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Eur J Cardiothorac Surg 2006;29:760-766
© 2006 Elsevier Science NL

Neutrophil transendothelial migration potential predicts rejection severity in human cardiac transplantation

^a Prof Eoin O’Malley National Centre for Cardiothoracic Surgery, Mater Misericordiae University Hospital, Dublin, Ireland
^b UCD School of Medicine and Medical Science, Mater Misericordiae University Hospital, University College Dublin, Ireland
^c Department of Pathology, Mater Misericordiae University Hospital, Dublin, Ireland
^d Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Ireland

Received 29 September 2005; received in revised form 21 December 2005; accepted 26 January 2006.

^_* Corresponding author. Address: Department of Surgery, Conway Institute of Biomolecular & Biomedical Research, Belfield, University College Dublin, Dublin 4, Ireland. Tel.: +353 1 7166733; fax: +353 1 7166887. (Email: william.watson{at}ucd.ie).

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Appendix A References

 
Objective: Transplant rejection remains a clinical problem despite therapies that focus on lymphocyte suppression, with little attention focused on the neutrophil. Neutrophils are however the first leukocyte to infiltrate the allograft, are capable of causing myocardial damage and may facilitate lymphocytes recruitment. We hypothesised that an early allograft neutrophil infiltration influences rejection severity. Methods: Myocardial neutrophil infiltration was assessed using CD15 and myeloperoxidase immunohistochemistry of rejection surveillance endomyocardial biopsy specimens from human cardiac transplant recipients (n = 18). In patients undergoing cardiac transplantation (n = 10), neutrophils were isolated from multiple perioperative blood samples using a ficoll-based density gradient centrifugation method. The expression of the neutrophil adhesion protein CD11b was then assessed using flow cytometry and compared to subsequent endomyocardial biopsy rejection grades. The effects of contemporary immunosuppressive agents on human neutrophil CD11b were also assessed using healthy control volunteers. Results: Myeloperoxidase staining of endomyocardial biopsies from human heart transplant recipients demonstrated a positive correlation between the degree of neutrophil infiltration and rejection severity at the first postoperative biopsy. Rejection severity was unrelated to ischaemic time. Functional assessment of neutrophils obtained from recipients was then performed. Perioperative transplant sampling demonstrated a significant correlation between the preoperative expression of CD11b and rejection grade at the first postoperative biopsy. In addition, dynamic changes in CD11b expression in the first 24 h positively correlated with subsequent rejection severity. In vitro experiments showed that transplant immunosuppression did not alter neutrophil CD11b expression. Conclusion: This study demonstrates a potentially greater role for neutrophils in cardiac transplantation than previously recognised, and suggests that blockade of the early allograft neutrophil infiltration might prevent subsequent lymphocyte recruitment and attenuate rejection.

Abbreviations: CPB = cardiopulmonary bypass • ISHLT = International Society for Heart Lung Transplantation

Key Words: Transplantation • Cardiac • Neutrophil • Rejection • Adhesion molecules • CD11b

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Appendix A References

 
Allograft rejection remains a significant factor in survival following cardiac transplantation. Our current understanding of the rejection process describes it as a delayed type hypersensitivity reaction primarily involving the lymphocyte. Consequently, contemporary and emerging therapies target lymphocyte suppression rather than other components of immune function, such as the neutrophil. The neutrophil is a major component of innate immunity and is the first leukocyte to infiltrate the cardiac allograft [1]. Their presence may then contribute to tissue damage by the production of oxygen radicals and the release of proteolytic enzymes. Neutrophils may in addition have a wider role than direct myocyte toxicity, as there is increasing evidence suggesting an interaction between the innate immune system and the ability of lymphocytes to cause tissue damage [2–5].

If neutrophil infiltration correlates with the rejection process, the identification of factors influencing their migration may represent an earlier marker or even a predictive marker of rejection. Tissue infiltration by circulating leukocytes is facilitated by adhesion molecules. The process of neutrophil transendothelial migration involves endothelial rolling, mediated by selectins, and firm adhesion facilitated by neutrophil ß₂-integrins, including the CD11b/CD18 complex. In animal models using cardiac xenografts and allografts, CD11b/CD18 inhibition reduces neutrophil infiltration of the myocardium and prolongs graft survival [6,7].

Cytoimmunological monitoring in heart transplantation has been performed using lymphocytes but has not been attempted using neutrophils [8,9]. We hypothesised that an early postoperative neutrophil infiltration of the allograft influenced the rejection severity and that this is dependent on an individualised neutrophil migratory response. A clearer understanding of the interrelationships between allograft rejection, and neutrophil migratory function, might aid our understanding of why rejection remains a clinical problem despite potent lymphocyte suppressive therapies.

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Appendix A References

 
2.1 Patient selection
The study was approved by the Mater Misericordiae University Hospital ethics committee with written informed consent obtained. The Irish Heart Transplant Program has been delivered through this site since 1985 and a total of 228 transplants have been performed.

2.2 Patient population
Group 1: Immunohistochemical analysis of endomyocardial biopsies was performed retrospectively on 18 transplant recipients.

Group 2: Neutrophil isolations were performed from consecutively recruited patients undergoing cardiac transplantation (n = 10). There were four females and six males, with a mean age of 51.1 years (SD ± 16.2). Six of the recipients had a preoperative dilated cardiomyopathy pathology and four an ischaemic pathology. Neutrophil isolation was performed from six perioperative samples, taken; preoperatively, at release of aortic cross-clamp, and 1, 6, 12 and 24 h after aortic cross-clamp release. These results were compared to rejection severity at the first postoperative endomyocardial biopsy.

Group 3: Neutrophils from normal healthy controls (n = 32, 14 females, mean age 28.9 ± 5 years) were isolated to perform investigations of standard transplant immunosuppressive drugs under in vitro conditions.

2.3 Transplantation management
Donor recipient matching was performed based on blood group and size. HLA matching is not performed, however, recipients with high levels of panel reactive antibodies (>20%) underwent a prospective crossmatch. Cardioplegic arrest of the donor heart is achieved with antigrade administration of 2 l of cold (4 °C) St Thomas solution and the heart is additionally cooled with topical slush and transported in an icebox. Cardiopulmonary bypass is utilised using a polyvinyl circuit, roller pump and membrane oxygenator. Orthotopic implantation is performed using the bi-caval technique. Immunosuppression is managed by a standardised protocol. Methylprednisolone is administered prior to the release of the aortic cross-clamp and continued on a 5 mg/kg/day in divided doses intravenously until the patient is able to take oral prednisolone. Cyclosporin is introduced on day 1 with target trough levels of 100–200 ng/ml. Induction therapy is not used routinely in our centre. However, patients with acute renal injury are managed with delayed introduction of cyclosporin and are instead treated with basiliximab (20 mg on days 1 and 4). Mycophenolate (2–3 mg/day) is introduced on day 2.

2.4 Rejection surveillance
Rejection surveillance is performed with a percutaneous endomyocardial biopsy. Biopsy analysis was performed by pathologists blind to the neutrophil study data and scored using the ISHLT endomyocardial biopsy rejection grade system [10]. The first biopsy is performed 7–10 days following transplantation. Subsequent biopsies are performed at increasing intervals, subject to clinical parameters.

2.5 Reagents
Dulbecco's modified eagle's medium (DMEM), penicillin and streptomycin solution, L-glutamine, and phosphate buffered saline (PBS) were purchased from Gibco life Technologies Ltd. Dextran and ficoll were purchased from Amersham Biosciences. Foetal calf serum (FCS), Cyclosporin A, methylprednisolone-21-hemisuccinate sodium and mycophenolic acid were supplied by Sigma. All other chemicals were supplied by Sigma-Aldrich, Dorset, UK, unless otherwise stated.

2.6 Endomyocardial biopsy immunohistochemistry
Specimens were fixed in 10% formaldehyde and embedded in paraffin. Formalin-fixed paraffin-embedded sections of 4 µm were mounted on glass slides. Immunohistochemical staining for myeloperoxidase was performed using a standard avidin–biotin complex method (Vector Laboratories, CA, USA). Antigen retrieval was carried out using a 0.1% trypsin solution in PBS/Tween for 15 min at 37 °C. Sections were incubated with the myeloperoxidase antibody (DakoCytomation), using a 1:300 dilution for 45 min at room temperature. Human tonsil specimens were used as a positive control. The substrate chromagen solution, 3,3'-diaminobenzidine (Sigma), was prepared and applied to the slides for 5 min. The slides were counter stained with hematoxylin and eosin. Finally, the slides were dehydrated and coverslipped. For CD15 measurement, the primary antibody was obtained from www.bdbiosciences.com and the antigen unmasking step consisted a 10 min microwave heating at 850 W in 0.01M citrate at pH 6.0. Slides were assessed in a blinded fashion by a consultant pathologist. The slides were viewed at 200x, the visual field selected was the area of maximal staining intensity within the myocardial tissue of the biopsy, and the score was generated by counting the number of cells staining positively in that single field.

2.7 Neutrophil isolation
Venous blood samples were obtained with 1 ml of 0.106 mmol/l sodium citrate solution per 10 ml of whole blood. Neutrophils were isolated by dextran (3%) sedimentation and centrifugation through a discontinuous ficoll gradient. Remaining red blood cells were lysed using 0.8% NH₄Cl. Isolated neutrophils at a concentration of 1 x 10⁶ were resuspended in DMEM supplemented with L-glutamine, penicillin/streptomycin and 10% FCS. Cells were incubated at 37 °C in a humidified CO₂ incubator (5% CO₂) in polypropylene tubes to prevent adherence. Neutrophil purity was assessed by size and granularity on flow cytometry and was consistently greater then 95%.

2.8 Neutrophil surface markers
Neutrophil cell surface marker measurement was performed on isolated neutrophils as described above. Surface expression of CD11b (Becton Dickinson) was assessed by flow cytometry as previously described [11]. Briefly 500,000 neutrophils in 500 µl of medium were incubated with 10 µl of antibody at 4 °C for 20 min, washed and analysed by flow cytometry using a Coulter ELITE cytofluorometer (Coulter Electronics, Bedfordshire, UK) and were compared to an unstained control.

2.9 Immunosuppression and the neutrophil
Isolated human control neutrophils were stimulated with LPS (1 ng/million) or TNF (10 ng/million) for 1 h in an incubator. Cyclosporin (1 µM), methylprednisolone (1 µM) and mycophenolate (1 µM) were then added for 3 h prior to measurement of CD11b expression.

2.10 Statistical analysis
Correlations among non-parametric data were performed with a Spearman rank correlation coefficient. Parametric data were subjected to a one or two-factor ANOVA as appropriate followed by post hoc comparison with a Fischer's least significant difference test. Survival was calculated by the method of Kaplan & Meier.

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Appendix A References

 
3.1 Neutrophil infiltration of the first post-transplant endomyocardial biopsy
We histologically examined the first post-transplant endomyocardial biopsy of 18 patients (Group 1) from our transplant recipient population for the presence of neutrophils. Initial examination of the biopsy specimens with H&E staining identified little neutrophil infiltration and no relationship with rejection grade (P = 0.708). Additional immunohistochemical staining of the same samples with the granulocyte cell surface marker CD15 also demonstrated few neutrophils and no relationship with rejection (P = 0.236). As these markers only measure the presence of existing neutrophils, we next wanted to detect evidence of any recent passage of neutrophils into the biopsy. Myeloperoxidase is a product specific to the neutrophil and its detection in tissue is a marker of the presence of neutrophils [12]. We next wanted to determine if there was an increase in myeloperoxidase staining in our biopsy samples. There was a positive correlation between myeloperoxidase staining in the biopsy and rejection grade (n = 18, R = 0.477, P = 0.011) (Fig. 1 , Table 1 ). The myeloperoxidase staining seen in these slides may reflect an earlier infiltration of neutrophils, which has undergone clearance by surrounding phagocytes in the myocardium. Analysis of the second biopsy again identified few viable neutrophils by H&E or CD15 staining. However, at this stage there was no significant relationship between myeloperoxidase and rejection severity (P = 0.615).

View larger version (128K): [in this window] [in a new window]   Fig. 1. Allograft neutrophil infiltration: endomyocardial biopsies performed on the allograft 7–10 days after transplantation were assessed using H&E staining and CD15 and myeloperoxidase (MPO) immunohistochemistry to assess the presence or recent passage of neutrophils. A single-blinded observer selected the area of maximum staining intensity within the myocardial biopsy at 200x magnification. The number of positively staining cells was then counted within that single field. No relationship was seen between rejection severity and neutrophil presence as determined by H&E or CD15 (n = 18, P = 0.236). However, a positive correlation was seen between MPO staining and rejection (n = 18, P = 0.011, R = 0.477).

View larger version (7K): [in this window] [in a new window]   Fig. 2. Perioperative CD11b measurement and rejection: neutrophil CD11b expression was measured in blood samples obtained at multiple time points at transplantation. These measurements were compared with the ISHLT rejection grade severity of the endomyocardial biopsy performed 7–10 days later. (A) This represents the relationship between neutrophil CD11b expression measured preoperatively and subsequent rejection (n = 10, P = 0.011, R = –0.746). (B) The change in CD11b expression over the first 24 h after the time of aortic cross-clamp release was compared with rejection (n = 10, P = 0.048, R = 0.803). The mean channel fluorescence (MnX) measured by flow cytometry is used to quantify CD11b expression.

View larger version (49K): [in this window] [in a new window]   Fig. 3. Neutrophil CD11b and immunosuppression: isolated human control neutrophils were stimulated with LPS (1 ng/million) or TNF (10 ng/million) for 1 hr in an incubator. Cyclosporin (1 µM), methylprednisolone (1 µM) and mycophenolate (1 µM) were then added for 3 hrs prior to measurement of CD11b expression. The mean channel fluorescence (MnX) measured by flow cytometry is used to quantify CD11b expression. A significant rise in CD11b expression is seen with LPS and TNF stimulation; however, none of the immunosuppressive agents had any significant effect on CD11b expression.

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Appendix A References

This early neutrophil infiltration may play a direct role in tissue destruction. However, there is no doubt that the acquired immune sensitivity of the cytotoxic lymphocyte is fundamental to allograft rejection. Rejection grade is generated based on histological evidence of myocyte damage and mononuclear leukocyte infiltration with the timing of the first biopsy at 7–10 days postoperatively designed to detect the first evidence of the delayed sensitisation of the acquired immune system [15]. An initial neutrophil infiltration may play a significant role in the subsequent activation of the lymphocyte [4]. Clavein et al. [16] have shown lymphocyte activation by oxygen radicals of which neutrophils are a major source. Innate immune responses also promote T-cell mediated adaptive immunity by activating antigen presenting cells involved in lymphocyte priming. In a mouse lymphocyte-mediated myocarditis model, depletion of neutrophils at the time of lymphocyte infusion prevents myocardial damage and improves animal survival. This occurs even when neutrophil counts normalise 48 h later [5]. Engeman et al. [2] have demonstrated in a contact dermatitis model that hapten stimulation is associated with the generation of neutrophil chemokines within 6–9 h. Neutrophil depletion or neutrophil chemokine antagonism at the time of hapten re-exposure significantly attenuates lymphocyte infiltration, and this response is augmented by neutrophil supplementation [2]. In rodent cardiac allograft transplantation, graft survival is prolonged if neutrophil allograft infiltration is prevented through either depleting neutrophils or antibody blockade of neutrophil chemoattractants. This has been shown to prevent the subsequent production of other leukocyte chemoattractants and the subsequent infiltration of lymphocytes [4].

Having shown that myeloperoxidase staining correlated with rejection in the first postoperative biopsy, and that this could be explained by an early neutrophil infiltration, we next wanted to identify a less invasive systemic blood marker to profile individual vulnerability to this neutrophil infiltration. We targeted the ß-2 integrin, CD11b/CD18 complex, because of its facilitative role in neutrophil transendothelial migration and the fact that, although limited to leukocytes, it is not found on lymphocytes [17,18]. Transendothelial migration depends on interactions between neutrophil integrins and endothelial adhesion molecules. ICAM-1 is a common endothelial target for neutrophil CD11b and increases in ICAM-1, seen with cardiac transplantation, are associated with neutrophil infiltration of the allograft and graft failure [19]. Blockade of CD11b has successfully improved survival in animal models of xenograft and allograft transplantation [6,7]. Not only does CD11b play a role in neutrophil adhesion, but it also influences the generation of reactive oxygen intermediates by neutrophils in the adherent state [20]. Specifically, it has been shown that CD11b is not only involved in cellular adhesion and migration but rather is also fundamental to adhesion dependent delivery of destructive reactive oxygen intermediates to the myocyte's intracellular compartment. Preventing neutrophil myocyte interactions with antibodies to CD11b and CD18 prevents this damage [21].

We have shown an inverse correlation between CD11b expression prior to surgery and rejection. This appears contradictory to the finding of a positive correlation between rejection and the rise in CD11b over the first 24 h following surgery. However, this could represent the perioperative inflammatory response and recognition of the allograft by the innate immune system, reflecting the importance of the neutrophils ability to increase CD11b expression following contact with the allograft. The observation that higher CD11b expression prior to contact with the allograft was protective against rejection may alternatively reflect an unrecognised preconditioning effect on neutrophil excitation. This may consequently suppress an individual's ability to mount a subsequent response on graft exposure. Collectively, these results suggest that the individual neutrophil CD11b response may be fundamental to the severity of rejection at the early postoperative biopsy.

In this study we have demonstrated that CD11b expression is unaffected by contemporary immunosuppressive agents. While lymphocytes and some aspects of neutrophil function are modified by immunosuppression, the expression of CD11b remains unaffected. The measurement of a neutrophil adhesion molecule from a peripheral blood sample may reflect the most relevant point of action of this aspect of neutrophil function, i.e. readiness to migrate from the circulating blood. Even though aspects of neutrophil function other then CD11b adhesion are modified by immunosuppressive therapy, the ability of large numbers of neutrophils to infiltrate the allograft may alone facilitate lymphocyte sensitisation.

This study has attracted our attention to the potential contribution of neutrophil migratory response to allograft rejection, an area largely neglected in comparison to our understanding of the role of the lymphocyte. This study suggests that neutrophil CD11b measurement is a potential predictor of early rejection following surgery and may represent a non-invasive test that could be carried out preoperatively or perioperatively. An ability to predict the result of the first biopsy would allow an opportunity to individualise immunosuppressive therapy from the time of surgery. The observation that contemporary immunosuppressive agents lack efficacy against this aspect of neutrophils function suggests a potential therapeutic gap. Leumedin, an amino acid derivative which inhibits neutrophil CD11b dependent migration, has already been shown in animal experiments to improve allograft survival [7]. Prevention of the early allograft neutrophil saturation may prevent neutrophil-mediated damage and attenuate lymphocyte activity. Stopping this initial neutrophil influx in animal allografts has a survival benefit [4]. Endeavours to antagonise this initial neutrophil influx in humans may also prove rewarding.

	Appendix A

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Appendix A References

 
Conference discussion

Dr J. Tsai (Pingtung, Taiwan): Would you kindly tell us about your examination, the first day, second day, third day, seventh day, the lymphocyte number, and the lymphocyte number correlated with your rejection. First day and maybe seventh day, if you have this data.

Dr Healy : We do indeed.

Prior to our investigations, researchers have looked at lymphocytes primarily and found no relationship between rejection and total lymphocyte number, although there are correlations with lymphocyte subsets.

In relation to our studies, we did look at perioperative neutrophil, lymphocyte, eosinophil counts, and we found no significant relationship between neutrophil numbers and rejection severity.

In the prospective data presented here, we have an N of 10, but we also looked retrospectively at our records of previous transplantations and again we saw no relationship between neutrophil number and rejection in the perioperative period.

However, I think that what our studies show is that it is not the number of neutrophils in circulating blood, but rather it is their ability to get out of that circulating blood and get into the allograft tissue that is important. That is why we focused very much on the measurement of adhesion markers, which are obviously important in terms of transendothelial migration and infiltration of our allograft.

	Acknowledgments

 
This work was supported by grants from the Irish Heart Foundation and in part by the Mater College.

	Footnotes

 
Presented at the joint 19th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 13th Annual Meeting of the European Society of Thoracic Surgeons, Barcelona, Spain, September 25–28, 2005.

	References

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Appendix A References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): David G. Healy James F. McCarthy John Hurley Alfred E. Wood Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Healy, D. G. Articles by Wood, A. E. Search for Related Content PubMed PubMed Citation Articles by Healy, D. G. Articles by Wood, A. E. Related Collections Transplantation - heart