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Summary: production.1–3. Experiences with IL-10 knock-out mice show that IL-10 may contribute to the control of inflamma- tory reactions because these ...
Bone Marrow Transplantation, (1997) 20, 365–368  1997 Stockton Press All rights reserved 0268–3369/97 $12.00

High interleukin-10 serum levels are associated with fatal outcome in patients after bone marrow transplantation L Hempel1 , D Ko¨rholz2, P Nußbaum3, H Bo¨nig2, S Burdach2,4 and F Zintl1 1

Department of Pediatric Hematology and Oncology, Friedrich-Schiller University Medical Center, Jena; 2Department of Pediatric Hematology and Oncology, Heinrich-Heine University Medical Center, Du¨sseldorf; 3Institute of Clinical Pathology, Osnabru¨ck; and 4 Biomedical Research Center, Heinrich-Heine University, Du¨sseldorf, Germany

Summary: IL-10 plays an important role in the control of immune reactions during systemic infection. Here, IL-10 serum levels were investigated in patients after BMT. The IL10 levels correlated with the clinical course of the patients and with serum levels of C-reactive protein (CRP) and neopterin (NP). A total of 26 patients with AML (7), ALL (12), CML (2), NHL (3) and multifocal Ewing’s sarcoma (2) had received autologous (10) or allogeneic (16) BMT from related (9) or unrelated donors (7). Routine serum samples were obtained prior to BMT and at days 46 and 100 after BMT. However, in patients with severe complications additional samples were drawn at individual points in time. Prior to BMT, IL-10 serum levels were not detectable in 24/24 patients. Post-BMT, 11 patients developed elevated IL-10 levels, of these eight died of complications (DOC), whereas only one of 15 patients with undetectable IL-10 died of complications, indicating that high IL-10 levels were significantly correlated with severe life-threatening complications (x2, P , 0.01). To determine the pathomechanism and role of the increased IL-10 levels, they were correlated to the respective NP and CRP serum concentrations. CRP and NP concentrations were found significantly elevated in patients with detectable IL-10, indicating a severe acute phase reaction associated with macrophage activation. In conclusion, high IL-10 serum levels in patients after BMT were significantly associated with fatal outcome. Since IL-10 is a strong suppressor of T cell immunity, high IL-10 production in patients with severe complications such as septic shock or GVHD .grade II after BMT might lead to functional immunodeficiency contributing to the poor prognosis of these patients. Keywords: sepsis; bone marrow transplantation; interleukin 10; neopterin

IL-10 is a potent macrophage and T cell-deactivating cytokine, leading to a significant suppression of cytokine Correspondence: L Hempel, Department of Pediatric Hematology and Oncology, Friedrich-Schiller University Medical Center, Kochstr 2, D07740 Jena, Germany Received 21 March 1997; accepted 15 May 1997

production.1–3 Experiences with IL-10 knock-out mice show that IL-10 may contribute to the control of inflammatory reactions because these animals showed severe gastrointestinal inflammation.4 In mouse models, elevation of IL10 during systemic infection protected the animals from an overwhelming immune reaction. Furthermore, mice with high endogenous or exogenous IL-10 levels survived systemic shock syndromes, while application of anti-IL-10 led to a fatal reaction.5–7 In contrast, in humans elevated IL-10 levels during systemic shock correlated with an increased TNF-a production, increased severity of septic shock and a high risk of disseminated intravascular coagulation.8–10 The association between high IL-10 levels and fatality of septic shock was explained by the fact that IL-10 might not be able to inhibit production of proinflammatory cytokines, while suppressing T cell reactions which might otherwise have supported pathogen elimination. In this study the role of IL-10 in patients after BMT was investigated under the hypothesis that in post-BMT patients, as in sepsis, increased IL-10 might be correlated to poor outcome. IL10 serum levels correlated with the clinical course of the disease, the severity of acute phase reactions and macrophage activation. Patients and methods Patients Twenty-six children and adolescents were enrolled into this study. Patients’ ages ranged from 4.0 to 22.4 years (median: 13.4 years). The male to female ratio was 18 to 8. Patients had previously been treated for ALL (12), AML (7), CML (2), NHL (3) and Ewing’s sarcoma (2). Five patients had received an allogeneic transplant from a matched-related donor, four patients had been transplanted with bone marrow from an haploidentical related donor and seven patients had had unrelated donors. In addition, 10 patients had received autologous bone marrow or peripheral blood stem cell transplants. Blood was drawn from patients prior to BMT and at days 46 and 100 after BMT. However, in patients with multi-organ failure (MOF) additional blood samples were taken during these complications. At the time of study, all patients with allogeneic transplants received immunosuppressive treatment. Acute GVHD was seen in only six of 16 patients (Table 1). However, six of 10 patients without acute GVHD died in the early post-transplant period.

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Table 1 No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Patients characteristics

Initials Sex DH AK DS KS FT CK RK CK SM KR SR FZ TB CL NS CS RB RF EG MG BH HH DS JS NS PW

F M M M M M M M F F M M F F M F M M M M F M M F M M

Age

Diagnosis

17.2 14.6 14.7 4.0 11.5 14.6 8.7 10.4 7.1 22.4 18.1 17.4 11.2 10.8 13.7 14.3 12.8 19.9 17.7 17.9 7.5 17.5 15.4 11.0 7.3 12.8

ALL CML ALL ALL AML CML AML NHL ALL ALL AML AML ALL ALL ALL ALL AML ALL AML NHL ALL AML EWS EWS NHL ALL

Status at BMT Type of transplant CR PR CR CR CR PR relapse PR CR relapse CR relapse CR CR relapse relapse CR CR CR CR CR CR PR PR CR CR

matched related matched related matched related matched related matched related matched unrelated matched unrelated matched unrelated matched unrelated matched unrelated matched unrelated matched unrelated mismatched related mismatched related mismatched related mismatched related autologous autologous autologous autologous autologous autologous autologous autologous autologous autologous

The study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Patients or their parents gave their consent. The study was authorized by the local ethics committee.

Acute GVHD

Status

GVHD none none none none none none none GVHD none GVHD none GVHD GVHD GVHD none none none none none none none none none none none

CCR CCR DOC, day +67 CCR relapse day +126, DOC, day +34 DOC, day +20 DOC, day +26 CCR DOC, day +27 DOC, day +103 DOC, day +21 CCR CCR DOC, day +83 DOC, day +19 relapse day +293, relapse day +192, CCR CCR CCR CCR relapse day +161, CCR CCR CCR

II

I–II III II II II–III

died day +183

alive died day +220

alive

Statistical analysis of data The Wilcoxon and x2 tests were used for statistical analysis. Results

Cytokine analysis Concentrations of IL-10 were assessed by commercially available ultrasensitive ELISA kits (BioSource, Camarillo, CA, USA) as described previously.3 The lowest concentration detected by this assay was 200 fg/ml. Neopterin (NP) determination Serum neopterin levels were measured by HPLC. Briefly, 200 ml samples or standards were added to a mobile phase containing 0.1 m KH2PO4, (Merck, Darmstadt, Germany), 0.005 m octansulfonic acid (Fluka, Bux, Switzerland; pH was adjusted to 3.0 with 1 N HCl) and 50 ml of 3.4 m HClO4. After vortexing the mixtures for 30 s, samples were centrifuged at 11 500 r.p.m. for 5 min. Supernatants (50 ml) were applied to a HPLC (Li-Chros-Pher 100 RP 8E; Merck). Neopterin concentrations were determined at Ex wavelength of 360 nm compared with Em wavelength of 430 nm. CRP determination CRP values were determined by particle-enhanced immunonephelometry. 40 ml serum were applied to a fully automatic nephelometer (Behringwerke, Marburg, Germany).

To determine the role of IL-10 in patients after BMT, IL10 serum levels were correlated with the clinical course of the patients. Prior to BMT (‘day 0’) serum levels of IL10 were undetectable in all patients. Post-BMT 11 patients developed detectable IL-10 at various points in time. Of these, eight patients died of complications within 1–14 (median 4) days after an elevated IL-10 had been found. The three surviving patients with elevated IL-10 serum levels presented with acute GVHD grade II or severe CMV infection. One patient received systemic IL-2 treatment at the time of IL-10 determination. In 15/26 patients IL-10 remained undetectable throughout the observation period. Of these, only one succumbed to complications. The last IL-10 measurement in this patient had been done 21 days prior to his death so that an increased IL-10 may have been missed. Thus, we found detectable IL-10 levels to be significantly correlated with DOC (Figure 1; Table 2; x2, P , 0.01). Next, the mechanism of in vivo IL-10 production was investigated. It was found that in patients with detectable IL-10, CRP levels (median: 8.57 mg/dl) were significantly elevated as compared to patients with undetectable IL-10 (median: 0 mg/l; Figure 2, Wilcoxon test, P , 0.001). Thus, in our patient population the IL-10 serum levels were associated with a severe acute phase reaction.

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DOC no DOC

60

IL-10 serum levels (ng/1)

Since IL-10 is produced by both T cells and macrophages,11–13 we determined the serum concentrations of neopterin as an indicator of macrophage activation.14–16 It was shown that high IL-10 levels were positively correlated with increased neopterin levels (Figure 3, Wilcoxon test, P , 0.01). Thus, increased IL-10 levels in post-BMT patients might be produced by macrophage activation during an acute phase reaction.

50 40 30 20

Discussion

10

After bone marrow transplantation, different parts of the immune system recover at different times. One of the earliest detectable functions is the production of macrophagederived cytokines.17 Increased macrophage activation might be observed at the time of engraftment.14 Since most of the patients with increased IL-10 levels in this study were found within the first month after BMT, it is most likely that the IL-10 was derived from activated monocytes rather than from T cells. Additional evidence for this point of view is presented by the observation that increased IL-10 levels were associated with a significantly increased production of neopterin, a protein released from macrophages upon activation.14–16 In patients after BMT, elevated neopterin and an abnormal monocyte activation has been observed from the time of engraftment until 6 weeks after BMT.14 Since macrophage activation did not correlate with complications such as GVHD or veno-occulusive disease (VOD), it was suggested that the monocyte activation might predispose to, rather than result from, the development of these complications. In our study we could demonstrate macrophage activation in patients with life-threatening complications after BMT, which correlated with an increased release of IL-10. Since increased IL-10 production has been reported to lead to a suppression of hematopoiesis18 as well as antigen-specific T cell activation,1,19 the increased IL-10 pro-

0

n=9

day

17

3 17

17

46

100

0

Figure 1 IL-10 serum levels in patients after BMT. IL-10 serum levels were determined in 26 patients prior to and post-BMT. Figure 1 shows time after BMT on the x-axis, and IL-10 concentrations on the y-axis. Patients who died of complications (DOC) are depicted as open squares, patients who did not succumb to complications as solid circles.

Table 2

Clinical course of patients with elevated IL-10 levels

No.

IL-10 level

Complications

6 7 8 10 11 12 15 16

46.6 64.4 8.5 8.9 21.0 62.2 35.5 62.0

sepsis, MOF, VOD, ARF sepsis, MOF, VOD, ARF sepsis, VOD VOD, ARF GVHD III–IV sepsis, MOF, VOD, ARF sepsis, MOF, GVHD III, ARF sepsis, MOF, VOD, ARF

DOC, DOC, DOC, DOC, DOC, DOC, DOC, DOC,

9 14 23

40.7 2.0 7.1

CMV infection GVHD II systemic IL-2 treatment

CCR CCR relapsed, alive

Patients without elevated IL-10

Status day day day day day day day day

34 20 26 27 103 21 83 19

Patients without elevated IL-10

Patients with elevated IL-10

Patients with elevated IL-10

30

Neopterin serum levels (µg/l)

CRP serum levels (mg/ dl)

40

30

20

10

25

20

15

10

5

0 0

n=15

n=15

n=11

n=11

Figure 2 Correlation between IL-10 and CRP levels in patients after BMT. CRP serum levels were determined in 26 patients after BMT on the same days that IL-10 was measured. Elevated CRP was significantly associated with detectable IL-10 (Wilcoxon test, P , 0.001).

Figure 3 Correlation between IL-10 and neopterin serum levels in patients after BMT. Neopterin serum levels were determined in 26 patients after BMT on the same days that IL-10 was measured. Elevated neopterin was significantly associated with detectable IL-10 (Wilcoxon test, P , 0.01).

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duction in these critically ill patients might have led to an immune-paralysis. This has previously been reported for patients with septic shock syndromes.8–10 In addition, enhanced stimulation of IL-10 increases susceptibility towards certain pathogens such as candida.20 Thus, although it could not be determined from our results whether high IL-10 levels were preceding the development of severe complications and thereby could be predictive of fatal outcome, it might be conceivable that IL-10 prevented patients from recovery by inhibiting immune-mediated elimination of pathogens. Therefore, future studies should investigate whether neutralization of IL-10 might help patients with severe GVHD or sepsis early after BMT.

9 10

11

12

Acknowledgements 13 This study was supported by DFG grants Ko 971/3-2 and He 2202/2-2. 14

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