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J Thorac Cardiovasc Surg 1995;110:1023-1029
© 1995 Mosby, Inc.

CARDIAC AND PULMONARY REPLACEMENT

AGE-RELATED DEVELOPMENT OF HUMAN ANTI-PIG XENOANTIBODY

He Xu, MD, Nillo Edwards, MD, Jonathan M. Chen, MD, Xu Dong, RN, Robert E. Michler, MD


New York, N.Y.

From the Cardiac Transplantation Research Laboratory, Division of Cardiothoracic Surgery, Columbia Presbyterian Medical Center, New York, N.Y.

Presented in part at the Sixty-sixth Scientific Sessions of the American Heart Association, Atlanta, Ga., November 1993.

Received for publication Sept. 1, 1994. Accepted for publication Dec. 23, 1994. Address for reprints: Robert E. Michler, MD, Director, Cardiac Transplant Service, and Director, Cardiac Transplantation Research Laboratory, Division of Cardiothoracic Surgery, Columbia Presbyterian Medical Center, 177 Fort Washington Ave., Milstein Hospital Building, Room 7435, New York, NY 10032.

Abstract

Human cytotoxic natural xenoantibodies are believed to be of the immunoglobulin M class in nature. However, a thorough understanding of the development of these natural antixenodonor xenoantibodies remains incomplete. In this study, serum samples were obtained from newborn, infant, and adult human beings. An enzyme-linked immunosorbent assay was used to determine the binding of human natural immunoglobulin M xenoantibodies to pig aortic endothelial cells and pig lymphocytes. A complement-mediated cytotoxicity assay was used to measure the cytotoxicity of newborn, infant, and adult serum to cultured pig aortic endothelial cells and pig lymphocytes. Adult human serum contained both natural immunoglobulin M and immunoglobulin G xenoantibodies to pig endothelial cells and lymphocytes, whereas newborn infant serum contained only immunoglobulin G xenoantibodies. Only adult human serum was cytotoxic to pig endothelial cells and lymphocytes. Human immunoglobulin M xenoantibodies became detectable by age 1 month. By age 2 months these natural anti-pig xenoantibodies reached serum levels equivalent to those in the human adult and resulted in similar cytotoxicity to that of adult human serum. These findings indicate that (1) natural anti-pig immunoglobulin M xenoantibodies are absent from newborn infant human serum, (2) newborn human serum is not cytotoxic to pig endothelial cells and lymphocytes despite the presence of immunoglobulin G xenoantibodies that bind to pig endothelial cells and lymphocytes, and (3) natural anti-pig immunoglobulin M xenoantibodies begin to develop as early as age 1 month and by age 2 months attain a circulating level comparable to that found in the adult. (J THORAC CARDIOVASC SURG 1995; 110:1023-9)

The critical shortage of donor hearts for cardiac allotransplantation had led to a search for alternative sources of suitable organs. Go Go 1,2 The need for grafts is particularly desperate among newborn infants with congenital malformations, for whom the long wait for an available organ not infrequently results in death. Closely related species such as nonhuman primates are a possible source of donor organs on the basis of their humanlike anatomy and physiologic makeup and their genetic proximity to man. However, as donors these animals rarely fall in the universal donor blood type group O and they harbor zoonoses infectious to man.

An alternate donor pool would be animals that are available in all human sizes, do not express ABO antigens or express all blood types commonly, and harbor few agents infectious to man. As a result of these logistic considerations, the pig has been proposed as an alternative to the nonhuman primate donor. The pig is available in all human sizes and pig cardiac anatomy and physiologic makeup are strikingly similar to those of human beings. Go 3 Pigs can be raised to have minimal risk of epizootic disease transmission and pig endothelial cells express ABH antigens weakly. Go 4 More than 90 million pigs are slaughtered annually as a food source, which makes these animals a more ethically acceptable alternative donor organ source. Go 5

Organs transplanted between distantly related species such as the pig and the baboon are rejected in a rapid and violent manner termed hyperacute rejection. This rejection reaction is believed to be initiated by the binding of naturally occurring xenoantibodies to donor organ endothelium leading to activation of the complement cascade. Go Go 6,7 Natural xenoantibodies have been shown to bepresent in all mammalian species. Go 8 The activation of complement by the binding of natural antibody causes endothelial cell dysfunction characterized by cell swelling, interstitial hemorrhage, inflammation, fibrin and platelet thrombus formation, and graft death in minutes to hours. Go Go 6,7 Limited prolongation of distantly related xenograft survival has been achieved by depletion of these natural antibodies with plasma exchange, Go 9 plasmapheresis, Go 10 donor-organ adsorption, Go Go 11,12 and inhibition of complement with cobra venom factor Go 13 or soluble complement receptor type 1. Go 14

Investigations regarding human and primate natural xenoantibodies have suggested that they are of the immunoglobulin M (IgM) class of immunoglobulin. Go Go Go Go 6,7,15,16 We have previously identified these natural xenoantibodies to be present only at low levels in the serum of newborn baboons. Go 17 To further our understanding of the development of natural human xenoantibodies, we examined serum samples obtained from newborn, infant, and adult human beings to evaluate the existence and cytotoxicity of human natural anti-pig xenoantibodies.

MATERIALS AND METHODS

Preparation of serum
Pooled human serum samples were obtained from the following age groups: newborn (term newborn infant cord blood, n = 10), 1 month (n = 5), 2 months (n = 5), 4 months (n = 5), 8 months (n = 5), 1 year (n = 5), 2 years (n = 5), 5 years (n = 5), 10 years (n = 5), and adult (>18 years, n = 10). All serum samples were heat-treated for 30 minutes at 56°C to inactivate complement and were subsequently stored at -80°C.

Target cells
Pig aortic endothelial cells were isolated with the use of collagenase and primary culture of these cells was done in Dulbecco's modified Eagle medium (DMEM) (Gibco, Grand Island, N.Y.) supplied with 10% fetal calf serum (Gibco) according to the method described by Rosiers, Nees, and Gerlach. Go 18 These endothelial cells were detached by trypsin and transferred into new tissue culture flasks. Subculture of pig aortic endothelial cells was done in DMEM containing 10% fetal calf serum. These endothelial cells were used for immunoassay before the tenth generation of cell division.

Once they obtained confluence, the cultured endothelial cells were transferred to flat-bottomed 96-well plates and allowed to grow until the formation of endothelial cell monolayers. Both pig lymphocytes and cultured pig aortic endothelial cells were used as target cells.

Pig lymphocytes were isolated from peripheral blood by flotation on a Ficoll-hypaque gradient (Gallard Schleseaga Co., New York, N.Y.). The cells were washed twice with phosphate-buffered saline solution (PBS) (Sigma Chemical Co., St Louis, Mo.). The viability of pig lymphocytes was measured by trypan blue exclusion and standardized to exceed 95%.

Immunoassay for natural xenoantibodies
An enzyme-linked immunosorbent assay (ELISA) was used to detect the binding of natural anti-pig xenoantibodies to cultured pig vascular endothelial cells according to the method described by Platt and colleagues. Go 19 In brief, cultured pig aortic endothelial cell monolayers were fixed with cold 0.01% glutaraldehyde for 5 minutes at 4° C and then washed three time with cold PBS. After the final wash, the plates were incubated with 1% bovine serum albumin in PBS at 4° C for 1 hour to block the nonspecific binding of antibodies and then were exposed to a variety of diluted serum samples (primary antibodies) for 60 minutes at 4° C. Cells were washed three times with PBS and incubated with secondary antibodies (goat anti-human IgM conjugated to alkaline phosphatase and goat anti-human IgG conjugated to alkaline phosphatase) for another 60 minutes. After the final washing with PBS, developer solution was added to each well and color development progressed in the dark. Finally, absorbance at 405 nm was measured by an ELISA reader. One percent bovine serum albumin was used as the negative control, and pooled adult human sera were used as positive controls. The methods for ELISA analysis of fresh pig lymphocytes were identical to those previously described herein for cultured pig aortic endothelial cells.

A complement-mediated cytotoxicity assay was done to measure the cytotoxicity of newborn and adult human serum to porcine lymphocytes. A total of 10 x 10 6 porcine lymphocytes were labeled with100 µCi of chromium 51 (Amersham Corp., Arlington Heights, Ill.) at 37° C for 60 minutes and then washed three times with Hanks solution (Gibco) to remove free chromium 51. A total of 1 x 10 6 pig lymphocytes were added to a 96-well round-bottom microtiter plate with 50 µl newborn or adult human serum samples and 50 µl of diluted rabbit complement (1:1 dilution), and the plate was incubated for 60 minutes at 37° C. After the final incubation, the plate underwent centrifugation at 1500 rounds per minute for 10 minutes and 100 µl of supernatant was collected with the radioactivity of released chromium 51 from the pig lymphocytes determined by gamma counter analysis.

Cultured pig endothelial cells were transferred into 24-well tissue culture plates and grown to confluence. The endothelial cell monolayers were labeled with 4 µCi per well of chromium 51 at 37° C for 2 hours and then washed three times with DMEM to remove free chromium 51. After the final wash, endothelial cell monolayers were incubated with 100 µl serum and 100 µl rabbit complement (1:1 dilution) at 37° C for 4 hours. Supernatants (100 µl) were collected, and the chromium 51 released from the pig endothelial cells was determined with use of a gamma counter. Pig serum was used as a negative control and cell lysis buffer as a positive control (Fisher Scientific, Orangeburg, N.Y.).

Percent cytotoxicity was calculated as follows:


where cpm is the counts per minute of chromium 51 determined in the assay.

RESULTS

Endothelial cells
Adult human IgM and IgG natural anti-pig endothelial cell xenoantibodies were demonstrated to bind to cultured pig aortic endothelial cells and at equivalent levels (Fig. 1).



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  		Fig. 1. Binding of adult human natural IgM and IgG xenoantibodies to cultured pig aortic endothelial cells was measured by ELISA.

 
The age-related binding of human natural IgM xenoantibodies to cultured pig endothelial cells demonstrated barely detectable levels at birth, which increased only slightly by age 1 month (Fig. 2). By age 2 months IgM xenoantibody binding was at a level comparable to that measured in pooled adult human sera (Fig. 2). In contrast, the binding of natural IgG xenoantibodies to pig aortic endothelial cells was readily detectable at birth and by age 1 month had achieved levels comparable to those of the adult (Fig. 3).



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  		Fig. 2. ELISA demonstrates age-related binding of human natural IgM xenoantibodies to cultured pig endothelial cells. Unlike adult human serum, newborn human serum contains low levels of natural anti-pig IgM xenoantibodies.

 


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  		Fig. 3. ELISA demonstrates age-related binding of human natural IgG antibodies to cultured pig aortic endothelial cells.

 
Pooled adult human serum was markedly cytotoxic to cultured pig aortic endothelial cells. In contrast, newborn human serum was not cytotoxic to pig aortic endothelial cells, which suggests that IgM is the cytotoxic xenoantibody. However, cytotoxicity of infant serum to pig endothelial cells occurred by age 2 months (Fig. 4).



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  		Fig. 4. Age-related development of cytotoxicity of human serum to cultured pig endothelial cells was determined by complement-mediated cytotoxicity assay. Newborn human serum demonstrated no cytotoxicity to pig endothelial cells.

 
Lymphocytes
The age-related binding of human natural IgM xenoantibodies to pig lymphocytes demonstrated barely detectable levels at birth, which increased only slightly by age 1 month (Fig. 5). By age 2 months IgM xenoantibody binding was at a level comparable with that measured in pooled adult human sera (Fig. 5). Insufficient serum was available to perform IgG binding studies (ELISA).



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  		Fig. 5. Age-related binding of natural IgM xenoantibodies to pig lymphocytes was measured by ELISA.

 
Significant cytotoxicity by adult human serum to pig lymphocytes was demonstrated, whereas no cytotoxicity was demonstrated by newborn human serum to pig lymphocytes (Fig. 6).



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  		Fig. 6. Complement-mediated cytotoxicity assay demonstrates high level of cytotoxicity of adult human serum to pig lymphocytes.

 
DISCUSSION

Hyperacute rejection of discordant xenografts is believed to be caused by the binding of natural xenoantibodies to donor organ endothelial cells, a process that ultimately results in the activation of the complement cascade and subsequent graft loss. Go Go Go 6,7,11 Investigations that characterized the nature and function of natural xenoantibodies have been reported previously by our transplantation research laboratory and by other investigators. Go Go Go Go 6,7,15,16 In light of our previous finding that human cytotoxic natural xenoantibodies are IgM in nature and are either absent or barely detectable in newborn baboon serum, Go 17 we undertook the present investigation to better understand the process by which natural IgM xenoantibodies develop in human beings.

It has been documented that the selective placental transfer of maternal immunoglobulin from mother to fetus can provide immediate passive protection from bacterial infection. By this mechanism, IgG is transferred during gestation, whereas IgM, IgA, and IgD are not because of their large molecular weight (molecular weight 900,000 for IgM). Go Go 19-21 Our data demonstrate that newborn human serum contains barely detectable levels of natural anti-pig IgM xenoantibodies and intermediate levels of IgG xenoantibodies. Newborn human serum is not cytotoxic to pig lymphocytes or cultured pig endothelial cells despite the binding of natural IgG xenoantibody. Human cytotoxic natural anti-pig IgM xenoantibodies begin to develop at age 1 month and ultimately attain levels comparable to those of the adult by age 2 months.

IgM antibody synthesis has been described as early as the eleventh week of gestation in the spleen and the colon. Go Go 22,23 Stiehmand Fudenberg Go 20 have suggested that human IgM antibodies develop rapidly after birth and have demonstrated IgM antibody levels in human infants between 1 and 3 months old to be 30% ± 11% of adult levels, those between 4 and 6 months to be 43% + 17% of adult levels, and those between 7 and 12 months to be 55% ± 23% of adult levels, with levels increasing slowly after the first year of life. However, at present, it is unclear whether natural xenoantibodies exhibit developmental patterns similar to those of these previously investigated IgM antibodies. Preliminary results indicate that the production of these natural xenoantibodies after birth may be induced by both living and killed bacteria. Go 24

We have previously demonstrated barely detectable levels of natural anti-pig IgM xenoantibody in newborn baboon serum Go 17 and prolongation of cardiac xenograft survival in a newborn pig-to-baboon model. Go 25 Kemp and associates Go 26 demonstrated delayed hyperacute rejection in a newborn rabbit-to-pig kidney discordant xenotransplantation model in which natural xenoantibodies were absent from newborn recipients.

In summary, human cytotoxic natural anti-pig IgM xenoantibodies are barely detectable at birth. These xenoantibodies (as measured by ELISA) begin to develop at approximately age 1 month. These cytotoxic IgM xenoantibodies develop rapidly between age 1 and 2 months and ultimately attain levels comparable to those of the adult by age 2 months. Human natural IgG xenoantibodies are found in all human sera, although newborn sera express lower levels of binding to pig aortic endothelial cells than do adult sera. The nature and function of these IgG xenoantibodies, however, remain poorly characterized. These results offer promising evidence for the potential success of clinical newborn cardiac xenotransplantation; however, further investigations are indicated regarding the mechanism of natural xenoantibody development and the long-term results of experimental neonatal xenotransplantation.

References

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  26. Kemp E, White D, Dieperink H, et al. Delayed rejection of rabbit kidneys transplanted into baby pigs. Transplant Proc 1987;19:1143-4.[Medline]



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