ITx is the standard of care for patients with life-threatening complications due to intestinal failure. In post-transplant phase patients frequently develop graft dysfunction, manifested by the acute onset of diarrhea or fever. The biologic elements leading to ACR in ITx are incompletely understood, and their delineation may be useful for early diagnosis and new strategies for clinical management. We used ostomy effluents, multiplex cytokine assay, and high-throughput proteomics analysis as a noninvasive biomaterial and biologically robust technology for longitudinal assessment of ITx patients. This was done in response to the unmet need for understanding the mechanism and determining biomarkers of rejection. In this study, we found that increased proinflammatory cytokines and HD5 are detectable in the ostomy effluent early posttransplant and progressively decline over a period of 8 weeks irrespective of the occurrence of rejection. More interestingly, HNP 1 and 2 are increased at the time of biopsy-diagnosed rejection and this is associated with increased neutrophils in the intestinal crypts. These results support the hypothesis that early “innate” tissue injury is an important component of the early graft rejection response.
Transplantation is a complex immunological state in which there is constant interaction between the graft and the recipient's immune system (19). Epithelial cells play an active role in mucosal immune activity by secretion of innate immune cytokines in response to microbial or metabolic stimuli, resulting in the molecular stress response (15, 20). The transplantation event is a major metabolic stress to the intestinal graft, putting the epithelium at risk for a molecular stress response. Consistent with this, four innate immune cytokines (TNF-α, IL-1β, IL-8, and G-CSF) typical of the epithelial stress response were detectable in ostomy effluents early in the posttransplant period, declining thereafter. IFN-γ remained at high levels throughout the posttransplant phase, perhaps reflecting its probable origin from CD8+ T cells and natural killer (NK) cells (21), which are physiologic lamina propria residents and constitutively express IFN-γ (22). More interestingly, IL-8, TNF-α, and IL-1β (and other epithelial stress response chemokines) are chemotactic for neutrophils and induce both neutrophil and Paneth cell-derived defensins (23, 24).
Paneth cells are another critical cellular component of innate immunity, and make up part of the epithelial barrier at the base of intestinal crypts. Impairment of Paneth cell-derived defensin, notably HD5, is a feature of genetic susceptibility in Crohn's disease (25), a disease with some clinical and pathologic features of intestinal rejection (26). In ITx recipients with ACR, we observed that HD5 levels were higher immediately posttransplant (Fig. 3C), and also after treatment for rejection in two patients (data not shown), perhaps due to steroid induction of HD5 in maturing Paneth cells (27). Along with other animal model studies, our study suggested that restoring HD5 is important for improving mucosal barrier function and protection from deleterious microbial commensals (28–30).
Several innate cell populations have been indicated to play critical roles in animal ACR models, including NK cells (21, 31), dendritic cells (32, 33), and neutrophils (34–36). Neutrophil-derived anti-microbial peptides are known not only to contribute to oxygen-independent microbial cytotoxicity, but also serve as chemotaxins for macrophages, dendritic cells, and T lymphocytes (37, 38). In our study, HNPs were elevated in ITx recipients with rejection as an early graft feature posttransplant (Figs. 2A,B and 3A,B). Fecal calprotectin, another neutrophil-derived protein, has also been reported to be elevated in patients with rejection (6). Neutrophils mediate tissue damage by an array of cytotoxic and proinflammatory mechanisms (34). Thus, neutrophils may participate in the balance of transplant integrity both through microbial protection during and after injury (3), and paradoxically by potentially augmenting adaptive immunity and exacerbating rejection. To our knowledge, this is the first human study of the function and distribution of neutrophils in ACR patients.
Although high-throughput MALDI-MS technology allows for the detection of hundreds of proteins simultaneously, there are several limitations that preclude its direct clinical use. It is expensive, time consuming, and requires proficiency in proteomics for sample analysis. In addition, interpretation of MALDI-MS data requires bioinformatics analysis and identification of peptides/proteins. Accordingly, molecules identified by MALDI-MS need development of conventional assays for clinical applications. For this reason, we performed ELISA to validate HNPs in ostomy effluent samples, and evaluated the correlation of ELISA measurements with the proteomic data. The results showed excellent correlation between the two modalities, suggesting that a simple immunoassay (e.g., ELISA) test of ostomy effluent could potentially complement sequential biopsies for ACR diagnosis and monitoring in clinical application.
This study is unique in using ostomy effluents (rather than biopsies or serum) as a biospecimen to delineate ACR in human ITx recipients. We emphasize that this study was not designed for transplant rejection biomarker validation. Preliminary results of biomarker discovery have been promising, but the dataset has been small due to many confounding demographic and clinical variables, and there is a dearth of preliminary data to estimate the sample size (19). Also, with only 200 ITx performed per year worldwide, a larger multicenter study and the use of less complex protein quantitation methods is necessary for suitable clinical monitoring. However, this study reveals an early and underappreciated role of innate immunity in ACR, and suggests that monitoring the HNP levels in ostomy effluents has the potential to permit quantitative assessment of ITx in a noninvasive manner.
MATERIALS AND METHODS
Institutional review board approval was obtained in compliance with the Health Insurance Portability and Accountability Act guidelines, for this study including all patients undergoing ITx between July 2008 and September 2009 at the Dumont-University of California, Los Angeles (UCLA) Transplant Center. Demographics, type of transplant, endoscopic procedure, postoperative immunosuppression, feeds, infectious status, and histopathology were recorded. ACR was defined according to the consensus statement that was developed at the 8th International Small Bowel Transplant Symposium in 2003 (4).
Our standard immunosuppression regimen included induction with an IL-2 receptor antagonist such as daclizumab (Zenapax, Roche Laboratories, Nutley, NJ) or rabbit antithymocyte globulin (Thymoglobulin, Genzyme, Cambridge, MA). Maintenance therapy included tacrolimus (Prograf, Fujisawa USA, Deerfield, IL), corticosteroids, and mycophenolate mofetil (CellCept, Roche Laboratories, Nutley, NJ). Target tacrolimus trough levels were 10 to 18 ng/mL. ACR was diagnosed by histopathology and treated with high dose steroids and antithymocyte globulin. In sensitized patients or patients suspected of antibody mediated rejection, a combination of Rituximab 375 mg/m (2) for one dose, plasmapheresis for 5 days, and immune globulin (Gammagard, Baxter, Westlake Village, CA) 1 g/kg was used both pre- and posttransplant.
Protocol endoscopy and biopsy of the transplanted intestine were obtained once weekly beginning posttransplant day 6 to 14 for the first 3 to 4 weeks, then once every 2 weeks until 8 weeks after the operation. Endoscopy was also performed, and biopsies obtained, when patient presented with clinical symptoms, such as diarrhea or fevers, indicating abnormal allograft function. Multiple biopsies from various portions of the transplanted graft were obtained, and paraffin-embedded sections stained with H&E were graded for ACR using standard rejection criteria (4) by UCLA gastrointestinal pathologists. Abnormal results prompted further endoscopic surveillance as indicated.
Ostomy Effluent Collection and Proteomic Evaluation
Ostomy effluents were freshly collected from the ostomy pouch an hour before all endoscopic procedures. The effluent specimens were immediately placed on ice, centrifuged at 2000 rpm for 7 min at 4°C, and the supernatant were stored at −80°C for batch processing at the UCLA High Throughput Clinical Proteomics core laboratory. The detailed procedure for sample processing and proteomic evaluation is illustrated in the supplemental material (see Supplemental Digital Content 1, http://links.lww.com/TP/A421). Briefly, each sample was processed identically for protein extraction, fractionation, and MALDI-MS analysis. Proteomic data were analyzed by various statistical methods.
Luminex Evaluation of Cytokines
An aliquot of protein extraction from ostomy effluent was used for Luminex evaluation, with the Bio-Rad human cytokine 17-plex assay (G-CSF, granulocyte-macrophage-CSF, IFN-γ, IL-10, IL-12, IL-13, IL-17, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, MCP-1, MIP-1β, and TNF-α) according to the manufacturer's instructions (Bio-Rad, Hercules, CA). All samples were mixed with a matrix of 2 mg/ml bovine serum albumin, 2 mM phenylmethylsulfonyl fluoride, 20 mM ethylenediaminetetraacetic acid and 1% Triton X-100 in equal volume to the sample volume. This same matrix was used to reconstitute one tube of cytokine standard. Data were acquired and analyzed on a Bio-Plex 200 system (Bio-Rad, Hercules, CA), and cytokine levels were normalized to protein concentrations to account for the different protein amount in each sample.
ELISA and IHC of HNPs
The total amount of HNP1 to 3 in protein extractions were quantified in duplicates by an ELISA kit following manufacturer's instructions (Cell Sciences, Canton, MA), using a standard curve constructed for each plate. Protein concentrations were also determined by BCA assay for each sample, and HNP1 to 3 levels were normalized to total protein (pg/mg).
Standard IHC procedures were used on biopsy sections (5 μm). Each section was incubated with anti-HNP 1 to 3 antibody (rabbit, 1:2000) or preimmune serum (rabbit, 1:2000) as negative control. Anti-Rabbit IgG biotinylated antibody was used as secondary and streptavidin-peroxidase conjugated antibody was used as tertiary (Vector Laboratories, Burlingame, CA), according to the manufacturer's protocol, and the sections were developed by diaminobenzidine tetrahydrochloride substrate (Pierce, Rockford, IL). For quantitation, 10 crypts in 3 different fields on 1 biopsy slide were evaluated for positivity of HNP staining. The number of HNP-positive crypts per 10 crypts was counted and averaged.
All data analyses were conducted in R software (http://www.r-project.org). To correct the pseudoreplicate effect from repeated sampling over time and the noisy nature of the human study, a mixed-effects model (package nmle) was used to evaluate each protein feature in proteomic or Luminex data. For the initial screening of the protein candidates associated with rejection, we set subject level as the random effect in the mixed-effect model, and the fixed effects included rejection status, posttransplant days, type of transplant, immunosuppressant, feeds, infection, and age. For confirmation of the significance of the HNP in rejection, we only included posttransplant days and rejection status as fixed effects, and subject level as random effect to optimize the mixed-effect model. Kruskal-Wallis test was used for group comparison in ELISA. Fisher's exact test was used to compare HNPs-positivity between rejection groups in IHC. Pearson's correlation coefficient was calculated to assess the correlation between the measurements of HNPs using ELISA and MALDI.
The authors thank Rita Kern for assisting with the Luminex assays, the UCLA Translational Pathology Core Laboratory for preparation of biopsy sections, Drs. Tomas Ganz and Erika Valore for providing anti-HNP 1–3 and anti-HD5 antibodies, Dr. Marvin Ament for his support as Division chief, and Drs. Martin Martin, Suzanne McDiarmid, and Robert Venick for editorial insight.
1.Kaufman SS, Atkinson JB, Bianchi A, et al. Indications for pediatric intestinal transplantation
: A position paper of the American Society of Transplantation. Pediatr Transplant
2001; 5: 80.
2.Hanto DW, Fishbein TM, Pinson CW, et al. Liver and intestine transplantation: Summary analysis, 1994–2003. Am J Transplant
2005; 5: 916.
3.Fishbein TM. Intestinal transplantation
. N Engl J Med
2009; 361: 998.
4.Ruiz P, Bagni A, Brown R, et al. Histological criteria for the identification of acute cellular rejection
in human small bowel allografts: Results of the pathology workshop at the VIII International Small Bowel Transplant Symposium. Transplant Proc
2004; 36: 335.
5.Wu T, Abu-Elmagd K, Bond G, et al. A schema for histologic grading of small intestine allograft acute rejection
2003; 75: 1241.
6.Sudan D, Vargas L, Sun Y, et al. Calprotectin: A novel noninvasive marker for intestinal allograft monitoring. Ann Surg
2007; 246: 311.
7.Mayer L. Mucosal immunity and gastrointestinal antigen processing. J Pediatr Gastroenterol Nutr
2000; 30(suppl): S4.
8.Iwaki Y, Starzl TE, Yagihashi A, et al. Replacement of donor lymphoid tissue in small-bowel transplants. Lancet
1991; 337: 818.
9.Newell KA. Transplantation of the intestine: Is it truly different? Am J Transplant
2003; 3: 1.
10.LaRosa DF, Rahman AH, et al. The innate immune system in allograft rejection
and tolerance. J Immunol
2007; 178: 7503.
11.Farmer DG, McDiarmid SV, Kuniyoshi J, et al. Intragraft expression of messenger RNA for interleukin-6 and tumor necrosis factor-alpha is a predictor of rat small intestine transplant rejection
. J Surg Res
1994; 57: 138.
12.Christopher K, Mueller TF, Ma C, et al. Analysis of the innate and adaptive phases of allograft rejection
by cluster analysis of transcriptional profiles. J Immunol
2002; 169: 522.
13.He H, Stone JR, Perkins DL. Analysis of robust innate immune response after transplantation in the absence of adaptive immunity1. Transplantation
2002; 73: 853.
14.Farmer DG, Shen X-D, Amersi F, et al. CD62 blockade with P-selectin glycoprotein ligand-immunoglobulin fusion protein reduces ischemia-reperfusion injury after rat intestinal transplantation
2005; 79: 44.
15.Kaser A, Blumberg RS. Endoplasmic reticulum stress and intestinal inflammation. Mucosal Immunol
2010; 3: 11.
16.Nilsson T, Mann M, Aebersold R, et al. Mass spectrometry in high-throughput proteomics
: Ready for the big time. Nat Meth
2010; 7: 681.
17.Li Q, Zhang Q, Xu G, et al. Metabonomics study of intestinal transplantation
using ultrahigh-performance liquid chromatography time-of-flight mass spectrometry. Digestion
2008; 77: 122.
18.McDiarmid SV, Farmer DG, Kuniyoshi JS, et al. Perforin and granzyme B. Cytolytic proteins up-regulated during rejection
of rat small intestine allografts. Transplantation
1995; 59: 762.
19.Sarwal MM. Deconvoluting the ‘omics' for organ transplantation. Curr Opin Organ Transplant
2009; 14: 544.
20.Hedges SR, Agace WW, Svanborg C. Epithelial cytokine responses and mucosal cytokine networks. Trends Microbiol
1995; 3: 266.
21.Obara H, Nagasaki K, Hsieh CL, et al. IFN-gamma, produced by NK cells that infiltrate liver allografts early after transplantation, links the innate and adaptive immune responses. Am J Transplant
2005; 5: 2094.
22.Yadavalli GK, Auletta JJ, Gould MP, et al. Deactivation of the innate cellular immune response following endotoxic and surgical injury. Exp Mol Pathol
2001; 71: 209.
23.Shi J. Defensins and Paneth cells in inflammatory bowel disease. Inflamm Bowel Dis
2007; 13: 1284.
24.Wehkamp J, Schwind B, Herrlinger KR, et al. Innate immunity
and colonic inflammation: Enhanced expression of epithelial alpha-defensins. Dig Dis Sci
2002; 47: 1349.
25.Wehkamp J, Wang G, Kubler I, et al. The Paneth cell alpha-defensin deficiency of ileal Crohn's disease is linked to Wnt/Tcf-4. J Immunol
2007; 179: 3109.
26.Fishbein T, Novitskiy G, Mishra L, et al. NOD2-expressing bone marrow-derived cells appear to regulate epithelial innate immunity
of the transplanted human small intestine. Gut
2008; 57: 323.
27.Nanthakumar NN, Young C, Ko JS, et al. Glucocorticoid responsiveness in developing human intestine: Possible role in prevention of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol
2005; 288: G85.
28.Salzman NH, Underwood MA, Bevins CL. Paneth cells, defensins, and the commensal microbiota: A hypothesis on intimate interplay at the intestinal mucosa. Semin Immunol
2007; 19: 70.
29.Salzman NH, Hung K, Haribhai D, et al. Enteric defensins are essential regulators of intestinal microbial ecology. Nat Immunol
2010; 11: 76.
30.Vaishnava S, Behrendt CL, Ismail AS, Eckmann L, Hooper LV. Paneth cells directly sense gut commensals and maintain homeostasis at the intestinal host-microbial interface. Proc Natl Acad Sci USA
2008; 105: 20858.
31.Kitchens WH, Uehara S, Chase CM, et al. The changing role of natural killer cells in solid organ rejection
and tolerance. Transplantation
2006; 81: 811.
32.Pêche H, Trinité B, Martinet B, et al. Prolongation of heart allograft survival by immature dendritic cells generated from recipient type bone marrow progenitors. Am J Transplant
2005; 5: 255.
33.Fu F, Li Y, Qian S, et al. Costimulatory molecule-deficient dendritic cell progenitors (MHC class II+, CD80dim, CD86−) prolong cardiac allograft survival in nonimmunosuppressed recipients. Transplantation
1996; 62: 659.
34.Jaeschke H, Farhood A, Smith C. Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo. FASEB J
1990; 4: 3355.
35.Morita K, Miura M, Paolone DR, et al. Early chemokine cascades in murine cardiac grafts regulate T cell recruitment and progression of acute allograft rejection
. J Immunol
2001; 167: 2979.
36.Farmer DG, Anselmo D, Shen XD, et al. Disruption of P-selectin signaling modulates cell trafficking and results in improved outcomes after mouse warm intestinal ischemia and reperfusion injury. Transplantation
2005; 80: 828.
37.Yang D, Chen Q, Chertov O, et al. Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells. J Leukoc Biol
2000; 68: 9.
38.Grigat J, Soruri A, Forssmann U, et al. Chemoattraction of macrophages, T lymphocytes, and mast cells is evolutionarily conserved within the human alpha-defensin family. J Immunol
2007; 179: 3958.
Intestinal transplantation; Rejection; Proteomics; Innate immunity
Supplemental Digital Content
© 2011 Lippincott Williams & Wilkins, Inc.