Serum amyloid A as a marker of inflammation in xenotransplantation

Abstract

Increasing evidence indicates that inflammation plays a role in pig-to-baboon organ xenotransplantation. We have evaluated serum amyloid A (SAA) as a marker of inflammation in baboons with various pig xenografts. We measured SAA levels in recipient baboons with pig artery patch (n = 5), life-supporting kidney (n = 5), heterotopic heart (n = 2), or hepatocyte (n = 1) grafts and using an OmniChek-SAA for Inflammation & Infection kit. C-reactive protein (CRP), another marker of inflammation (e.g. D-dimer), was also measured.SAA indicated increased inflammation when baboons developed consumptive coagulopathy (CC; e.g. thrombocytopenia) or infection. SAA also indicated that treatment of the recipient with tocilizumab reduced inflammation. There was significant positive correlation between SAA with changes in CRP (r = 0.6, P < 0.05) and with D-dimer (r = 0.8, P < 0.01), but SAA appeared at times to more accurately reflect the clinical state of the baboon. In sum, measurement of SAA proved simple and quick, and indicated (1) significant inflammation when CC or infection was present, and (2) reduced inflammation when treatment with tocilizumab was administered.

Introduction

Similar to C-reactive protein (CRP), serum amyloid A (SAA) is a nonspecific inflammatory reactive protein that has been shown to increase under various pathological conditions, for example, systemic infection. ¹ It has also been reported to increase during acute allograft rejection. ¹ It may play a role in the inflammatory process through its cytokine-like property, rather than as a passive responder, ² for example, by stimulating the production of IL-8, TNF-α, and IL-1β. ² SAA can also induce tissue factor expression and inhibit expression of tissue factor pathway inhibitor in endothelial cells. ³ To our knowledge, there has been no investigation of SAA in xenotransplantation.

In this pilot study, we measured SAA, CRP, and D-dimer in baboons with pig organ, cell, or artery patch grafts.

Materials and methods

Animals

Baboons (n = 14, Papio species, Oklahoma University Health Sciences Center, Oklahoma City, OK) received either an artery patch (n = 5), ⁴ a life-supporting kidney (n = 5), ⁵ a heterotopic heart (n = 2), ⁶ or a hepatocyte (n = 1) ⁷ graft from a genetically engineered pig (Revivicor, Blacksburg, VA; Table 1). Anesthesia, intravascular catheter placement in baboons, and pig-to-baboon surgical procedures have been described previously.^4-7

Table 1.

Immunosuppressive and anti-inflammatory therapy and changes in serum amyloid A (SAA), CRP, and D-dimer in baboon recipients of pig grafts.

Organ Baboon	Maintenance IS regimen	IL-6R blockade a (±)	Status on day of SAA measurement	SAA	Day of SAA measurement	CRP c (mg/dL)	D-dimer d (µg/mL)
Artery patch b (n = 5)
B12912	Anti-CD40mAb + CTLA4-Ig	−	Stable	Significant	48	1.70	1.46
B5412	Anti-CD40mAb + CTLA4-Ig	−	Stable	Significant	28	1.96	1.44
B12612	CTLA4-Ig	+	Stable	No	48	0.15	0.64
B9512	Anti-CD40mAb + CTLA4-Ig	+	Stable	No	48	0.19	1.81
B6414	None	−	Rejection/sensitized	Moderate	48	0.36	0.94
Kidney (n = 5)
B9313	Anti-CD40mAb	+	Infection e	Significant	136	1.24	2.38
B17315	Anti-CD40mAb	+	Stable	No	180	0.02	0.7
B17615	Anti-CD40mAb	+	Stable	No	180	0.03	0.36
B17415	Anti-CD40mAb	+	CC	Significant	12	0.17	2.31
B17515	Anti-CD40mAb	+	CC	Significant	12	0.22	2.38
Heart (n = 2)
B18013	Anti-CD40mAb + CTLA4-Ig	+	Rejection	No	118	0.44	0.9
B19510	Anti-CD154mAb	−	Infection f	Significant	15	3.14	2.39
Hepatocytes (n = 1)
B14014	Tacrolimus + rapamycin	−	Rejection/sensitized	No	35	0.42	1.22

a

Tocilizumab (10 mg/kg, every 2 weeks).

b

Electively euthanized.

c

Normal level of CRP = < 0.5 mg/dL.

d

Normal level of D-dimer = < 0.45 µg/mL.

e

Systemic Myroides spp.

f

Systemic Enterococcus faecium.

All animal care was in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Research Council (8^th edition, revised 2011), and was conducted in an AAALAC-accredited facility. Protocols were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

Immunosuppressive and anti-inflammatory therapy

For the artery patch, kidney or heart xenotransplantation experiments, immunosuppressive therapy was based on costimulation blockade using either (1) anti-CD154mAb (National Institutes of Health (NIH) NHP Reagent Resource, Boston, MA), or (2) CTLA4-Ig (BMS, Princeton, NJ) ± anti-CD40mAb (2C10R4; NIH NHP Reagent Resource), or (3) anti-CD40mAb (Table 1).^4-6 Eight immunosuppressed baboons with pig artery patch (n = 2), kidney (n = 5), or heart (n = 1) grafts received interleukin-6 receptor (IL-6R) blockade with tocilizumab (10 mg/kg; Actemra, Genentech, South San Francisco, CA). For the hepatocyte xenotransplantation, immunosuppressive therapy was based on tacrolimus/rapamycin (Table 1). ⁷

Measurement of SAA, CRP, and D-dimer

Whole blood and serum samples were obtained from recipients before and serially after transplantation. SAA was measured with a Rapid Test for Inflammation & Infection kit (OmniChek-SAA: Accuplex Diagnostics, Maynooth, Co Kildare, Ireland), as per the manufacturer’s instructions. Briefly, serum was drawn up into an applicator and introduced into the port on the test strip; the result was indicated in the test window within 5–10 min (Figure 1). The test took <10 min to carry out in the animal facility. D-dimer and serum CRP was measured by standard methods (Central Laboratory of Presbyterian Hospital, Pittsburgh, PA), and the result of CRP was usually available within 2–3 h, though sometimes it took longer.

Figure 1.

Representative results of assay for SAA showing (a) 3 lines (negative), indicating no active inflammation, (b) 2 lines, indicating mild-to-moderate inflammation, and (c) 1 line, indicating significant inflammation.

Statistical analysis

Monotonic association analysis was evaluated using Spearman’s rank-order correlation. Statistical analysis was performed using social sciences software GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Values of P < 0.05 were considered statistically significant.

Results

Clinical course and graft survival (Table 1)

Baboons with pig artery patch grafts were electively euthanized at 28 (n = 1) or 48 (n = 4) days post-transplantation. Baboons with kidney grafts were euthanized or died from complications of consumptive coagulopathy (CC; n = 2) or infection (n = 3), though the SAA was measured in two of these before infection developed (when the baboons were healthy and stable; Table 1). Baboons with pig heart grafts were euthanized either for graft failure (n = 1) or infection (n = 1). The baboon with pig hepatocytes was electively euthanized 35 days’ post-transplantation. CRP, D-dimer, and SAA were measured in all baboons before the transplants. CRP and D-dimer were within the normal ranges (CRP < 0.5 mg/dL; D-dimer < 0.45µg/mL), and SAA showed no inflammation.

SAA, CRP, and D-dimer levels in baboons that developed CC, Infection, or rejection (Table 1)

The following observations were made.

1. There was significant positive correlation between CRP and SAA (r = 0.6, P < 0.05; Figure 2). Significant positive correlation was also observed between D-dimer and SAA (r = 0.8, P < 0.01; Figure 3).

2. In immunosuppressed baboons with pig artery patch grafts, both SAA and CRP indicated a persistent inflammatory state, as reported previously, though the CRP could be suppressed by treatment with tocilizumab.^5,8

SAA demonstrated significant or moderate inflammation whenever CC or infection developed (r = 0.7, P < 0.05). When baboons developed CC despite receiving tocilizumab therapy, SAA and D-dimer continued to show evidence of an inflammatory state, whereas CRP was within the normal range, suggesting that SAA and D-dimer may have been more accurate. Examples are the two baboons with kidney grafts (B17415, B17515) that were euthanized on post-transplant day 12 for rapid-onset CC. SAA indicated significant inflammation and D-dimer were high levels, whereas the CRP remained within the normal range (Table 1 and Figures 2 and 3); we would anticipate that inflammation was present in these two baboons.

• 3. In two of the three baboons in which rejection of the graft was confirmed (B18013, B14014), neither SAA nor CRP indicated an inflammatory state. However, D-dimer showed high levels. In the third (B6414), SAA indicated moderate inflammation, D-dimer showed high, whereas the CRP did not; this baboon was the only one of the three that was not receiving immunosuppressive therapy, making it more likely that some inflammation may well have been present, suggesting that SAA and D-dimer may have provided more reliable data.

Figure 2.

Correlation between SAA and CRP in baboons (n = 13). A significant positive correlation between CRP and SAA was observed (r = 0.6, P < 0.05). The extent of inflammation was determined by SAA as none, moderate, or significant. However, in three recipient baboons that developed consumptive coagulopathy (B17415, B17515; n = 2) or rejection (B6414; n = 1), SAA indicated increased inflammation, whereas CRP was within the normal range (indicated by red lines). The horizontal blue line indicates the upper limit of the normal range of CRP (<0.5 mg/dL).

Figure 3.

Correlation between SAA and D-dimer in baboons (n = 13). A significant positive correlation between SAA and D-dimer was observed (r = 0.8, P < 0.01). The extent of inflammation was determined by SAA as none, moderate, or significant. SAA indicated increased inflammation and D-dimer levels were high in four recipient baboons that developed consumptive coagulopathy (B17415, B17515; n = 2) or infection (B9313, B19510; n = 2, indicated by red lines). In two recipient baboons that were stable and did not receive tocilizumab (B12912, B5412), SAA indicated significant inflammation and D-dimer and CRP showed increased levels (indicated by blue lines). The horizontal blue line indicates the upper limit of the normal range of D-dimer (<0.45µg/mL).

Discussion

A systemic inflammatory response has been documented after pig-to-baboon organ or artery patch transplantation in baboons,^8,9 as indicated by a persistent increase in CRP^8,9 and changes in serum cytokines and chemokines. ⁹ SAA is an inflammatory marker that is elevated in different pathological states (e.g. rheumatoid arthritis, ¹⁰ lung cancer, ¹¹ cardiovascular disease, ¹² and in recipients of kidney or heart allografts when undergoing rejection or infection ¹ ). However, to our knowledge, SAA has not been investigated previously in recipients of pig xenografts.

This study demonstrated that the SAA assay indicated a significant or moderate inflammatory state in baboon recipients of pig grafts when CC or infection developed. D-dimer also showed high levels when CC or infection developed. Both SAA and D-dimer correlated with CRP in becoming elevated in pig artery patch recipients that were not receiving tocilizumab.^8,9 When tocilizumab was being administered, whereas the CRP did not indicate inflammation while the baboon recipients were stable, the SAA did. ⁸ The mechanism by which tocilizumab results in a reduction in CRP is likely to be related to the effect of IL-6. ⁸ Our observations suggest that these assays might be able to differentiate between rejection and inflammation/infection.

In sum, the SAA assay proved simple and quick, may have some advantages over CRP, and may be a useful marker to indicate that infection or CC is developing, allowing more-specific investigations to be initiated.