Serum levels of macrophage-colony stimulating factor (M-CSF): a marker of kidney allograft rejection
Yannick Le Meur1,
Valérie Leprivey-Lorgeot2,
Sandrine Mons1,
Mattew José3,
Jacques Dantal4,
Brigitte Lemauff4,
Jean-Claude Aldigier1,
Claude Leroux-Robert1 and
Vincent Praloran5
1 Service de Néphrologie, Centre Hospitalier Universitaire Dupuytren, Limoges, France, 2 Laboratoire de Physiologie, Faculté de Médecine, Limoges, France, 3 Renal Laboratory, Monash Medical Centre, Melbourne, Australia, 4 Service de Néphrologie et Immunologie Clinique, Centre Hospitalier Universitaire, Nantes, France and 5 Laboratoire Universitaire dHématologie, Université Victor Segalen, Bordeaux, France
Correspondence and offprint requests to: Yannick Le Meur, Service de Néphrologie, Centre Hospitalier Universitaire Dupuytren, 2, rue Martin Luther King, F-87042 Limoges Cedex, France. Email: yann.lemeur{at}chu-limoges.fr
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Abstract
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Background. Macrophage-colony stimulating factor (M-CSF) is the principal factor for survival of monocytes and macrophages that play an important role in allograft rejection. We studied M-CSF serum levels during successful renal transplantation and acute graft rejection.
Methods. A total of 114 kidney allograft recipients were assessed for M-CSF levels by enzyme-linked immunosorbent assay (ELISA).
Results. M-CSF serum levels were elevated in pre-transplant haemodialysis patients (611±355 IU/ml vs 168±61 in normal controls, P<0.01). Following successful renal transplantation, M-CSF decreased in the first month, stabilizing at 257±222 IU/ml (not significantly different from normal controls) in 52 post-transplant stable patients. There was no correlation between M-CSF level and creatinine clearance. M-CSF levels increased significantly (25 times) during biopsy-proven acute rejection episodes in 20 of 25 patients. All rejection episodes were successfully treated and serum M-CSF decreased rapidly to pre-rejection levels in 17/20 patients. In contrast, in five patients with cyclosporin toxicity and four patients with other causes of allograft dysfunction, M-CSF serum levels did not change.
Conclusions. M-CSF serum level might be a specific marker of acute rejection. The source of increased production during rejection warrants further investigation, with infiltrating T cells and resident kidney cells being likely candidates.
Keywords: acute rejection; kidney graft; macrophage; macrophage-colony stimulating factor
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Introduction
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Macrophage-colony stimulating factor (M-CSF) is a pleiotropic molecule involved in haematopoiesis, bone and lipid metabolism, fertility and pregnancy, and immune system regulation [1]. It is the principal factor for survival, proliferation and differentiation of monocytes and macrophages. This growth factor also has a role in cytokine production, cytotoxicity and phagocytosis [2]. Monocytes, macrophages, endothelial cells, fibroblasts [2] and activated T lymphocytes [3] can produce M-CSF in vivo and/or in vitro. Within the kidney, tubular cells [4], mesangial cells [5] and podocytes [6] also produce M-CSF. In serum, M-CSF is present as a secreted soluble glycoprotein of 85 kDa and is measurable at a stable concentration in normal healthy subjects [7]. Patients with chronic renal failure have increased serum M-CSF [8], however the effect of renal transplantation on M-CSF levels is unknown. In two different animal models of acute rejection namely graft vs host reaction [9] and mouse cardiac allograft rejection [10], both tissue production and serum concentration of M-CSF were increased.
In the present work we studied the evolution of M-CSF serum levels following successful renal transplantation and during acute allograft rejection.
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Subjects and methods
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This retrospective study conducted in two centres includes 114 kidney allograft recipients. Sixty-two patients were followed during the first year after transplantation; among them, 22 patients without delayed graft function were specifically studied during the first month. A group of 52 stable patients defined by absence of acute or chronic rejection, infection and cyclosporin toxicity and > 1 year post-transplant was also analysed. In addition a group of 55 healthy volunteers were used as normal controls.
The immunosuppressive regimen was quadruple and sequential for 90% of the patients and consisted of thymoglobulin, corticosteroids, cyclosporin A (CsA) and azathioprine. The remaining 10% of the patients did not receive thymoglobulin. Thymoglobulin (1.5 mg/kg/day) (Institut Mérieux, Lyon, France) was administered intravenously for the first 512 days and adjusted daily to the CD3 lymphocyte counts. A 500 mg methylprednisolone bolus was given the day of transplantation followed by 1 mg/kg/day prednisone for 7 days. This was then tapered weekly by 10 mg until it reached 20 mg/day, then by 5 mg to reach 10 mg/day. CsA was introduced at a dose of 68 mg/kg/day 2 days before thymoglobulin discontinuation. Targets for through levels of Ca (as measured by monoclonal RIA) were fixed at 200250 ng/ml for the first month, 150200 ng/ml at 3 months, 125150 ng/ml at 6 months and 100 ng/ml thereafter.
All patients with deteriorating graft function (serum creatinine elevated by 20%) underwent a graft biopsy. Rejection episodes (n = 25) were classified according to the 1997 Banff classification [11]. Treatment of acute rejection consisted of methylprednisolone boluses and of thymoglobulin or OKT3 as rescue therapy. Acute cyclosporin toxicity (n = 5) was diagnosed if the serum creatinine returned to baseline following a reduction in cyclosporin dose and if there was no evidence of rejection on biopsy. Diagnosis of cytomegalovirus (CMV) infection (n = 7) was done by antigenaemia and/or schell vial culture.
Blood samples were collected in dry tubes before transplantation, twice per week during the first month and as part of a routine follow-up after 1 month post-transplantation. A blood cell count and differential were performed in all patients at the same time. Serum was rapidly collected after centrifugation of tubes and immediately stored at 20°C until determination of M-CSF concentration.
Serum concentrations of M-CSF were determined with an immunoassay procedure (ELISA) developed in our laboratory, using a polyclonal rabbit anti-M-CSF antibody [7]. The lower detection limit of the assay is 10 IU/ml. Intra-assay coefficient of variation was 10% and inter-assay coefficient of variation was 9.5% [7].
Statistical analysis were performed using Statview for Windows software (SAS Institute Inc., Cary, NC). All results were expressed as mean±SD. Comparisons were made using Student's t-test. Differences were considered to be significant when P was < 0.05.
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Results
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Pre-transplant haemodialysed patients (n = 22) had M-CSF serum levels that were 3 times higher than normal controls (611±355 vs 168±61 IU/ml, P<0.01). In contrast, in the group of 52 stable transplant patients (mean creatinine 140±40 µmol/l), M-CSF serum levels (257±222 IU/ml) were significantly reduced compared with the pre-transplant haemodialysed group, and were not significantly different from the normal controls. In fact, as seen in the group of 22 patients followed just after transplantation, the reduction in M-CSF serum levels occurred within the first month (Figure 1). The reduction in serum M-CSF lags behind the reduction in serum creatinine, which reached a minimum level by day 10 at which time M-CSF levels were still significantly elevated and continued to decrease until day 30. No correlation was found between the M-CSF serum level and creatinine clearance, either in this group of 22 patients, or in the total group of 114 kidney graft recipients. Neither was there any correlation between M-CSF level and the number of circulating monocytes, lymphocytes or CD3 counts.

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Fig. 1. Evolution of M-CSF and creatinine serum levels before transplantation and in the first month after transplantation. The measurements were performed by ELISA in 22 patients.
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We then analysed the clinical situations in which M-CSF serum levels were modified within the first year after transplantation. In this period, acute rejection was the most common situation associated with elevated M-CSF serum levels significantly different from those observed in 28 stable transplant patients (no rejection, no cyclosporin toxicity, no infection) (1371±974 vs 185±92 IU/ml, P<0.01) (Figure 2). In fact, M-CSF increased in 20 of 25 patients with acute rejection. This increase was important (up to 5-fold compared with their pre-rejection levels) and occurred concomitantly to that of creatinine in 16 patients, before in two, and after in two. All patients were successfully treated with corticosteroids, thymoglobulin (three cases) or OKT3 (one case). The rise in M-CSF levels during rejection was followed by a subsequent fall to subnormal levels after successful treatment in most patients. However some patients (five cases) still had elevated serum M-CSF levels after treatment, and when followed for a further 3 months after the rejection episode, their M-CSF either slowly decreased to reach pre-rejection levels (two cases) or remained moderately elevated (three cases).

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Fig. 2. Comparison of M-CSF serum levels in 62 patients during the first year after transplantation. Patients were divided into four groups: 28 stable transplant patients (no rejection, no infection, no cyclosporin toxicity), 25 patients with a biopsy-proven acute rejection, five patients with cyclosporin toxicity and four patients with other causes of allograft dysfunction (two ischaemic and two obstructive ARF). The measurements were performed by ELISA.
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We compared the group of 25 patients with acute rejection with a group of five patients with cyclosporin toxicity and four patients with other causes of allograft dysfunction (two ischaemic and two obstructive acute renal failure (ARF)). While M-CSF serum levels increased 5-fold in patients with acute rejection, they did not change with acute cyclosporin toxicity (167±95 IU/ml) or in these other causes of renal dysfunction (431±213 IU/ml) (Figure 2). Yet, despite differences in serum M-CSF, all three groups had similar levels of creatinine. If we consider the peak of M-CSF serum levels (that is the highest concentration measured during the acute rejection or the cyclosporin toxicity episodes), none of the stable patients or patients with cyclosporin toxicity had a peak value > 500 IU/ml compared with 20 out of 25 patients with acute rejection (Figure 3). In addition, patients are more likely to have higher M-CSF serum levels with increasing severity of rejection according to the Banff classification. All cases of the grade II (6 cases) or III (2 cases) rejections had a peak of M-CSF serum levels > 500 IU/ml as compared with two out of four for the borderline group and 10 out of 13 for the grade I rejections.

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Fig. 3. Peak of M-CSF serum level during episodes of acute rejection or cyclosporin toxicity. Peak of M-CSF was defined as the highest concentration measured during the episode. Results were compared with peak of M-CSF in 28 stable transplant patients (no rejection, no cyclosporin toxicity, no infection) followed during the first year after transplantation. The measurements were performed by ELISA.
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We also found high (and transient) M-CSF serum levels in CMV diseases (seven cases) (735±568 IU/ml) and serum sickness due to treatment with thymoglobulin (six cases) (1903±1147 IU/ml), and a moderate increase in one case of Escherichia coli septicaemia (402 IU/ml). However, these patients did not have renal dysfunction as shown by their normal creatinine levels and clearance. Thus, acute rejection is the only situation to associate increased M-CSF serum levels with allograft dysfunction within the first year after transplantation.
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Discussion
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In this study we showed that M-CSF serum levels, elevated in haemodialysis patients as already shown [8], decreased in the first month after transplantation and stabilized close to normal levels. This reduction in M-CSF after transplantation is not mainly due to the increase of its clearance by the transplant kidney for the following reasons: (i) M-CSF is a high molecular weight molecule (85 kDa), whose metabolism in vivo is independent of the renal function; (ii) there was no correlation between M-CSF and creatinine clearance in transplant patients or patients with chronic renal failure [8]; (iii) serum levels increased in patients with acute rejection but did not in cyclosporin toxicity or other causes of allograft dysfunction. One hypothesis is that particular cell types (monocytes, lymphocytes and endothelial cells) were up-regulated to produce M-CSF in patients with chronic renal failure as a part of the dysregulated immune system of uraemia [12]. This situation could be reversed by transplantation. Alternatively, the immunosuppressive drugs might play a role on M-CSF production. Cyclosporin is known to modify M-CSF expression and its production by T cells in vitro [13]. In addition, the depleting effect of thymoglobulin on lymphocyte numbers could also play a role, as activated lymphocytes are important producers of M-CSF [3]. However, some patients in our study did not have induction therapy and still showed a reduction in M-CSF serum levels after transplantation.
M-CSF serum levels could be valuable as a marker of acute rejection in kidney allograft as first shown in a murine model of graft vs host [9]. In our study, M-CSF serum levels increased in 20 out of 25 patients with acute rejection and this increase was discriminative in allograft dysfunctions due to other causes. Numerous markers of acute rejection, especially cytokines or their soluble receptors, have been analysed previously [14]. Most of them failed to show sufficient specificity and sensitivity to be used routinely. In the present study the specificity of M-CSF is good but the sensitivity is only 80% when using a threshold of 500 IU/ml. Most of the patients had a maximum of two samples collected per week, and as the increase of M-CSF during acute rejection occurs very quickly (over a few days), the recorded peak M-CSF level may have been falsely low. A daily or alternate day sample collection could increase the sensitivity of the method and needs to be investigated in a prospective study. As M-CSF serum levels also increase in some patients with chronic allograft nephropathy even though in a lower range than in acute rejection (personal data), M-CSF serum values should be considered as an useful additional parameter to the renal biopsy for the diagnosis of acute rejection and for monitoring the responsiveness to corticosteroid therapy. Additionally, M-CSF measurement in the urine during renal allograft rejection has to be studied.
The source and role of the increased M-CSF production during rejection warrant investigations. M-CSF is produced by cells of the monocyte-macrophage lineage [2], activated T cells [3] and tubular epithelial cells [4]. These in vitro studies together with animal and human studies in various nephropathies [4,15] suggest that infiltrating T cells and/or macrophages, and tubular cells are likely to produce M-CSF in acute rejection. Recently, in a rat model of acute rejection, we proved this hypothesis showing an up-regulation of M-CSF expression by both tubular and infiltrating cells [16]. This increased expression at the site of inflammation could explain high M-CSF serum levels as it was shown in MLR-lpr mice using a kidney transplant approach [17].
Studies of the infiltrating cells from acutely rejecting allograft showed a high proportion of macrophages (2060%) [18] and infiltration by macrophages is a poor prognostic sign for transplant survival [19]. Recent studies suggest that M-CSF is an important factor for macrophage accumulation and proliferation in experimental models as well as in human glomerulonephritis [4,15]. These results have been reinforced in our study in rat allograft rejection [16]. More recent data, using a specific functional blockade gave a definitive proof of the role of local production of M-CSF in driving in situ macrophage proliferation at sites of inflammation. In a model of unilateral ureteric obstruction in mice, anti c-fms administration (M-CSF receptor) reduces macrophage accumulation by 70% by blocking macrophage proliferation [20]. In a mouse model of acute renal allograft rejection, administration of anti c-fms reduces the severity of tubulointerstitial rejection [21]. Our hypothesis is that M-CSF drives the recruitment, activation and proliferation of macrophages in human acute renal allograft rejection and this might contribute to the subsequent kidney damage.
Conflict of interest statement. None declared.
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Received for publication: 1. 7.03
Accepted in revised form: 10. 3.04