoglycans was digested with heparinase.

Tissue preparations for immunohistochemistry

Fragments of lung, kidney, heart, liver, intestine, skin, skeletal muscle, brain, eye, pancreas, peritoneal serosa, and fat were obtained immediately after sacrifice. Fresh frozen sections were stained, or double-stained, with FITC- or TRITC-conjugated antibodies¹³. The sections were examined in a Nikon epifluorescence and a phase-contrast microscope or a Zeiss LSM 410 Laser scanning confocal microscope. Control experiments were performed with baboon anti-Gal absorbed with Gal/BSA or rabbit erythrocytes, sheep anti-BSA, substitution of the primary antibody with PBS, and digestion of tissue sections with - or -galactosidase. This last test was performed with a two-hour incubation with 1 unit of -galactosidase and 2 units of -galactosidase in 100 mmol/L NaCl and 50 mmol/L sodium acetate, pH 5, in a closed, wet chamber at 37°C. As control, buffer without enzyme was added. After digestion, the sections were washed and stained with FITC-conjugated baboon anti-Gal.

RESULTS

Serological characterization of baboon anti-Gal

Immunoreactivity was studied by ELISA on Gal/BSA; IgG titers were increased 64-fold, and IgM titers were increased two- to fourfold, as compared with naive baboon or preimmune baboon. Similar results were obtained with Gal-bearing proteins, such as pig thyroglobulin and mouse laminin. Sheep anti-BSA was not reactive Figure 1a. Immunoreactivity of baboon anti-Gal/BSA IgG on porcine endothelial cells was 20 to 30 times higher than preimmune baboon IgG (data not shown).

Figure 1.

Serological characterization of baboon anti-Gal. (A) Reactivity of baboon anti-Gal IgG and IgM by ELISA with Gal/BSA. The titer of baboon anti-Gal IgG () is increased 64-fold as compared with preimmune baboon IgG (). The titer of baboon anti-Gal IgM () is increased twofold to fourfold. Symbols are: (), normal baboon IgM; (X) sheep anti-BSA IgG. (B) ELISA reactivity of baboon anti-Gal and preimmune baboon -globulins with Matrigel. The binding of baboon anti-Gal () is 328-fold higher than that of preimmune baboon -globulin (); (), normal rabbit g-globulin. (C) Binding of baboon anti-Gal to Gal/BSA, pig thyroglobulin, and to single components of the extracellular matrix, measured by ELISA. The highest binding is for Gal/BSA (), followed by mouse laminin (), porcine thyroglobulin (), mouse heparan sulfate proteoglycans (), and bovine fibronectin (). The antibody does not react with human laminin (X) and the nephritogenic tubular basement membrane antigen or with bovine chondroitin sulfate, hyaluronate, bovine collagen I, II and III, pig collagen I and II, and BSA (data not shown). (D) Western blot analysis of components of the extracellular matrix. Baboon anti-Gal antibody recognizes antigens expressed on mouse laminin (lane 1), mouse heparan sulfate proteoglycans (lane 4), and bovine fibronectin (lane 6), but not on human laminin (lane 3) and human fibronectin (lane 8). Absorption of Gal antibody with rabbit erythrocytes abolishes the reactivity with mouse laminin (lane 2), mouse heparan sulfate proteoglycans (lane 5), and bovine fibronectin (lane 7).

Full figure and legend (141K)

Reactivity with the extracellular matrix was studied by ELISA on Matrigel, showing binding to mouse laminin, mouse heparan sulfate proteoglycans, and bovine fibronectin. Binding of baboon anti-Gal was 328-fold higher than that of preimmune baboon -globulin Figure 1b. The presence of Gal epitopes in single components of the extracellular matrix was also studied by ELISA; mouse laminin and mouse heparan sulfate proteoglycans were most reactive, and minimally bovine fibronectin Figure 1c.

Western blot analysis Figure 1d showed binding of Gal antibody to immobilized mouse laminin (lane 1), mouse heparan sulfate proteoglycans (lane 4), and bovine fibronectin (lane 6), but not to human laminin (lane 3) and human fibronectin (lane 8). The reactivity for mouse laminin (lane 2), mouse heparan sulfate proteoglycans (lane 5), and bovine fibronectin (lane 7) was abolished by absorption of baboon anti-Gal antibody with rabbit erythrocytes.

Binding of baboon anti-Gal to normal pig tissues

The highest dilution producing unequivocal immunofluorescence in pig tissue sections was 0.63 mg/mL. The staining titer of baboon anti-Gal IgG was 200-fold higher than that of preimmune baboon IgG, which mainly bound to tubular brush border. This IgG and affinity-purified baboon anti-Gal antibody produced the same staining pattern. The IgM fractions of naive pigs or preimmune sera did not stain the tissues, whereas IgM of baboon anti-Gal stained similarly to IgG, but weaker. In tissues of all pigs tested the staining patterns were similar, although with varied intensity.

In the kidney, Gal was localized in the brush border of renal proximal tubules, tubular basement membranes, and basolateral compartments in proximal tubules, in the endothelium of peritubular capillaries and, less intensely, in the glomerular basement membranes Figure 2a,Figure 2b,Figure 2c. The fibroblast-like cells in the medullary interstitium were strongly stained Figure 2e. With the exception of tubular epithelium, Gal colocalized with laminin and heparan sulfate proteoglycans Figure 2c,Figure 2dFigure 2eFigure 2f, and it also colocalized with podocalyxin in endothelia and in the podocytes. Fibronectin, which is mainly present in the mesangium and tubular basement membranes, colocalized with Gal in these structures.

Figure 2.

Localization of Gal in the kidney. (A) Localization of Gal in glomerular capillary walls and Bowman's capsule. (B) Strong expression of Gal in the brush border of proximal tubules and in tubular basement membranes. (C and D) Renal cortex. Gal (C) and laminin (D) antibodies colocalize in tubular and, with less intensity, glomeular basement membranes; in contrast, only Gal antibodies stain tubular brush border. (E and F) Inner stripe of the renal medulla. Gal antibody (E) and Griffonia Simplicifolia B4 (F) bind to fibroblast-like cells and to capillaries. (G and H) -galactosidase digestion abolishes the ability of baboon anti-Gal to stain the kidney tissue (G), whereas -galactosidase does not (H). (I) Absorption of baboon anti-Gal with Gal-rich rabbit erythrocytes abolished the ability to stain the kidney. A and B, 800; C to I, 400.

Full figure and legend (515K)

In the lung, immunofluorescence for Gal was mainly in: alveolar epithelium, and alveolar macrophages, septal fibroblast-like cells and septal matrix; bronchial vessels and plasma membranes of bronchial epithelial cells, lamina propria, and peribronchial extracellular matrix; chondrocytes and ground substance of bronchial cartilage Figure 3a,Figure 3b,Figure 3c,Figure 3d. Gal colocalized with laminin and heparan sulfate proteoglycans in the basement membranes of alveoli (Figure 4a,Figure 4bFigure 4c,Figure 4d,Figure 4e,Figure 4f, Figure 4j, and Figure 4k), bronchi, serous and mucous bronchial glands, and vessels. Fibronectin partially colocalized with Gal in alveolar basement membranes, but was mainly expressed in the septa Figure 4g,Figure 4h,Figure 4i and in the media of the arteries, where Gal was not, or only weakly, detected.

Figure 3.

Localization of Gal in the lung. (A and B) Alveolar capillary walls (a, indicates an alveolar space) and wall of a large vessel (arrow). (C and D) Medium sized bronchus. Gal (C) and perlecan (D) antibodies colocalize in the chondrocytes, in epithelial, vascular, and glandular basement membranes, and in the lamina propria. A and B, 400; C and D, 600.

Full figure and legend (232K)

Figure 4.

Colocalization of Gal with laminin and heparan sulfate proteoglycans. Sections of normal pig lung stained with two antibodies, one labeled with Texas red, and the other with FITC, and they were studied by confocal microscopy. Gal (A, D, and G) is mainly localized in the alveolar epithelium, and at the surface of an alveolar macrophage (A, arrow). Laminin (B) is localized in the alveolar basement membrane and in the alveolar septa, but not in the alveolar macrophage (arrow); heparan sulfate proteoglycans (E) is localized in the peripheral alveolar basement membrane. Fibronectin (H) is mainly in the septa (arrow) and, weakly, in the alveolar basement membrane. In C and in F, the yellow/orange color indicates basement membrane colocalization of Gal and laminin (arrow), and Gal and heparan sulfate proteoglycans, respectively. In (I), the orange color indicates a weak basement membrane colocalization of Gal and fibronectin. (J and K) These are enlargements of the areas indicated by rectangles in C and F. A to I, 1500; J and K, 3000.

Full figure and legend (362K)

In the liver, Gal was detected in the walls of sinusoids, in the matrix of the septa, and in the walls of septal and portal vessels and of central veins. With double staining and confocal microscopy, Gal was visualized in the endothelium of sinusoids and colocalized with laminin and heparan sulfate proteoglycans, in the basement membranes of the vessels, and in Disse's space. With fibronectin, it colocalized in the matrix of the septa and in Disse's space, but not in the basement membranes.

In the heart, Gal was strongly expressed in endothelium, basement membranes, and smooth muscles of all vessels, in the sarcolemma of cardiomyocytes, and with weaker intensity, in the connective tissue around muscle fibers. Laminin, heparan sulfate proteoglycans, and fibronectin colocalized with Gal in the basement membranes and sarcolemma, but were also expressed in the transverse striations.

In large vessels, Gal was localized in the surface endothelium and in the vasa vasorum and, in lesser amount, in smooth muscle cells and connective tissue matrix of aorta. Gal was present in endothelium of pulmonary vein but in that of pulmonary artery was only minimal and focal; it was also present in endothelium and smooth muscle cells of medium-sized muscular arteries and veins. By dual-fluorescence confocal microscopy, laminin, and heparan sulfate, proteoglycans colocalized with Gal in the basement membranes and smooth muscle cells. Fibronectin colocalized in only the connective tissue.

The specificity of baboon anti-Gal for Gal was shown by their colocalization with Griffonia Simplicifolia B4 (Figure 2 E, F) and by examination of sections predigested with - or -galactosidase. Digestion with Gal actosidase, but not with -galactosidase, abolished the staining (Figure 2 G, H). Moreover, absorption with Gal/BSA, but not with BSA, and absorption with Gal-rich rabbit erythrocytes abolished the ability of baboon anti-Gal to stain Figure 2i. Sheep anti-BSA, used at the concentration of 300 mg/mL, did not stain (data not shown).

DISCUSSION

We have studied the distribution of baboon anti-Gal antibodies in pig kidney and other organs most likely to be used for human transplantation, and provide new serological and morphological information on the expression of Gal epitopes on some glycoproteins of the extracellular matrix and cells that make and remodel it.

Immunization of baboons with Gal/BSA resulted in the production of increased levels of Gal IgG and IgM, as shown by increased binding to immobilized Gal/BSA and to Gal epitope-rich mouse laminin and pig thyroglobulin. These results are consistent with the immune responses of monkeys transplanted with porcine or bovine cartilage¹⁴ and patients transplanted with fetal porcine cells¹⁵ or treated with porcine liver perfusion¹⁶.

By ELISA, baboon anti-Gal react with Matrigel (which contains laminin, heparan sulfate proteoglycans, and type IV collagen, with a ratio of 1:0.6:00.3)¹⁷ more strongly than preimmune baboon serum. These results confirm that laminin expresses Gal epitopes^2,18, and they provide new serological evidence that Gal antibodies recognized epitopes on heparan sulfate proteoglycans. The reactive epitope of the glycoproteins identified by human natural xenoantibodies is Gal^19,20,21, which is broadly represented in phylogeny⁵. Therefore, baboon anti-Gal is a reagent suitable for the study of events that may occur in porcine grafts when human hosts develop a memory, high titer and high avidity, anti-Gal immune response.

In agreement with previous studies, mainly performed with Griffonia Simplicifolia B4 and Gal antibody isolated from normal human sera^22,23, we found that Gal is expressed on vascular endothelial and some epithelial cells. However, baboon anti-Gal also stained the basement membranes and the extracellular matrices where they colocalized with laminin, heparan sulfate proteoglycans, and partially with fibronectin antibodies. Moreover, the Gal antibodies strongly bound to all mesenchymal cells, including various types of fibroblasts, chondrocytes, and monocyte/macrophages, especially alveolar macrophages. In general, the staining was more diffuse than that obtained with antibodies to single components of the extracellular matrix, such as laminin, heparan sulfate proteoglycans, and fibronectin, probably because of the widespread distribution of Gal epitopes on glycoproteins and glycolipids². The principal immunologic target was Gal because the staining was identical to that obtained with Griffonia Simplicifolia B4, which is specific for Gal epitopes^18,24, was eradicated by digestion with -galactosidase, but not by -galactosidase, was abolished by absorption of baboon anti-Gal with Gal/BSA, but not with BSA, and was almost completely abolished by absorption with Gal-rich rabbit erythrocytes. The binding of baboon anti-Gal to the extracellular matrix and to cells that synthesize is congruent with the results of experiments in which pig organs, extracorporeally perfused with human blood, bound IgG and IgM to basement membranes and extracellular matrix²⁵.

In conclusion, the serologic study demonstrates reactivity of Gal antibodies for matrix glycoproteins, especially laminin and heparan sulfate proteoglycans, and these results are complemented by immunohistologic studies showing that in pig tissues, anti-Gal, antilaminin, and antiheparan sulfate proteoglycans antibodies are colocalized. The reactivity of Gal antibodies with extracellular matrix may have consequences if all the epitopes visualized in tissue sections are accessible to Gal antibodies in vivo and if pig organs will be made to survive in humans for long periods, a prospect that now seems increasingly probable³. In these conditions, Gal antibody may contribute to acute vascular rejection and later elicit basement membrane changes, similar to those induced in laboratory animals by antilaminin^26,27 and antiheparan sulfate proteoglycans^28,29 antibodies.

References

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Acknowledgments

The work was supported by grants from the National Institutes of Health: DK-36807-25/27 (G.A.) and HL-42507-PERC (D.S.). We thank Dr. Marilyn G. Farquhar and Dr. Robert A. Orlando for heparan sulfate proteoglycans and podocalyxin antibodies; Drs. Ralph J. Butkowski and Aristidis S. Charonis for tubular basement membrane antigen; Dr. Jeffrey L. Platt for reading the manuscript, providing helpful advice, and suggestions; and Ms. Theresa Swayne for expert technical help.