Abstract
Laryngeal transplantation is an increasingly viable proposition for patients with irreversible diseases of the larynx. One human transplant has been performed successfully, but many questions remain before routine transplantation can begin. In order to measure the immunological changes in mismatched transplants, it is first necessary to know the immediate combined effects of ischaemia-reperfusion injury (IRI) plus the added insult of major surgery in a fully matched setting. We measured the changes in immunologically active mucosal cells following 3 h of cold ischaemia and 8 h of in situ reperfusion in a major histocompatibility complex (MHC)-matched minipig model (n = 4). Biopsies were prepared for quantitative, multiple-colour immunofluorescence histology. The number of immunologically active cells was significantly altered above (supraglottis) and below (subglottis) the vocal cords following transplantation and reperfusion (P < 0·05, P < 0·001, respectively). However, the direction of the change differed between the two subsites: cell numbers decreased post-transplant in the supraglottis and increased in the subglottis. Despite the statistical evidence for IRI, these changes were less than the large normal inter- and intrapig variation in cell counts. Therefore, the significance of IRI in exacerbating loss of function or rejection of a laryngeal allograft is open to question. Longer-term studies are required.
Keywords: larynx, pig, transplant
Introduction
Although experimental studies into laryngeal transplantation began in the 1960s [1–4], it is not yet a routine clinical procedure. However, recent advances have made laryngeal transplantation an increasingly viable proposition for patients with irreversible laryngeal disease [5]. One successful human laryngeal transplant has been performed and the 40-month follow-up report was encouraging [6].
Dense populations of immunologically active cells, including cells expressing high levels of major histocompatibility complex (MHC) class II, have been reported within the normal laryngeal mucosa in man [7,8], pig [9] and rat [10]. The presence of these cells is likely to increase the risk of immunological rejection [11]: skin grafts and mucosal sites such as lung and intestine, which also contain MHC class II positive cells, have poorer survival rates than solid organ transplants [12]. Consistent with this hypothesis, laryngeal grafts underwent massive cellular infiltration and subsequent graft necrosis in dog and rat models without appropriate immunosuppression [13,14]. For this reason, detailed studies of graft rejection are required in fully functional and surgically accessible models, prior to clinical trials [15,16].
A second major factor likely to exacerbate graft rejection is post-transplant ischaemia [17]. Ischaemic-reperfusion injury appears to trigger early influx of dendritic cells and local up-regulation of MHC antigens, thus further increasing the immunogenicity of the grafted organ [18,19]. The severity of this acute phase is likely to determine strength of the later, alloantigen-dependent rejection [20]. In the early phase after transplantation (up to 8 h), cells of the myeloid series (neutrophils and monocyte/macrophages) will predominate [21].
To examine early reperfusion events, we used an in vivo re-vascularized laryngeal transplantation model using MHC-homozygous NIH minipigs [22].
Materials and methods
Animals
The haplotype of the individual minipigs used is shown in Table 1. Operative details have been described previously by our group [16]. Briefly, pigs were premedicated with intramuscular ketamine (10 mg/kg; Vetco, Dublin, Ireland) and azaparone (2 mg/kg; Jansse-Cilag, Pfarrgasse, Austria). Anaesthesia was induced by intravenous propofol (Abbott Laboratories, Kent, UK) and maintained with isofluorane (Baxter Healthcare Corporation, Cambridge, UK), 50 : 50 oxygen : nitrous oxide (BOC, UK) administered through an endotracheal tube. Atracurium (Glaxo, Middlesex, UK) was used as the muscle relaxant at an initial dose of 1 mg/kg then subsequently by an infusion as required. Intra-operative monitoring included nasal temperature, electrocardiograph (ECG) monitoring, invasive arterial pressure monitoring, pulse oximeter, inspired and expired gases and capnographic monitoring using Datascope Gas Module II™ and Passport™ 2. Intra-operative analgesia was maintained using intravenous morphine (0·2 mg/kg every 4 h; Martindale Pharmaceuticals, Essex, UK) and intravenous fentanyl (2 µg/kg at 20–30-min intervals as required; Janssen). These studies were approved by the appropriate local and national ethical boards (UK Home Office PPL 30/1786).
Table 1.
In vivo laryngeal transplant pig data.
| Exp. no. | Donor/recipient | Sex | Weight | Haplotype |
|---|---|---|---|---|
| 1 | Donor | Female | 10·0 kg | CC |
| 1 | Recipient | Male | 9·8 kg | CC |
| 2 | Donor | Female | 9·2 kg | CC |
| 2 | Recipient | Female | 9·6 kg | CC |
| 3 | Donor | Female | 30 kg | CC |
| 3 | Recipient | Male | 32 kg | CC |
| 4 | Donor | Female | 32 kg | CC |
| 4 | Recipient | Male | 35 kg | CC |
Pre-retrieval biopsies (donor)
After anaesthesia, laryngeal biopsies were taken from the laryngeal subsites cranial and caudal to the vocal cords (‘supraglottis’ and ‘subglottis’). This separation is important as these sites are different anatomically and functionally. Biopsy tissue was covered in octreotide (OCT) and snap-frozen in isopentane cooled over liquid nitrogen as described previously [23]. The cores were stored at −70° until further processing.
Operative details (donor)
In brief, the larynx and trachea were identified and supplying vessels isolated. Specifically, the axillary artery was isolated with a vasiloop (Bard, Crawley, UK). The venous vascular bundle was slooped independently, but not tied, to prevent venous obstruction and resulting limb oedema. The nerves of the brachial plexus were identified but not cut until after the pig had been killed, in order to minimize the physiological response to the nociceptive stimuli. Soft tissue dissection was continued cranially to the level of the hyoid bone. There, the common carotid artery was identified and slooped, retaining the fine arterial supply to cranial and caudal larynx. The internal jugular vein and vago-sympathetic trunk were identified and slooped together.
The sternum was opened in the midline, allowing access for ligation of vessels entering or leaving the great vessels within the thorax. The aorta was isolated and the animal given 5000 IU of intravenous heparin (CP Pharmaceuticals, Wrexham, Flintshire, UK). Five minutes following the heparin administration the animal was killed with pentabarbitone (100 mg/kg, Rhone Merieux Ltd, Harlow, UK). Arterial clamps were placed across the proximal ascending aorta and the proximal descending aorta. The graft was perfused under gravity for 10 min via an aortic root cannula (14G, Medtronic Inc., Minneapolis, MN, USA) with 900 ml of University of Wisconsin solution at 4°C (Viaspan®; Dupont Pharma, UK).
During this time sloops on the graft vessels were tied in order to isolate the graft. An ice pack was placed over the graft to keep it cool. Cutting diathermy and ties were used to remove the graft from the carcass. Once the graft was free from the animal it was placed in an Alden intestinal bag (330 × 254 mm, Rush, N. Ireland), filled with 100 ml of cold University of Wisconsin solution and stored on ice to complete the 3-h cold ischaemic time.
Operative details (recipient)
Following cold ischaemia the graft was reperfused by the recipient animal. Prior to vessel anastomosis the recipient animal was heparinized as described. The recipient proximal common carotid artery was anastomosed end-to-end to the graft’s proximal subclavian artery. The distal graft subclavian vessel was anastomosed end-to-end to the distal common carotid recipient artery. Following the second anastomosis, all the arterial clamps were removed, cold ischaemia ended, and the time of graft reperfusion was recorded. Venous flow was identified leaving the graft through the cut end of the superior vena cava. The venous anastomosis was end-to-side, the end of the superior vena cava of the graft to the side of the jugular venous confluence.
Following 8 h of reperfusion the graft was removed and laryngeal tissue taken from the supra- and subglottis. This tissue was also covered in OCT, snap-frozen in isopentane cooled over liquid nitrogen and stored at −70° until further processing. The animal was then killed with pentabarbitone (100 mg/kg).
Immunohistology
The samples were processed and prepared for multiple colour immunofluorescence, as described previously by our group [24,25]. Briefly, frozen sections (4–6 µm) were incubated with a series of solutions including 5% goat and 5% pig serum, optimally diluted primary antibody and secondary, fluorochrome- or biotin-conjugated isotype-specific anti-mouse antibody (Southern Biotechnology Associates Inc., Birmingham, AL, USA). Tertiary staining was achieved by incubation with AMCA avidin D (Vector Laboratories Inc., Burlingame, CA, USA). The tissue sections were visualized and images captured as described for cytospins. For each biopsy approximately five fields were captured. Numbers of leucocytes stained with combinations of monoclonal antibodies were counted as described [23]. Leucocytes in the epithelium and in the lamina propria were counted separately and the results expressed as cells/mm2.
Monoclonal antibodies
Different epitopes were identified with the monoclonal antibodies, as shown in Table 2. In order to survey cells of the innate, adaptive (cell-mediated and humoral) immune response, these antibodies were chosen. Nuclear material was stained using 4,6-diamidino-2-phenylindole (DAPI, Sigma) 0·08 mg/ml.
Table 2.
Primary monoclonal antibodies used for analysis of laryngeal allografts.
| Epitope | Isotype | Tissue distribution | Clone |
|---|---|---|---|
| MHC class II | IgG2a | Macrophages, dendritic cells, B cells | MSA 3 (DR) |
| CD172 | IgG2b | NP, monocytes and macrophages | 742215 |
| CD163 | IgG1 | Monocytes and macrophages | 2A10/11 |
| CD16 | IgG1 | NK, monocytes, macrophages and NP | G7 |
| CD14 | IgG2b | Monocytes, macrophages, dendritic cells | Mil 2 |
| CD21 | IgG2b | Mature B cells | CC51 |
| CD79α | IgG1 | B cells | HM57 |
| Light chain | IgG2a | Light chain of antibodies | 1393E1 |
| CD4 | IgG1 | T helper cells | STH293 (CD8/MHC II work) |
| CD4 | IgG2b | T helper cells | MIL 17 (activation work) |
| CD8 | IgG2a | Cytotoxic T cells | MIL 12 |
| γδ T cell receptor | IgG2b | γδ T cells | PPT27 |
| CD25 | IgG1 | Activated T cells | K231·3B2 |
| CD45 | IgG1 | Leucocytes | K252.E4 |
| CD45RC | IgM | Naive leucocytes | Mil 15 (288D2) |
| SwC9 | IgG1 | Macrophages (and possibly monocytes) | C4 |
Statistics
The data were analysed using paired t-tests and the χ2 test (spss; SPSS Inc., Chicago, IL, USA).
Results
Four in vivo laryngeal transplants were performed using eight animals. All donor–recipient pairs were matched at the MHC locus (CC). No epithelial cell denudation or necrosis was observed histologically. There was an abundance of immunologically active cells both before and after transplantation (Fig. 1 and Table 3). The number of cells expressing different combinations of myelomonocyte-associated antigens was counted and a subset of the data is shown in Fig. 2. Univariate analysis using paired t-tests demonstrated that numbers of some cell subsets increased significantly in the epithelium and lamina propria of the subglottis, while no subsets decreased significantly. By contrast, in the supraglottis, the numbers of some subsets decreased significantly while no subsets increased (Table 3). However, although the direction of the changes was consistent within a subsite, the cell types involved were not always consistent between subsites. In order to assess overall changes in myeloid cells and T cells, the direction of change between 0 and 8 h was categorized for each subset in each subsite in each animal, into + (increase) or – (decrease) or * (no change). The directions of change in numbers of myeloid cells and T cells were then analysed by χ2 test against an expected null hypothesis of an equal number of increases and decreases for each subsite (Table 3). Again, significantly greater numbers of T cell and myeloid subsets increased in the epithelium and lamina propria of the subglottis and decreased in the supraglottis than predicted by the null hypothesis.
Fig. 1.
Expression of major histocompatibility complex (MHC) class II and myelomonocytic cell antigens in the pig laryngeal mucosa after 3 h of cold ischaemic time and 8 h of reperfusion. Blue, MHC class II; green, CD14; red, CD16.
Table 3.
Overview of the change in cell numbers in the subglottis and supraglottis following laryngeal transplantation showing the difference in cell numbers between induction of anaesthesia of the donor animal and the end-point, which was following 8 h of reperfusion within the recipient animal.
| Subglottis | Supraglottis | |||||||
|---|---|---|---|---|---|---|---|---|
| Epithelium | Lamina propria | Epithelium | Lamina propria | |||||
| Change | P-value | Change | P-value | Change | P-value | Change | P-value | |
| CD172+CD163–MHCII– | + + − + | + + + + | – − – + | – − – + | ||||
| CD172+CD163+ MHC II+ | + − + + | + + + + | – − – – | + − + + | ||||
| CD172+CD163– MHC II+ | ** + + | + + + + | 0·003 | – * − + | – − – + | |||
| CD172–CD163– MHC II+ | + + + + | 0·018 | + + + + | – * − + | – + − + | |||
| MHCII+CD14–CD16– | + + + + | 0·036 | + + + + | 0·015 | – − – – | 0·024 | – − – – | 0·021 |
| MHCII–CD14+CD16+ | + + + + | + + − + | – − + – | – + + + | ||||
| MHCII+CD14+CD16+ | + − + + | + + + + | 0·032 | – − – + | – + − + | |||
| Myeloid cells χ2 | Value 15·4 | P-value < 0·01 | Value 24·1 | P-value < 0·001 | Value 9·8 | P-value < 0·05 | Value 0·5 | P-value n.s. |
| CD4+CD25–CD45RC– | + − – + | + + + + | 0·017 | *– − + | – − – + | |||
| CD4+CD25+CD45RC– | + − + – | – − – + | – − – + | – − – – | ||||
| CD4+CD25–CD45RC+ | * + + * | + + + + | – * + * | – –+ + | ||||
| CD4+CD25+CD45RC+ | **** | *** + | **** | – –* – | ||||
| CD8+CD25–CD45RC– | + + − + | – + + + | – − – – | – − + – | ||||
| CD8+CD25+CD45RC– | + − – + | + + * + | – − – – | 0·010 | + + – – | |||
| CD8+CD25–CD45RC+ | + + + + | + + + + | 0·015 | – − – – | 0·041 | – − + – | ||
| CD8+CD25+CD45RC+ | **** | * + ** | – *** | **** | ||||
| CD4+CD8–γδ– | + + − + | + + − + | – − – + | + − + − + | ||||
| CD4–CD8+γδ– | + + + + | – –+ + | – − – – | + – − – – | ||||
| CD4–CD8–γδ+ | + + + – | + + + + | – − – – | 0·014 | – − – – | |||
| CD4–CD8+γδ+ | + + − + | + + − + | – − – – | – –* – – | ||||
| Lymphoid cells χ2 | Value 8·5 | P-value < 0·05 | Value 20·6 | P-value < 0·001 | Value 23·7 | P-value < 0·001 | Value 11·5 | P-value < 0·01 |
| Both myeloid and lymphoid cells χ2 | Value 22·6 | P-value < 0·001 | Value 37·7 | P-value < 0·001 | Value 33·1 | P-value < 0·001 | Value 9·7 | P-value < 0·05 |
+: Indicates an increase in cell numbers (per mm2); –: indicates a decrease.
No change. Statistically significant data sets are marked on the table; n.s.: not significant.
Fig. 2.
Expression of myeloid and lymphoid antigens within the laryngeal epithelium before and after transplantation. (a) Major histocompatibility complex (MHC) class II+CD163+CD172+ cells within the supraglottis. (b) MHC class II+CD163+CD172+ cells within the subglottis. (c) CD8+CD4–γδ– T cells within the supraglottis. (d) CD8+CD4–γδ– T cells within the subglottis. Each symbol and line represents results from a single animal.
Discussion
The results from these short-term laryngeal transplants showed that the content of T cells (CD4 and CD8) and myeloid cells (MHC class II, CD14, CD172) within the mucosa of the MHC-matched pig laryngeal grafts was significantly altered following transplantation and reperfusion. However, as the magnitude of changes seen was less than interpig variation in each parameter, the occurrence of clinically significant ischaemia-reperfusion injury in this model is questionable.
This experiment demonstrated further the utility of the pig model in experimental studies of laryngeal transplantation. All the grafts were well-perfused at the time the animals were killed, as confirmed by gross appearance and by using laser-Doppler fluxmetry (data not included). Although it may have been possible to study reperfusion injury simply by temporarily ligating and perfusing the larynx, we wished to study the effect of the combined challenges of transplantation surgery, perfusate, ischaemia and reperfusion. These observations are a prerequisite to interpreting later changes in both matched and unmatched animals.
Given the time scale (8 h) and the full MHC-matching, although mismatched for minor antigens, the probable mechanisms of these changes included a combination of surgical, ischaemic and reperfusion injury. However, there was considerable interindividual variation in the numbers of cells present in all of the anatomical sites, consistent with our previous studies [26]. Taken together with the inconsistency in cell types undergoing significant change, our results suggest that the effects of ischaemia-reperfusion injury, although present, are of lower magnitude than the interindividual variation. It is therefore arguable that the level of injury incurred by operation, ischaemia and reperfusion may not be biologically or clinically significant, at least over the time scale observed. Although short, 8-h reperfusion has been sufficient to demonstrate immediate reperfusion injury in a range of other transplants [21,27]. Our results suggest, therefore, that progression to longer-term, recovery laryngeal transplants would be an acceptable experimental procedure.
Interestingly, discrete anatomical subsites of the laryngeal mucosa were affected differently. Myeloid and lymphoid cell numbers increased both in the subglottis and trachea (data not shown for the trachea) while decreases were observed in the supraglottis. In other systems, increases in cell numbers are associated with histological and/or functional injury [27,28], suggesting that the supraglottis may have been less affected by the insults applied here. However, this does not explain the observation that cell numbers actually decreased in the supraglottis, suggesting active mobilization from the site.
Because the number of cells in tissues at a single time-point is a balance between influx, efflux and transit time, differences in any of these between subglottis and supraglottis may account for the observed differences. First (the hypothesis we favour), the different epithelial types (supraglottis: squamous; subglottis: respiratory) could directly influence cells in the lamina propria, affecting tolerance to injury and expression of specific molecules, particularly chemokines and α4β7 [29,30]. Secondly, there may be differences in microvessel density. We used MIL11 (expressed by pig endothelial cells) to check for differences in microvascular density between the two subsites and found no significant difference (data not shown). Thirdly, the technical design of graft revascularization may preferentially support leucocyte recruitment to the caudal larynx. This hypothesis assumes that the venous outflow from the distinct anatomical subsites, in particular the supraglottis, was not similarly unevenly distributed, for which there was no clinical evidence. The latter observation also does not support the converse possibility that the observed results were due to impaired venous drainage caudally compared to cranially. Finally, there may have been differences in the efflux of leucocytes into lymphatics. In support of this hypothesis, a study of rat larynxes demonstrated that inflammation can increase lymphatic trafficking of dendritic cells [31].
Having identified changes in cell numbers in these studies the use of CD45 allelically mismatched pigs could be used, in future work, to determine the direction of movement of the leucocytes [32]. In addition, the use of male-into-female grafts and the use of the pig allelic antigen mismatched donor–recipient combinations may provide a more detailed description of cell movements [33].
Because only same-sex or female-into-male transplants were performed, sex-related sources of minor antigen mismatch were minimized. Although we cannot exclude the possibility that minor antigen mismatches may contribute to cellular changes, the time-course of the work described here (8 h) would be unlikely to include the effects of anything other than pre-existing antibody. Formal exclusion of minor mismatch effects would require the use of highly or fully matched pigs, such as the Babraham line of Large Whites [34] or the more inbred derivatives of the NIH minipigs.
In summary, our results demonstrate that some reperfusion injury does appear to occur in laryngeal graft mucosa over the first 8 h of reperfusion as measured by significant changes in populations of immunologically active cells. However, the observed changes were less than the large inter- and intrapig variation also demonstrated. Thus, although the biological significance of the changes in leucocyte numbers and the extent to which these changes will compromise graft survival is not completely clear, we predict that short cold-ischaemic times as used here will not generate severe reperfusion injury and enhanced allorejection in transplanted larynxes in animals or humans progressing to recovery in an MHC-matched setting. Longer-term recovery studies are necessary to confirm these results.
Acknowledgments
Emma Barker was an MRC Research Training Fellow, Martin Birchall and experimental costs were supported by the Wellcome Trust (grant no. 061125/Z/00); Mick Bailey was supported by the BBSRC.
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