Pre-transplant plasma and cellular levels of CD44 correlate with acute renal allograft rejection
Kasper M. A. Rouschop1,*,
Joris J. T. H. Roelofs1,*,
Ajda T. Rowshani2,
Jaklien C. Leemans1,
Tom van der Poll3,
Ineke J. M. ten Berge2,
Jan J. Weening1 and
Sandrine Florquin1
1 Department of Pathology and 2 Department of Internal Medicine, Division of Clinical Immunology and Rheumatology and 3 Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, Amsterdam, The Netherlands
Correspondence and offprint requests to: K. M. A. Rouschop, MSc, Department of Pathology, Academic Medical Center, PO Box 22660, 1100 DD, Amsterdam, The Netherlands. Email: k.m.rouschop{at}amc.uva.nl
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Abstract
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Background. Since CD44 is involved in activation, proliferation, rolling and extravasation of lymphocytes, we hypothesized that it could be involved in the pathophysiology of acute renal allograft rejection.
Methods. Plasma and peripheral blood mononuclear cells (PBMCs) were collected from patients 24 h prior to transplantation and analysed retrospectively. Soluble CD44, interleukin-2 receptor (IL-2R), intracellular adhesion molecule-1 (ICAM-1) and C-reactive protein (CRP) in plasma were determined by enzyme-linked immunosorbent assay (ELISA). Cellular CD44 expression on peripheral lymphocytes was determined by flow cytometric analysis.
Results. Patients who later developed renal allograft rejection had statistically significantly increased soluble CD44 levels, but not soluble ICAM-1, IL-2R or CRP in plasma prior to transplantation. In addition, cellular CD44 on T-lymphocytes was decreased 24 h prior to transplantation in patients that would reject their allograft, compared with patients without rejection. Additionally, plasma CD44 and cellular CD44 revealed an inversely proportional correlation. Lipopolysaccharide (LPS)-induced immune activation did not influence plasma or cellular CD44 levels in healthy volunteers, suggesting that more specific factors influence the shedding of CD44 on T lymphocytes, leading to increased risk of renal allograft rejection.
Conclusion. Although the exact mechanism remains to be elucidated and further research is required, soluble CD44 levels and cellular surface CD44 on T lymphocytes prior to transplantation might be useful as predictive markers for the occurrence of acute renal rejection.
Keywords: acute rejection; CD44; lymphocyte; pre-transplantation
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Introduction
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Despite the introduction of successful immunosuppressive drug therapies, acute renal allograft rejection still occurs in 1020% of patients after cadaveric renal transplantation and causes graft loss in up to 6% in the first year after transplantation [1]. Histopathologically, acute cellular rejection is characterized by an inflammatory infiltrate consisting mainly of T lymphocytes located in the interstitium, tubular epithelium and, dependent on the type and severity of rejection, vascular endothelium [2]. Despite numerous efforts in the past, still no adequate risk markers for acute allograft rejection prior to transplantation are available that may predict the occurrence of acute renal allograft rejection.
CD44 is a family of type I transmembrane glycoproteins with a wide tissue distribution including expression on leukocytes, epithelial and endothelial cells. CD44 has been implicated in many physiological and pathological processes, such as cellcell and cellmatrix interaction, wound healing/scarring, cell migration, lymphocyte activation and binding/presentation of growth factors. CD44 is also involved in adhesion and extravasation of lymphocytes [3]. In renal diseases, expression of CD44 and its ligands is markedly enhanced. CD44 is upregulated in glomeruli, on injured renal tubular epithelial cells and capillary endothelial cells in human renal diseases [4,5] as well as in several animal models of renal diseases [68].
Growing evidence suggests that CD44 may be important in immune responses to allografts. For example, in a model of skin transplantation, anti-CD44 antibodies blocked homing of activated lymphocytes into the graft, delaying graft rejection [9]. Renal allograft survival was prolonged after administration of low molecular weight hyaluronic acid (HA), the major ligand of CD44, in an experimental model of renal allograft transplantation [10].
Since CD44 is involved in activation, proliferation, rolling and extravasation of lymphocytes [3,11], we hypothesized that it could be involved in the pathophysiology of acute renal allograft rejection.
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Materials and methods
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Patients
Twenty-four patients with biopsy-proven acute renal allograft rejection (16 type 1 rejection, 6 type 2 rejection, 2 type 3 rejection), according to the Banff 97 classification [2] (rejectors), were selected randomly from the renal transplant patient population of the Academic Medical Center of the University of Amsterdam. As control, 9 transplant patients who showed no clinical or histopathological evidence of acute or chronic allograft rejection (non-rejectors) and 10 healthy volunteers were included. Blood was collected 1 day before transplantation, at which time none of the patients received any immunosuppressive drugs. From 17 patients (12 rejectors and 5 non-rejectors), peripheral blood mononuclear cells (PBMCs) were isolated and frozen until flow cytometric analysis was performed. Plasma and cells were analysed in a retrospective manner.
Additionally, in order to determine CD44 kinetics under inflammatory conditions, 8 healthy volunteers received an intravenous bolus injection of Escherichia coli lipopolysaccharide (LPS), lot G (United States Pharmacopeial Convention, Rockville, MD), over 1 min in an antecubital vein at a final dose of 4 ng/kg body weight. Plasma samples and PBMCs of the volunteers injected with LPS were collected prior to and 24 h after injection. All protocols had been approved by the Medical Ethical Committee of the Academic Medical Center of the University of Amsterdam, and each patient and volunteer had given written informed consent.
ELISAs
Plasma levels of soluble CD44, soluble intracellular adhesion molecule-1 (sICAM-1; both Bender Medsystems, Vienna, Austria), soluble interleukin-2 receptor (sIL-2R, CD25) (Roche, Woerden, The Netherlands) and C-reactive protein (CRP) (Alpha Diagnostic, San Antonio, TX) were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions.
Flow cytometry
Cell surface CD44 [(CD44fluorescein isothiocyanate (FITC), Pharmingen, San Diego, CA] was determined by flow cytometric analysis of peripheral lymphocytes. Differentiation between CD4- and CD8-positive lymphocytes was done using CD4 (CD4-PerCP, Pharmingen)- and CD8 (CD8-APC, Pharmingen)-specific antibodies.
Statistical analysis
All parametric data passed the normality test (P>0.05) as determined by the KolmogorovSmirnov test. Univariate analysis was performed with either a
2 test or t-test when appropriate. Data were analysed by analysis of variance (ANOVA) followed by Bonferroni post hoc test. Analysis of correlation was performed using Spearman rho tests.
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Results
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Clinical and biochemical parameters of the included patients
No statistically significant differences were observed between the rejectors and non-rejectors concerning age and gender of the recipient, type of renal replacement therapy prior to transplantation, age of the kidney donor, donor source (cadaveric graft vs heart-beating donor kidney) or smoking behaviour (Table 1). The type of renal disease leading to renal failure may affect sCD44 levels. However, the diversity of primary renal diseases suggests that the observed differences in sCD44 levels are independent of the primary renal disease. Human leukocyte antigen (HLA) mismatching cannot influence sCD44 levels prior to transplantation but is a major determinant of renal rejection. In our patient populations, only a mismatch in HLA-A was higher in rejectors compared with non-rejectors. No differences were detected in ABO blood group incompatibility, delayed graft function and time in dialysis prior to transplantation.
Increased sCD44 levels in rejector patients prior to renal transplantation
Rejectors displayed significantly higher pre-transplantation plasma levels of sCD44, compared with non-rejectors (Figure 1A). No statistical differences were detected between healthy volunteers and non-rejectors (Figure 1A). To test whether sCD44 was derived from endothelium or lymphocytes through non-specific shedding of cell surface proteins, sIL-2R (Figure 1B) and sICAM-1 (Figure 1C) were determined in plasma of patients prior to transplantation. No statistically significant differences could be detected between rejectors and non-rejectors. Additionally, since inflammation can cause shedding of CD44, we determined CRP levels in plasma (Figure 1D). No differences were detected between rejectors and non-rejectors and no correlation was observed between sCD44 and CRP. This suggested a specific mechanism leading to CD44 shedding.

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Fig. 1. sCD44 levels are increased in rejectors compared with non-rejectors prior to transplantation. sCD44 levels of rejectors (filled squares) were increased compared with non-rejectors (filled triangles) (A). No statistically significant differences could be detected between non-rejectors and healthy volunteers (filled inverted triangles). No differences between rejectors and non-rejectors were detected in plasma sIL-2R (B), sICAM-1 (C) or CRP (D) levels. Plasma sCD44 (A), sIL-2R (B), sICAM-1 (C) and CRP (D) were determined in plasma by ELISA. Statistical analysis was performed using ANOVA followed by a Bonferroni post hoc test.
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Increased sCD44 levels correlate with decreased cellular expression on peripheral lymphocytes
Analysis of peripheral blood cells prior to transplantation revealed increased numbers of total lymphocytes in rejectors compared with non-rejectors (Figure 2A), whereas no differences could be detected in total leukocyte numbers, monocytes or neutrophils (data not shown). High numbers of lymphocytes correlated with higher plasma levels of sCD44 (Figure 2B) in rejectors.

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Fig. 2. Increased peripheral lymphocyte levels correlate with sCD44 levels. Numbers of peripheral lymphocytes are increased in rejectors (filled squares) compared with non-rejectors (filled triangles) (A) as determined by MannWhitney U-test. Increased numbers of lymphocytes correlate with elevated sCD44 levels in plasma (B) as determined by Spearman rho test.
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To determine whether sCD44 could still be derived from lymphocytes, cell surface CD44 was determined by flow cytometric analysis of peripheral lymphocytes 24 h prior to transplantation. Differentiation of CD4- and CD8-positive lymphocytes was made using CD4- and CD8-specific antibodies. Interestingly, CD44 expression on CD8-positive cells was significantly decreased in the rejectors compared with non-rejectors (P<0.05); a similar trend was noticed on CD4-positive cells between rejectors and non-rejectors (P<0.10) (Figure 3A). No differences in CD44 expression were observed on CD4- and CD8-positive T lymphocytes between the non-rejectors and healthy volunteers (data not shown).

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Fig. 3. Cellular CD44 is decreased on peripheral lymphocytes in rejectors compared with non-rejectors. Flow cytometric analysis of peripheral blood cells revealed decreased cellular expression of CD44 on CD8- (P = 0.05) and CD4- (P<0.10) positive T lymphocytes in the rejectors (filled squares) compared with non-rejectors (filled triangles) as determined by MannWhitney U-test (A). Plasma sCD44 correlates with cellular expression of CD44 on CD4- and CD8-positive peripheral T lymphocytes (Spearman rho test) of rejector and non-rejector patients (B).
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In addition, comparison of plasma sCD44 and cellular CD44 of rejectors and non-rejectors revealed an inversely proportional correlation (Figure 3B).
CD44 is unaffected by LPS injection in healthy volunteers
High levels of sCD44 in the plasma of rejectors with decreased cellular expression may reflect a state of non-specific inflammation. To study CD44 kinetics in inflammation, healthy volunteers received a bolus injection of LPS. Injection of LPS in healthy volunteers had no effect on plasma sCD44 levels (Figure 4A) or CD44 expression on T lymphocytes (Figure 4B). LPS injection induced high levels of plasma CRP and increased sICAM-1 levels (data not shown). sIL-2R levels remained unchanged. As expected, no correlation between sCD44 and cellular CD44 was found in the LPS volunteers (data not shown).

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Fig. 4. LPS challenge of healthy volunteers does not affect CD44. sCD44 levels in plasma of healthy volunteers (filled circles) and 24 h after bolus injection of LPS (filled diamonds) was comparable (A). LPS challenge did not change cellular CD44 expression on lymphocytes (B). Statistical analysis was performed using MannWhitney U-tests.
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Discussion
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Acute renal allograft rejection still causes a large number of graft losses. Early recognition of patients at risk for renal allograft rejection may lead to individualization of therapeutic regimens and ensure a more adequate follow-up of these patients.
In this retrospective study, we showed that sCD44 levels in the plasma are increased prior to renal transplantation in patients that would reject their allograft. Interestingly, sCD44 levels did not correlate with other well known predictors of renal rejection such as smoking, ABO blood group incompatibility, delayed graft function or the period of time that patients received dialysis. In our patient population, HLA-A mismatching in rejectors was higher than in non-rejectors. Not all mismatches for HLA antigens have equal weight. The major impact comes from the DR and B antigens, with little additional effect from the A antigens. This suggests that CD44 is an independent predictive marker of acute renal rejection. Increased plasma sCD44 levels have been described in immune activation, for example in primary Sjögrens' syndrome [12] and malignancies [13], whereas low plasma sCD44 levels are found in immunodeficiency [14], possibly related to proteolytic activity leading to shedding of CD44. Additionally, smoking increases plasma levels of sCD44 [15], but no differences in smoking behaviour in rejectors and non-rejectors were observed. At the molecular level, proteinases, such as membrane type 1 and membrane type 3 metalloproteinases (MT1-MMP and MT3-MMP), have the potential of shedding CD44 from the cellular surface. However, other intracellular mechanisms may be involved as well. Activation of the Rho family GTPases and Ras, all involved in cytoskeletal rearrangement, may induce CD44 shedding. Although the exact function of sCD44 is not fully understood, shedding of CD44 may influence cellular mechanisms by competing with cell-bound CD44 for binding of ligands, as reviewed by Cichy et al. [16].
To test whether CD44 could be derived from lymphocytes or from endothelial cells due to non-specific shedding, we determined sIL-2R and sICAM-1 in plasma of the patients prior to transplantation. sIL-2R, a marker for the activity of lymphocytes, was comparable in the plasma of rejectors and non-rejectors. Similar levels of sICAM-1, a marker for endothelial cells, in plasma were also detected in rejectors compared with non-rejectors. These data suggested that sCD44 was derived from a source other than lymphocytes and endothelium or that CD44 is shed from cells in a specific mechanism.
In acute renal rejection, lymphocytes exert a crucial role [2]. The type of renal replacement therapy (haemodialysis or peritoneal dialysis) influences lymphocyte numbers. Yet no differences in renal replacement therapy were observed between rejectors and non-rejectors. In both patient groups,
50% received haemodialysis and 50% peritoneal dialysis. Haemodialysis may also activate lymphocytes, but blood was obtained prior to dialysis treatment, excluding lymphocyte activation and CD44 shedding. No differences in lymphocyte counts and sCD44 were observed between patients on haemodialysis or peritoneal dialysis (data not shown). Therefore, it is more interesting that plasma levels of sCD44 correlated with the number of peripheral lymphocytes and decreased expression of cellular CD44 on these lymphocytes. Upon activation, lymphocytes express high cell surface levels of CD44 [17]. These T lymphocytes are highly proliferative, resulting in increased numbers of lymphocytes in the blood. Upon cytokine stimulation, CD44 is shed from the lymphocyte surface, after which the cells undergo activation-induced apoptosis [18].
We show that the shedding of CD44 from the lymphocytes seems to be regulated by specific mechanisms prior to transplantation since plasma levels of sIL-2R were comparable in rejectors and non-rejectors. Moreover, plasma CRP levels were comparable in rejectors and non-rejectors. This seems to exclude an overall shedding of cell surface molecules expressed by lymphocytes. Although we provide indirect evidence that sCD44 is derived from lymphocytes, we cannot exclude that sCD44 may also be derived from different sources.
Additionally, bolus injection of LPS in healthy volunteers increased CRP and sICAM-1 levels but was unable to change sCD44 and sIL-2R levels in the plasma (CRP, sICAM-1 and sIL-2R data not shown). Furthermore, LPS injection did not alter CD44 expression on T lymphocytes, suggesting that a more T cell-specific stimulus is required to influence lymphocyte-mediated CD44 kinetics.
Recent research has shown that other parameters already prior to renal transplantation may also predict renal allograft rejection. A large study observed that high serum sCD30, an activation marker of Th-2-type cytokine-producing T cells, prior to transplantation was associated with increased graft loss [19]. In addition, Rotondi et al. [20] showed that high CXCL10 levels, a potent chemoattractant for lymphocytes and dendritic cells, in serum of patients prior to transplantation may be a useful marker to identify patients at risk for allograft rejection and graft loss.
Our study suggests that determination of sCD44, possibly in combination with sCD30 and CXCL10, in the plasma of patients prior to transplantation could be a relatively simple and efficient method to identify transplant patients at risk for acute renal allograft rejection. Yet, this study should be undertaken in a much larger number of patients and prospectively rather than retrospectively. The decreased cellular expression of CD44 on T lymphocytes and the inverse correlation between CD44 expression on T lymphocytes and plasma sCD44 suggest that CD44 may exert specific functions on T lymphocytes that eventually lead to acute allograft rejection. However, the exact mechanism of CD44 shedding and the function of sCD44 prior to transplantation remain to be elucidated and require further research.
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Acknowledgments
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The authors would like to thank Mrs Si La Yong for technical assistance in performing the FACS analyses. This research was funded by the Netherlands Organization for Scientific Research (grants 907-00-004 and 015-001-061) and the Dutch Kidney Foundation (grant PC 125).
Conflict of interest statement. None declared.
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Notes
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*These authors contributed equally to this work. 
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Received for publication: 15. 2.05
Accepted in revised form: 20. 7.05