Plasma levels of soluble CD30 are increased in children with chronic renal failure and with primary growth deficiency and decrease during treatment with recombination human growth hormone

Giancarlo Barbano1,, Francesca Cappa1, Ignazia Prigione2, Vito Pistoia2, Amnon Cohen3, Sabrina Chiesa1, Rosanna Gusmano1 and Francesco Perfumo1

1 Department of Pediatric Nephrology, 2 Oncology Laboratory and 3 Department of Pediatrics, Giannina Gaslini Institute, Children's Hospital, Genoa, Italy



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Previous studies have suggested that in vivo Th2 lymphocyte activation is related to increased soluble CD30 (sCD30) plasma levels. As various hormones (dehydroepiandrosterone, glucocorticoids, progesterone) can regulate the Th1/Th2 balance, and because growth hormone (GH) enhances lymphocyte function, we measured sCD30 plasma levels, before and after treatment with recombinant human GH (rhGH), in children with growth failure due to chronic renal failure (CRF) or to isolated GH deficiency in order to evaluate the potential effects of rhGH treatment on Th1/Th2 balance.

Methods. sCD30 plasma levels were determined by ELISA assay in 30 children with CRF (mean age 10.7±3.7 years), in five children with isolated GH deficiency (mean age 11.4±2.6 years), and in 10 normal controls (mean age 10.1±3.5 years).

Results. sCD30 levels were higher in the 30 children with CRF than in the 10 controls (179.8±79.4 vs 11.3±10.9 U/ml, P<0.001) exhibiting an inverse correlation with glomerular filtration rate (GFR) (r=-0.7860, P<0.001). In 11 children with CRF, after 19.9±16.7 months of rhGH treatment, a decrease of sCD30 plasma level (170±50 vs 134±49 U/ml, P<0.01) was observed. The five children with primary GH deficiency had higher sCD30 plasma level than controls (mean 147±105 vs 11±10 U/ml, P<0.004) and sCD30 plasma levels decreased to 95.2±109.6 U/ml after rhGH treatment.

Conclusions. The finding that rhGH treatment decreased sCD30 plasma levels in children with CRF, and that children with primary GH deficiency had higher sCD30 plasma levels than controls, suggest that GH may regulate CD30 expression and possibly the balance of Th1/Th2. Whether the uraemia-induced increase in sCD30 is due to decreased renal excretion, to overproduction or both, remains to be determined.

Keywords: CD30; chronic renal failure; growth hormone therapy; primary growth hormone deficiency; T helper 2 (Th2) lymphocytes



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The neuroendocrine and immune systems are linked by a bi-directional relationship. Cytokines can stimulate or inhibit the secretion of hormones from both the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-ovarian (HPO) axes and hormones can influence immune function [1]. Moreover, the neuroendocrine system can influence the Th1/Th2 balance. In fact it has been shown that dehydroepiandrosterone (DHEA) promotes the differentiation of T helper 1 cells (Th1), which produce IFN-{gamma}, IL-2 and are mainly involved in delayed hypersensitivity. In contrast, glucocorticoids and progesterone promote the expansion of T helper 2 cells (Th2), which produce IL-4, IL-5 and provide help for humoral immune responses (mainly IgE and IgG4) [2].

Although Th1 and Th2 subtypes have been characterized on the ground of their cytokine secretion patterns, recent studies have reported preferential expression of the CD30 molecule on Th2-type T cells [3].

CD30 is a surface receptor (belonging to the tumour necrosis factor superfamily) originally described as a marker for Reed-Stemberg cells in Hodgkin's disease (HD) [4]. Although the physiological expression of CD30 is mainly restricted to activated T and B cells, virally transformed T and B cells (EBV, HTLV, HIV) can also bear CD30. Whereas little is known about CD30 functions, it has been reported that CD30–CD30 ligand interaction provides a co-stimulatory signal for T cell proliferation. Moreover, CD30 expression is associated with production of a soluble form of CD30 (sCD30), generated by proteolytic cleavage, and it has been shown that patients with autoimmune diseases, HIV infection, and atopy have increased serum levels of sCD30, suggesting a preferential activation of Th2 cells in these immune disorders. In addition plasma levels of sCD30 are increased in some malignant lymphomas (mainly HD) and correlate with disease activity [57].

There is also accumulating evidence that chronic renal failure (CRF) can affect both immune and neuroendocrine function. In particular, several immunological abnormalities have been reported in uraemic patients, such as defective cell-mediated and humoral immunity, monocyte dysfunction, chronic complement activation via the alternative pathway [8], and activation of circulating T cells [9].

In addition, uraemia itself can decrease the bioactivity of growth hormone (GH), of insulin-like growth factors (IGFs), and cause other hormonal dysfunctions which may cause growth failure in children with CRF [10,11].

Based on these data many children with CRF, who fail to grow after measures to correct malnutrition, acidosis, and disturbances in calcium-phosphate balance, have been treated with supraphysiological doses of recombinant human GH (rhGH) [10,11]. Treatment with rhGH has also been given to children with functioning renal transplant and growth retardation, but there are concerns that GH has effects on the immune system, such as stimulation of lymphocyte proliferation, cytotoxic activity, and IFN-{gamma} production in mixed leukocyte cultures [1217].

In a recent review, it was hypothesized that GH is not an obligate immunoregulator, but acts rather as an anabolic and stress-modulating hormone in most cells including those of the immune system [18]. However, GH administration in critically ill adults is associated with increased morbidity and mortality and the preponderance of multiple-organ failure as well as septic shock or uncontrolled infection. These effects of GH suggest that a modulation of immune function may have been involved [19].

We determined plasma levels of sCD30 in children with CRF or with primary GH deficiency, before and during treatment with rhGH, in order to investigate whether rhGH would influence immune activation pathways, and in particular the activity of the Th2 T cell subset as previously suggested by other authors [7,16].



   Subjects and methods
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 Subjects and methods
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 Discussion
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We measured sCD30 plasma levels in 30 children (19 males and 11 females, mean age 10.6±4.1 years), with preterminal CRF (see Table 1Go). The primary renal diseases were: reflux nephropathy (12), renal dysplasia (6), nephronophthisis (4), obstructive uropathy (4), and dominant polycystic kidney disease (4).


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Table 1. Characteristics of children with chronic renal failure (CRF) and plasma levels of soluble CD30

 
Patients included in the study had to meet the following criteria: (i) no immune system involvement in the pathogenesis of renal disease, (ii) no neoplastic or rheumatologic active disease, (iii) no active infectious disease, (iv) no history of allergic diseases, and (v) no sign of malnutrition. GFR was calculated by the Schwartz formula and ranged from 6 to 80 ml/min/1.73 m2 (see Table 1Go). In 11 out of 30 children (10 males and one female, mean age 9.8±3.1 years), sCD30 plasma levels were determined before and after a mean of 19.9±16.7 months treatment with rhGH (median dose 4 U/m2/day), and they were determined in five children (two males and three females, mean age 12.8±3.7 years) when treatment with rhGH had been already instituted for a mean of 13±3 months.

Plasma levels of sCD30 were also evaluated in five age-matched children (two males and three females, mean age 11.4±2.6 years) with growth failure due to isolated GH deficiency but normal renal function. GH deficiency was confirmed after at least two confirmatory tests (GH peak secretion of less than 10 ng/ml after insulin, clonidine, or arginine challenge). Plasma levels of sCD30 were determined in 10 age-matched healthy children with normal renal function (seven males and three females). Informed consent of parents was always obtained.

Detection of soluble CD30 plasma levels was obtained immediately after venipuncture. Blood samples were centrifuged and plasma was separated from cells, collected and stored at -80°C until further testing. Plasma levels of sCD30 were determined by ELISA assay (Dako CD30 ELISA kit, Dako Glostrup, Denmark). The coefficient of variation was 3.8% within-run and 6.6% between-run. As plasma levels of sCD30 were determined in children with CRF (and in particular in children with CRF studied before and during rhGH treatment) using the same ELISA assay kit, a within-run variation coefficient of 3.8% was assigned to these data.

We further investigated CD30 expression on mononuclear cells isolated from peripheral blood (PBMC) in five subjects with CRF, before treatment with rhGH and after a mean of 12±5 months of rhGH therapy. This was also determined in five age-matched normal subjects. Venous heparinized blood was collected in the morning from fasting patients and controls, and PBMC were separated on a Ficoll gradient and washed twice with Hanks' solution. Surface marker analysis was performed on PBMC by direct immunofluorescence using the CD30 monoclonal antibody (mAb), purchased from Dako. Cells were incubated with saturating amounts of mAb for 30 min at 4°C, washed twice with PBS and analysed by flow cytometry using a FACSCAN (Becton-Dickinson) with the gate set on the lymphocyte population. Isotype-matched fluorochrome-conjugated mAbs of unrelated specificities were used as controls to assess non-specific binding of test mAbs to each cell population and to set the threshold between positively and negatively staining cells.

Statistical analysis
Data were analysed by Mann–Whitney U tests, Wilcoxon matched pairs tests and by a linear regression model and multivariate linear regression model. Values of P<0.05 were considered statistically significant.



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 Results
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Plasma levels of sCD30 were ten to twentyfold higher in children with CRF compared with the control group (179.8±79.4 vs 11.3±10.9 U/ml, Mann–Whitney U test: P<0.001) (Figure 1Go). An inverse linear correlation was found with GFR (r=-0.7860, P<0.001) (Figure 2Go).



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Fig. 1. Plasma levels of sCD30 were higher in children with CRF than in controls (Mann–Whitney U test, P<0.001). Children with CRF under treatment with rhGH had lower sCD30 plasma levels than in untreated children with CRF (P<0.04). sCD30 plasma levels were also significantly higher in children with primary GH deficiency than in normal controls (P<0.004) and decreased after treatment with rhGH.

 


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Fig. 2. sCD30 plasma levels increased in patients with CRF. The figure shows an inverse correlation between sCD30 and GFR (r=-0.7188, P<0.001). sCD30 is expressed as U/ml and GFR as ml/min/1.73 m2.

 
Plasma levels of sCD30 were significantly lower in the 16 children with CRF under treatment with rhGH for a mean of 16.3±15.1 months than the 14 children with CRF not administered rhGH (122.4±59.6 vs 187.0±96.9 U/ml, Mann–Whitney U test: P<0.04) (Figure 1Go). Although the group of children treated with rhGH had a predominance of boys (13 males and four females vs six males and eight females in the untreated group), there were no differences in age, height, or and GFR between those treated with rhGH and untreated children (see Table 1Go). Moreover, when we measured sCD30 plasma levels in 11 children with CRF before and after treatment with rhGH (mean treatment duration 19.9±16.7 months), a significant decrease in sCD30 plasma levels was observed, from 170.6±50.3 U/ml before treatment to 134.7±49.4 U/ml during treatment (Wilcoxon matched pair test P<0.01) (Figure 3Go). During the same period, GFR decreased from a mean of 16 to 14 ml/min/1.73 m2.



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Fig. 3. sCD30 plasma levels in patients with CRF before (170.6±52.6 U/ml) and after treatment (134.7±49.4 U/ml) with rhGH (Mann–Whitney U test P<0.01).

 
The percentage of decrease in sCD30 ({Delta}sCD30=[T2-T1]/T1x100) was directly and significantly related to the duration, in months of rhGH therapy (r=0.6499, P<0.05). In addition, a multivariate linear regression model that included the percentage of height increment in cm observed in children treated with rhGH ([T2-T1]/T1x100), the percentage change in GFR ([T2-T1]/T1x100) and {Delta}sCD30, during the same period of follow up, showed that {Delta}sCD30 was inversely related to the increment of height (r=–0.580, P<0.048) but not to GFR change (r=–0.096, P<0.766).

Interestingly, the group of five age-matched children with primary GH deficiency and normal renal function had significantly higher sCD30 levels than in normal controls before treatment with rhGH (mean 147.6±105.0 vs 11.3±10.9 U/ml Mann–Whitney U test P<0.004) and, after a mean of 31.2±5.0 months of treatment with rhGH, the levels decreased to a mean of 95.2±109.6 U/ml (Figure 1Go).

The expression of surface CD30 on circulating PBMC in five uraemic children (before and after rhGH therapy) and in five age-matched normal controls was investigated by flow cytometry. Uraemic children had a negligible proportion of PBMC staining for CD30 and no significant difference from that found in PBMC values in age-matched normal controls. Moreover, there were differences of expression of CD30 on PBMC of uraemic children before and after rhGH treatment (data not shown).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Elevated sCD30 plasma levels have been previously related to different disorders of the immune system, including malignant lymphomas, SLE, connective tissue disease, atopic disorders and HIV infection [6]. Both activated T cells and activated B cells can express surface CD30, and it has been suggested that CD30 expression does not discriminate between Th1 and Th2 type T cells [20]. However, other in vivo and in vitro studies have supported the hypothesis that CD30 is mainly expressed in Th2 lymphocytes [3] and that sCD30 plasma levels may reflect the Th2 T cell activation status [7,16].

As there is accumulating evidence that neuroendocrine axes can influence the immune system, and in particular the balance between Th1-Th2 immune responses [2], the effect of rhGH treatment on plasma levels of sCD30 in children with CRF, who fail to grow under common conservative treatment, was evaluated in the present study.

Our data demonstrated that sCD30 plasma levels were higher in children with CRF than in controls (P<0.001), and were inversely related to GFR (P<0.001). We also found that plasma levels of sCD30 were significantly lower in children with CRF under rhGH treatment than in untreated children with CRF (P<0.04). Although the group of children treated with rhGH had a predominance of boys (13 males and four females vs six males and eight females in the untreated group), there were no differences in age or height between rhGH-treated and untreated children (see Table 1Go). However, as rhGH treated children had lower GFRs than untreated children (mean GFR 19 vs 28 ml/min/1.73 m2, P=0.338), this finding is important because we had expected higher sCD30 plasma levels in the former group. Most of the 14 children not treated with GH subsequently underwent rhGH treatment; however, plasma samples for CD30 were not available during the follow-up study.

In a group of 11 children with CRF, plasma samples were available after a mean of 19.9±16.7 months of treatment with rhGH, and a decrease in sCD30 plasma levels were observed (P<0.01) (Figure 3Go). The finding that the percentage of decrease of sCD30 plasma levels in that group was not related to GFR variations, but rather to the duration of rhGH therapy, in months, (r=0.6499, P<0.05) and to the increment of height (r=–0.580, P<0.048), supports the hypothesis that rhGH treatment may modulate sCD30 plasma levels. However, these observations were possible because our patients had rather stable renal function during the follow-up. In contrast, if we had observed a more rapid decline of GFR in our paediatric patients, we would probably not have detected a decrease in sCD30 plasma levels because the reduction in glomerular filtration should have increased plasma levels of sCD30 (see Figure 1Go). The finding that the single patient that showed an increase in sCD30 plasma levels during rhGH treatment (Figure 3Go) was the same that had a 61% decrease in GFR during the same period, is consistent with the hypothesis that uraemia-induced reduction in GFR cause an accumulation of sCD30 in plasma that is reduced in part by rhGH treatment.

Moreover, as previous studies demonstrated that uremia itself can activate T cells [9], we speculate that in CRF, an enhanced T cell expression of CD30 can occur, resulting in increased shedding of sCD30 molecules that may contribute to sCD30 accumulation in uraemia.

In this context, although it is conceivable that sCD30 accumulation in CRF is due mainly to reduced urinary excretion of the molecule, the finding of a progressive reduction of sCD30 plasma levels in children with CRF under treatment with rhGH suggests that rhGH can downregulate the expression of CD30 (and in turn sCD30 shedding) in activated T cells or in other unidentified cells.

As the percentage of PBMC staining for CD30 was comparable in uraemic children and controls, and did not change during treatment with rhGH, the issue of whether or not uraemia is responsible for enhanced expression of CD30 in T cells or in other cell types requires additional investigation.

Alternatively, it is possible that rhGH treatment may decrease sCD30 plasma levels by augmenting the renal excretion of sCD30. To reach firm conclusions about the renal handling of a molecule, additional data are needed, including charge, sieving coefficient, and reabsorption/degradation rate. Because these were not available for the sCD30 molecule, we cannot determine whether sCD30 accumulation in CRF was related to reduced urinary excretion or to overproduction, or whether sCD30 plasma levels decreased during rhGH treatment because of decreased sCD30 production or increased renal excretion.

However, the finding that in children with primary GH deficiency and normal renal function also had sCD30 plasma levels that were higher than in normal controls (P<0.04), and that decreased during treatment with rhGH, suggests that GH deficiency can increase sCD30 plasma levels and that rhGH treatment may play a direct role in the regulation of sCD30 plasma levels. These observations suggest that the per cent decrease of sCD30 plasma levels was related to rhGH treatment duration (P<0.05) and to the effects of rhGH on the growth of the children (P<0.048). In addition, they support the hypothesis that the reduced bioavailability of GH in uraemia may have effects not only on growth but also on the immune system.

Taken together, these data strongly suggest that GH can influence the mechanisms of T cell activation. However whether rhGH treatment can affect the balance between Th1 and Th2 subpopulations and whether it can down-regulate Th2 cells remains to be established [3,1821]. Although previous studies did not show significant differences in other immune activation parameters in children treated with rhGH [17], the finding that rhGH treatment in critically ill adults was associated with increased mortality due to rapid onset of septic shock and multiple-organ failure, further supports, on clinical grounds, the hypothesis that the hypermetabolic and proinflammatory effects of rhGH treatment can affect immune function [19,22].

In conclusion, our data suggest that GH may act as an important regulator of the immune system, and point to new approaches for the investigation of rhGH effects in the treatment of children with CRF and with primary GH deficiency.



   Acknowledgments
 
This research was supported by a grant from the Fondo Malaitie Renali del Bambino, Genova, Italy.



   Notes
 
Correspondence and offprint requests to: Giancarlo Barbano MD, Nephrology Department, Istituto Giannina Gaslini, Largo G. Gaslini 5, I-16148 Genova, Italy. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 25. 5.99
Revision received 2. 3.01.