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In vitro effect of human recombinant growth hormone on lymphocyte and granulocyte function of healthy and uremic children
Immunology Letters 63 (1998) 41 – 47
In vitro effect of human recombinant growth hormone on lymphocyte and granulocyte function of healthy and uremic children Beata Derfalvi a, Katalin Nemet b, Csaba Szalai c, Eva Kenesei d, Peter Sallay d, Tivadar Tulassay d, Andras Falus a,* a
Department of Genetics, Cellular- and Immunobiology, Semmelweis Medical Uni6ersity, Nagy6a´rad te´r 4, Budapest 1098, Hungary b Institute of Immunology and Haematology, Budapest, Hungary c Heim Pal Children’s Hospital, Budapest, Hungary d First Department of Pediatrics, Semmelweis Medical Uni6ersity, Nagy6a´rad te´r 4, Budapest 1098, Hungary Received 15 September 1997; received in revised form 6 April 1998; accepted 14 April 1998
Abstract The recombinant human growth hormone (rhGH), currently used in supraphysiological doses to promote growth acceleration in chronic renal failure children (CRF), also has the ability to influence their impaired immune functions. The effect of human growth hormone on the lymphoproliferative response in vitro was analyzed in the peripheral blood lymphocytes of 25 healthy and 11 uremic children. In 72% of the uremic cases and in 60% of the healthy individual children the hormone increased the lymphoproliferation alone, and/or when used in combination with phytohaemagglutinine. The range of the effective hormone concentrations differed individually. Using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) a great variation in the gene expression of growth hormone- (GH)-receptor in peripheral lymphocytes was detected. The respiratory burst activity of peripheral polymorphonuclear leukocytes (PMN) in vitro, in response to GH alone and when combined with suboptimal dose of phorbolester (PMA), was assessed by measuring luminol enhanced chemiluminescence in ten uremic and 18 healthy children. In six out of the ten of the CRF patients and in eight out of 18 of the healthy children the GH enhanced the oxidative burst activity of granulocytes provoked by a suboptimal dose of PMA. However, the effective doses (10, 50 and 300 ng/ml) and incubation times (0, 45 and 90 min) showed individual variations. Our data suggest that rhGH treatment in uremic children could be advantageous considering this population’s enhanced susceptibility to bacterial, viral and fungal infections. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Human recombinant growth hormone; Lymphocyte; Granulocyte
1. Introduction Growth hormone (GH), a potent regulator of somatic growth and metabolism plays a modulatory role in the development and function of the immune system. Mononuclear leukocytes from various tissues including spleen, bone marrow, Peyer’s patches, peripheral blood and thymus epithelial cells all are able to express the gene for GH and secrete immunoreactive GH [1,2]. * Corresponding author. Tel./fax: [email protected]
+36 1 2102929; e-mail:
0165-2478/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0165-2478(98)00052-2
Strong GH-receptor (GHR) expression was found in B-cells, whereas T-cells and NK cells exhibited lower levels of receptors. In granulocytes GH acts through prolactin receptor [3,4]. The GHR belongs to the cytokine/haematopoietic receptor superfamily; binding of GH to its receptor induces the dimerization and phosphorylation of JAK2 kinases. These molecules serve in signal transduction by phosphorylating signal transducers and activators of transcription factors (STAT 1,3,5). Mitogen-activated protein kinases have also been reported to be activated by the GHR after stimulating the tyrosine phosphorylation of Shc—a cytoplasmic sub-
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strate of tyrosine kinase receptors [5]. Transcription of early response genes c-fos and c-jun are rapidly stimulated by GH [6]. GH stimulates the growth and function of cells in the immune system. Specifically GH has been shown to augment the proliferation and differentiation of erythroid, megakaryocytic and myeloid progenitor cells [7]. The hormone stimulates phagocyte migration, primes neutrophils and macrophages to produce superoxide anion and cytokines [8,9]. Inhibited fMLP (formyl-muramyl-leucine-proline) stimulated chemotaxis in polymorphonuclear neutrophils suggests that GH stimulates polymorphonuclear leukocytes (PMN) adhesiveness; finally enhances opsonic activity both in vitro and in vivo [10,11]. Growth hormone augments multiple lymphocyte functions, such as antibody synthesis, cytolytic activity of T-lymphocytes, natural killer cell activity, production of TNF-a, IL-1 a, IFN-g, and the synthesis of thymic hormones like thymulin [12–14]. The recombinant form of this hormone, in addition to being used in the therapy of primary and secondary GH deficiencies, is also used in a broad spectrum of pediatric diseases related with short stature or growth failure: Down- and Turner-syndrome, chronic renal failure (CRF), juvenile rheumatoid arthritis, D-vitamin resistant rickets and osteochondrodystrophies [15]. The objective of this study was to determine whether recombinant human growth hormone (rhGH), already used in growing number of pediatric and adulthood diseases, has any effect on the spontaneous and mitogen induced proliferation of human peripheral blood lymphocytes and on oxidative burst of peripheral blood PMN leukocytes in both healthy and uremic children, in vitro. RhGH treatment in children suffering from growth retardation due to CRF is well regarded. However, there is limited information on how this treatment effects their altered immune system.
2. Patients, materials and methods
2.1. Patients The uremic patients were treated and followed at the haemodialysis center of the 1st Department of Pediatrics, Semmelweis University of Medicine. The uremic group consisted of 11 children (six girls and five boys) with a mean age of 12.18 93.37 years and serum creatinine levels averaging 5879244 mmol/l (normal value B100 mmol/l), white blood cell count 5.75 9 1.44 G/l, absolute number of lymphocytes: 2.139 0.74, monocytes: 0.3990.08, neutrophil granulocytes: 2.82 90.96. Five uremic patients were being treated by haemodialysis performed three times a week for 4 h, using cellulose acetate or Gambrane type of dialysis membranes. These patients were tested just before the
dialysis session. Three patients were being treated by peritoneal dialysis and another three were not on dialysis. The children were treated by recombinant human rhGH 4 U/m2/day, subcutaneously. The patients included in this trial were in a stable state of health without any clinical or laboratory (differential cell count, C-reactive protein) evidence of bacterial infections and were receiving no medication known to interfere with the immune system, such as immunosuppressive drugs. A group of 25 healthy children (10 girls and 15 boys) aged 11.45+ 3.3 years, with an average serum creatinine level of 619 9.1 mmol/l, waiting for same-day surgery, served as a normal control population. White blood cell count: 6.479 1.04, absolute number of lymphocytes: 2.269 0.54, monocytes: 0.419 0.11, granulocytes: 3.4190.97. To be included in the study, the absence of any immunological abnormalities, infections and/or renal dysfunction was required. This study was approved by the Ethical Committee of the Semmelweis Medical University according to the Helsinki Declaration. The parents of each patient gave written informed consent.
2.2. Lymphoblast proliferation test Venous blood samples (9 ml) were collected in heparinized (Vacuette, Greiner) tubes in sterile circumstances. Peripheral blood lymphocytes were separated by centrifugation on Ficoll-Hypaque (Sigma) gradient [16]. Cell viability, as shown by trypan blue exclusion, was over 97%. The cells were suspended in cell culture medium RPMI 1640 (GIBCO) supplemented with 10% heat-inactivated fetal calf serum, glutamine and gentamycine. Five parallel cultures containing 1.5×105 cells/200 ml culture medium in a 96-well plate were established and incubated for 72 h at 37°C in 5% CO2 in the presence of: rhGH 5, 25, 50, 100 and 500 ng/ml (kindly provided by Pharmacia & Upjohn) and PHA 3 mg/ml (Murex Diagnostics) in the last 48 h. Changes in the cell count, reflecting the proliferation were quantified by staining with 3-4,5-dimethylthiazol-2-yl2,5-diphenyltetrazolium bromide (MTT; Sigma). This dye binds to the mitochondrial succinyl dehydrogenase in the cells, producing photometrically measurable extinctions which are proportional to the cell counts [17].
2.3. Separation of granulocytes Polymorphonuclear cells were isolated (95% purity) from 9 ml heparinized sterile venous blood after harvesting the mononuclear cells by Ficoll-Hypaque density-centrifugation (Sigma). Sedimentation on 6% dextran (Dextran T500, Pharmacia & Upjohn) was followed by hypotonic lysis of erythrocytes. Neutrophil cell viability was greater than or equal to 95%, assessed by trypan blue exclusion method.
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Table 1 Effect of rhGH on spontaneous lymphoproliferation in healthy and uremic children Patient
Control mean 9S.D.
Effect of rhGH over or below mean 9 2 S.D.
The effective rhGH concentration ng/ml
CRF group n =11 Enhancement M.D. V.B. S.L. K.B. Zs.K.
0.133 90.008 0.126 90.007 0.101 90.008 0.07890.012 0.068 90.004
0.152 0.142 0.13 0.157 0.087
100 100 100 50 25
Healthy group n = 25 Enhancement SZ.T. M.K. F.B. L.N. K.B. W.A.
0.32690.023 0.5 90.03 0.03690.003 0.04490.001 0.10690.004 0.03190.003
0.451 0.606 0.047 0.049 0.164 0.039
100 50 50 50 25 25
0.10890.006 0.16690.018 0.13190.003 0.11190.008 0.27690.025
0.093 0.118 0.117 0.088 0.197
500 500 100 100 25
Inhibition B.N. M.SZ. K.B. M.T. S.CS.
CRF: chronic renal failure; GH: growth hormone.
2.4. Measurement of the PMN oxidati6e burst Reactive oxygen intermediers released by PMNs were quantified by measuring luminol (5-amino-2,3-dihydro1,4-phtalazinedione, SIGMA) enhanced chemiluminescence in a Bertold LB 952 type luminometer. Cells were diluted in Hank’s balanced salt solution (HBS) without phenol red to a concentration of 1×105/ml and stimulated with a suboptimal concentration of 2 nM PMA (phorbol-12-myristate-13-acetate, SIGMA) to induce a low chemiluminescence signal. Cells were preincubated with 10, 50 and 300 ng/ml concentration of rhGH (Genotropin, gift from Pharmacia & Upjohn) for 0, 45 and 90 min at 37°C. Chemiluminescence was monitored in the dark at room temperature in 7 min intervals for 45 min. The assay was conducted after the peak chemiluminescence relative light unit (RLU) for each vial had passed. Estimation of the induced chemiluminescence was counted by the integral of the time versus RLU curve. We considered at least 10% change in the integral, in the reactive oxidative metabolite synthesis, to be significant.
2.5. Re6erse transcriptase polymerase chain reaction (RT-PCR) analysis of the GH receptor mRNA RNA was prepared by lysis in guanidinium thiocyanate and extraction in acidic phenolchlorophorm. Complementary DNA was synthesized from total cellu-
lar RNA (1 mg) using cloned murine leukemia virus reverse transcriptase and oligonucleotide primers (Perkin Elmer). cDNA aliquots were amplified using the primers for hGHR: sense-(1594–1617) 5%-CCCGGAAATGGTCTCACTGCCA-3%; antisense-(1902 – 1878), 5%-TCTTTGTCAGGCAAGGGCAAGGCAG-3% (Pharmacia Biotech). The primers for the GHR correspond to a region coding for the intracellular domain of GHR, identifying only the transcript coding specifically for the membrane-associated receptor rather than the soluble GH-binding protein. Amplification with 40 cycles of PCR at 95°C (30 s), 55°C (1 min) and 72°C (1 min) using Taq polymerase (Promega) resulted in PCR products of the expected size. These products were characterized by Hind III restriction enzyme analysis to confirm that the amplification was faithful. PCR products were electrophoresed on 2% agarose gels [3].
2.6. Serum neopterin ELISA-assay The microwell strips as solid phase are coated with anti-neopterin antibodies. Neopterin from the sample competes with the conjugate of neopterin and horseradish peroxidase for binding sites on the solid phase. The substrate of the enzyme is 2,2%-Azino-di-[3ethyl-benzthiazoline sulfonate(6)]. Absorbance at 405 nm is read, results are obtained via a standard calibration curve.
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Table 2 Effect of rhGH on phytohaemagglutinin induced lymphoproliferation in healthy and uremic children Patient
Control mean 9 S.D.
Effect of rhGH over or below mean 9 2 S.D.
The effect of rhGH concentration ng/ml
CRF group n = 11 Enhancement L.T. B.ZS. A.J.
0.0459 0.011 0.069 9 0.01 0.0769 0.01
0.083 0.092 0.139
500 500 25
0.1829 0.013 0.2669 0.013
0.136 0.227
500 50
0.2559 0.073 0.0929 0.021 0.0349 0.003 0.0399 0.013 0.0529 0.012 0.1129 0.003 0.346 9 0.032 0.2629 0.026
0.46 0.172 0.044 0.073 0.152 0.141 0.423 0.395
500 500 500 500 500 50 50 25
0.3449 0.074 0.1429 0.002 0.177 9 0.009 0.3119 0.011
0.107 0.119 0.152 0.253
500 500 100 100
Inhibition M.D. V.B. Healthy group n =25 Enhancement M.K. T.V. F.B. G.D. V.G. S.H. K.E. S.T. Inhibition S.CS. K.Be. B.N. K.Br.
CRF: chronic renal failure; GH: growth hormone.
2.7. Statistics A number of cases where the effect of GH on lymphoblast proliferation exceeded the mean9 2 S.D. range are shown. Multiple variance analyses (MANOVA) of the GH dose/response relationship, and the hormone-PHA interaction was performed. The differences between the healthy and uremic group were studied by x 2 test. Neopterin levels were calculated by Student’s t-test.
3. Results
3.1. Proliferati6e responses of peripheral blood lymphocytes to GH and PHA Tables 1 and 2 show how GH affected the lymphoblast proliferation. In 5/11 uremic and 6/25 healthy children GH had a direct proliferative effect on lymphocytes. In 5/25 cases in the healthy group the cell count decreased below the control mean decreased by 2 S.D. When combined with PHA the hormone increased the proliferation of lymphocytes in further 3/11 patients from the CRF group and 8/25 patients from the healthy group. However, in 2/11 and 4/25 cases GH
had a negative effect on the lectin induced lymphoblast proliferation. No significant differences between the two groups GH sensitivity to lymphoblast proliferation was found by x 2 test. Two-way ANOVA test revealed that in 15/25 (60%) of control children GH alone had a direct stimulatory effect on lymphoid proliferation, and/or further enhanced the effect of PHA significantly. In two cases we observed contradiction while the GH itself increased but when combined with PHA decreased the cell counts. In 6/25 (24%) healthy children the GH alone decreased the blastogenesis, or decreased the proliferative effect of the PHA. In six control children the GH had no effect (while the cells reacted to PHA). In two CRF patients neither the hormone, nor the lectin influenced the lymphocyte cell count.
3.2. PMN oxidati6e burst Fig. 1 shows a representative experiment of PMA induced chemiluminescence enhanced by GH dose dependently, after 45 min incubation time. Other experiments indicated that the GH alone did not stimulate the oxidative burst, but a combination of GH and PMA resulted in a synergistic activation typical for PMN-priming factors.
B. Derfal6i et al. / Immunology Letters 63 (1998) 41–47
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Fig. 1. PMA induced chemiluminescence-time curve of granulocytes separated from a healthy child, after 45 min of preincubation with increasing doses of rhGH. RLU: relative light unit; cpm: counts per minute.
Our results are summarized in Table 3. Granulocytes of healthy and uremic patients were primed by GH in the majority of the children. Six patients (60%) in the CRF group, but only eight individuals (44%) of the healthy group showed a dose- and time-dependent increase of free radical production. On the contrary, inhibition occurs less frequently in the CRF than in the healthy group, since only in 1/10 CRF patients was the oxidative burst slightly inhibited compared to 5/18 in the healthy group. Further, one of the uremic group and three of the healthy subjects the GH increased or decreased the oxidative burst depending on the incubation time. In both groups there were two cases in which granulocyte metabolic burst was not affected by the hormone. The optimal concentrations and incubation times of the hormone in the phorbolester induced metabolic burst differed individually. Day to day variation of the determination was excluded by using the cells of healthy control children each time. The ability of PMNs to produce free radicals to PMA without GH did not show significant difference in the two groups (P= 0.46). Chi-squared-probe did not indicate significant differences between the sensitivity to GH in the two groups.
3.3. Neopterin ELISA Serum neopterin levels were significantly higher in the uremic group, compared to the healthy children (healthy: 5.695.1, CRF: 130.2975, P = 10 − 9).
3.4. GHR gene expression by RT-PCR analysis Fig. 2 demonstrates the gene expression of GHR showing a high inter individual variance in childrens’ lymphocytes. According to our present findings the receptor expression did not correlate with the result on
the lymphoblast proliferation in the presence of GH and did not show any consequent differences in the two groups.
4. Discussion The neuroendocrine and immune system communicate bidirectionally through cytokines, peptide hormones, neurotransmitters, and in this process the receptors can be also shared. In addition to the endocrine actions of pituitary growth hormone, this hormone also appears to be involved in the autocrine/paracrine regulatory actions in various immune cells. Patients with end-stage renal disease and those treated with chronic dialysis have an immunodeficiency clinically manifested by: prolonged skin allograft survival, an increased susceptibility to intracellular pathogens, and about 7–10 times higher incidence of cancer, anergy in type four delayed type hypersensitivity tests, defective responses to T-cell-dependent antigens such as influenza and hepatitis-B virus. They also have abnormal antibody responses, decreased ability to seroconvertion after immunization and an increased incidence of autoantibodies [18]. Malnutrition, anaemia, blood transfusions, iron overload, which enhances bacterial growth and virulence, and the long-term exposure to dialysis membrane might all be involved in altering their immune system [18]. Uremic toxins are also responsible for the decreased immune responses, preincubation of human CD4 + T-cells with sera from haemodialysed and peritoneal dialysed uremic patients inhibited the capacity of the cells to be stimulated by phytohaemagglutinine and by anti-CD3 monoclonal antibody [18,19].
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Table 3 Effect of rhGH on granulocytes oxidative burst in control and uremic children
Healthy (n =18) Chronic renal failure (n =10)
Enhancement (%)
Inhibition (%)
ENH/INH (%)
No effect (%)
8 (44.4) 6 (60)
5 (27.8) 1 (10)
3 (16.7) 1 (10)
2 (11.1) 2 (20)
In the present study we found that in 60% of the control and 72% of the uremic children rhGH had a positive, stimulating effect on lymphoblast proliferation. However the grade of the effect and the range of the optimal concentrations of the hormone varied widely. Conflicting findings about the in vitro and in vivo effect of GH on the PHA induced lymphoproliferative response can be found in the literature [20 – 22], however this action is specific, because anti-hGH-serum completely blocks the modulating capacity of hGH [20]. The proliferation ability of peripheral lymphocytes to polyclonal antigens/mitogens of GH-deficient childrens after 9 months of rhGH treatment showed a transient elevation, however others found no significant changes [23,24]. In Europe, the incidence of end stage renal failure is 3.5 – 10 children per million children under the age of 15 years, and this prepubertal age-group is a candidate for rhGH treatment [25]. Regarding the low incidence of this disease we could involve only 11 children in this study. In our study two chronic renal patients showed blunted lectin induced proliferative responses. These results are consistent with the altered lymphocyte functions reported previously in renal failure patients [18]. These reduced responses can be partially explained by the lower IL-2 receptor density in CD4 + cells compared to those in healthy controls [26]. The present data indicate a preferential enhancement of lymphocytic mitogenesis in the CRF group with relative high individual variance. Despite the only partially understood cellular/molecular mechanisms, this simple blast transformation system used in this study might, in the
future, enhance the predictability of the previously unforeseen in vivo reactivity pattern to GH in individual children. Further, in the case of transplanted children, the results of this in vitro test might shed light on the controversy of whether rhGH treatment has a detrimental effect in acute and chronic rejections. We hypothesised that the differences in the responses to GH can be controled by the quantitative differences in the expression of GH receptors; however, we did not find a correlation between the receptor number and responsiveness by semiquantitative RT-PCR analysis. Therefore, GH mitogen responsiveness appears to be controled by other factors downstream to GH binding, such as at the level of the various steps of signal transduction. These individual differences could be explained by the incomplete maturation of the immune system in children, however we took into consideration that at least quantitatively the immune cells component are homogenous above 7 years. Several authors have investigated the monocytic production of superoxide and other reactive metabolites in response to GH. However, there has been little published regarding the effect of GH on PMN metabolic activity, especially characterized by chemiluminescence. The determination of chemiluminescence is an effective method for detection of the whole spectrum of reactive oxidative intermediers. Our results correlate with those of Spadoni et al., using FACS analysis, that the oxidative burst of PMNs can be significantly enhanced by GH in a time- and dose-dependent manner in vitro, and GH acts as a priming factor [27]. In contrast to PMN stimulators (e.g. phorbolesters) that cause a direct response in PMNs (such as chemotaxis, adhesion, ingestion, induction of metabolic burst, degranulation) PMN
Fig. 2. GH-receptor gene expression detected by reverse transcriptase polymerase chain reaction (RT-PCR). Initials of healthy (KBr, FB) and CRF (VB, VI, KBa, ZsK, SL) patients, (M) molecular weight marker, 308 base pair.
B. Derfal6i et al. / Immunology Letters 63 (1998) 41–47
priming factors enhance the neutrophil response to a stimulus. Patients with CRF have an increased susceptibility to infections due to diminished chemotactic response and impaired superoxide production of PMN cells [28,29]. One reason for that the granulocyte inhibitory proteins in patients sera, which block effective bacterial killing, chemotaxis and oxygen metabolism [30]. Each dialysis session triggers macrophage activation, mainly through the generation of activated complement components, following the contact of blood with dialysis membranes. The increased generation of basal reactive oxygen intermediers of neutrophils and monocytes contrasts with their reduced oxidative response to stimulating agents, such as invading microorganisms. In our results the higher neopterin levels in the CRF group, likely induced by interferon-g of activated T-cells indicate basal monocyte activation as well. In this study we have demonstrated that the GH in vitro could advantageously affect the lymphocyte and granulocyte function of the immunocompromised CRF patients, although it could have dangers in the later transplanted cases. The causes of the different reactivity to GH require further studies as well as the safety of the rhGH treatment in CRF since the pharmacological GH dose is higher than the substitution dose traditionally used as replacement therapy for GH deficient children.
[5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
[15] [16] [17] [18] [19]
[20] [21] [22]
Acknowledgements
[23]
The authors thank the financial support of the ‘Foundation for the Hungarian Science’ of the Hungarian Credit Bank.
[24] [25]
[26]
References
[27] [1] D.A. Weigent, J.E. Blalock, Cell. Immunol. 135 (1991) 55 – 65. [2] N. Maggiano, M. Piantelli, R. Ricci, L.M. Larocca, A. Capelli, F.O. Ranelletti, J. Hystochem. Cytochem. 42 (1994) 1349 – 1354. [3] R. Badolato, H.M. Bond, G. Valerio, A. Petrella, G. Morrone, M.J. Waters, S. Venuta, A. Tenore, J. Clin. Endoc. Metab. 79 (1994) 984 – 990. [4] Y.K. Fu, S. Arkins, G. Fuh, C. Cunningham, J.A. Wells, S.
.
[28] [29] [30]
47
Fong, M.J. Cronin, R. Dantzer, K.W. Welley, J. Clin. Invest. 89 (1992) 451 – 457. J.A. VanderKuur, N. Billestrup, G. Norstedt, S.C. Carter, J. Biol. Chem. 270 (1995) 7587 – 7593. M.C. Postel-Vinay, J. Finidori, Eur. J. Endocrinol. 133 (1995) 654 – 659. C.J. Wiedermann, N. Reinisch, C. Kahler, H. Braunsteiner, J. Ped. Endocrinol. 6 (1993) 85 – 91. W.J. Murphy, H. Rui, D.L. Longo, Life Sci. 57 (1995) 1–14. H. Saito, T. Inoue, K. Fukatsu, L. Ming-Tsan, T. Inaba, R. Fukushima, T. Muto, Horm. Res. 45 (1996) 50 – 54. J. Warwick-Davies, D.B. Lowrie, P.J. Cole, J. Immunol. 154 (1995) 1909 – 1918. C.J. Wiedermann, M. Niedermu¨hlbicher, D. Geissler, H. Beimpold, H. Braunsteiner, Br. J. Haematol. 78 (1991) 19 –22. K.W. Kelley, Ann. New York Acad. Sci. 584 (1990) 95–103. E.C. Snow, T.L. Feldbush, J.A. Oaks, J. Immunol. 121 (1981) 161 – 164. L. Sommese, G. Donnarumma, G. Cipollaro de L’Ero, A. Marcatili, M. Vitiello, M. Galdiero, J. Med. Microbiol. 45 (1996) 40 – 47. B.M. Lippe, J.M. Nakamoto, Rec. Progr. Horm. Res. 48 (1993) 179 – 235. A. Boyum, Scand. J. Clin. Lab. Invest. 97 (1968) 21 – 51. M.V. Berridge, A.S. Tan, K.D. McCoy, R. Wang, Biochemica 4 (1996) 15 – 20. B. Descamps-Latscha, A. Herbelin, Kid. Int. 43 (Suppl. 41) (1993) 135 – 142. R. Hershkovitz, Z. Korzets, M. Rathaus, D. Gilat, I. Drucker, Y.A. Mekori, J. Bernheim, Nephrol. Dial. Transplant. 10 (1995) 2065 – 2069. W. Kiess, H. Holtmann, O. Butenandt, R. Eife, Eur. J. Pediatr. 140 (1983) 47 – 50. R.M. Schimpff, A.M. Repellin, Acta. Endocrinol. Copenh. 120 (1989) 745 – 752. M. Bozzola, A. Valtorta, A. Moretta, D. Montagna, R. Maccario, G.R. Burgio, Thymus 12 (1988) 157 – 165. B.H. Petersen, R. Rapaport, D.P. Henry, C. Huseman, W.V. Moore, J. Clin. Endocrinol. Metab. 70 (1990) 1756 – 1760. G.L. Spadoni, P. Rossi, W. Rango, E. Galli, S. Cianfarani, C. Galasso, B. Boscherini, Acta Paediatr. Scand. 80 (1991) 75–79. Report from the European Dialysis and Transplant Registry 1. EDTA Registry Centre Survey, 1985, Nephrol. Dial. Transplant 2 (1987) 475 – 487. J. Stachowski, M. Pollok, M. Burrichter, C.A. Baldamus, J. Clin. Lab. Immunol. 34 (1991) 171 – 177. G.L. Spadoni, A. Spagnoli, S. Cianfarani, D. Del-Principe, A. Menichelli, S. Di-Giulio, B. Boscherini, Acta Endocrinol. Copenh. 124 (1991) 589 – 594. R.R. Martin, G. Eknoyan, C. Saenz, S.E. Hyde III, J. Olivero, J. Med. 10 (1979) 267 – 278. S.L. Lewis, D.E. Van-Epps, D.E. Chenowth, Clin. Nephrol. 30 (1988) 63 – 72. M. Haag-Weber, W.H. Horl, New-Horiz. 3 (1995) 669–679.
In vitro effect of human recombinant growth hormone on lymphocyte and granulocyte function of healthy and uremic children Beata Derfalvi a, Katalin Nemet b, Csaba Szalai c, Eva Kenesei d, Peter Sallay d, Tivadar Tulassay d, Andras Falus a,* a
Department of Genetics, Cellular- and Immunobiology, Semmelweis Medical Uni6ersity, Nagy6a´rad te´r 4, Budapest 1098, Hungary b Institute of Immunology and Haematology, Budapest, Hungary c Heim Pal Children’s Hospital, Budapest, Hungary d First Department of Pediatrics, Semmelweis Medical Uni6ersity, Nagy6a´rad te´r 4, Budapest 1098, Hungary Received 15 September 1997; received in revised form 6 April 1998; accepted 14 April 1998
Abstract The recombinant human growth hormone (rhGH), currently used in supraphysiological doses to promote growth acceleration in chronic renal failure children (CRF), also has the ability to influence their impaired immune functions. The effect of human growth hormone on the lymphoproliferative response in vitro was analyzed in the peripheral blood lymphocytes of 25 healthy and 11 uremic children. In 72% of the uremic cases and in 60% of the healthy individual children the hormone increased the lymphoproliferation alone, and/or when used in combination with phytohaemagglutinine. The range of the effective hormone concentrations differed individually. Using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) a great variation in the gene expression of growth hormone- (GH)-receptor in peripheral lymphocytes was detected. The respiratory burst activity of peripheral polymorphonuclear leukocytes (PMN) in vitro, in response to GH alone and when combined with suboptimal dose of phorbolester (PMA), was assessed by measuring luminol enhanced chemiluminescence in ten uremic and 18 healthy children. In six out of the ten of the CRF patients and in eight out of 18 of the healthy children the GH enhanced the oxidative burst activity of granulocytes provoked by a suboptimal dose of PMA. However, the effective doses (10, 50 and 300 ng/ml) and incubation times (0, 45 and 90 min) showed individual variations. Our data suggest that rhGH treatment in uremic children could be advantageous considering this population’s enhanced susceptibility to bacterial, viral and fungal infections. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Human recombinant growth hormone; Lymphocyte; Granulocyte
1. Introduction Growth hormone (GH), a potent regulator of somatic growth and metabolism plays a modulatory role in the development and function of the immune system. Mononuclear leukocytes from various tissues including spleen, bone marrow, Peyer’s patches, peripheral blood and thymus epithelial cells all are able to express the gene for GH and secrete immunoreactive GH [1,2]. * Corresponding author. Tel./fax: [email protected]
+36 1 2102929; e-mail:
0165-2478/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0165-2478(98)00052-2
Strong GH-receptor (GHR) expression was found in B-cells, whereas T-cells and NK cells exhibited lower levels of receptors. In granulocytes GH acts through prolactin receptor [3,4]. The GHR belongs to the cytokine/haematopoietic receptor superfamily; binding of GH to its receptor induces the dimerization and phosphorylation of JAK2 kinases. These molecules serve in signal transduction by phosphorylating signal transducers and activators of transcription factors (STAT 1,3,5). Mitogen-activated protein kinases have also been reported to be activated by the GHR after stimulating the tyrosine phosphorylation of Shc—a cytoplasmic sub-
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strate of tyrosine kinase receptors [5]. Transcription of early response genes c-fos and c-jun are rapidly stimulated by GH [6]. GH stimulates the growth and function of cells in the immune system. Specifically GH has been shown to augment the proliferation and differentiation of erythroid, megakaryocytic and myeloid progenitor cells [7]. The hormone stimulates phagocyte migration, primes neutrophils and macrophages to produce superoxide anion and cytokines [8,9]. Inhibited fMLP (formyl-muramyl-leucine-proline) stimulated chemotaxis in polymorphonuclear neutrophils suggests that GH stimulates polymorphonuclear leukocytes (PMN) adhesiveness; finally enhances opsonic activity both in vitro and in vivo [10,11]. Growth hormone augments multiple lymphocyte functions, such as antibody synthesis, cytolytic activity of T-lymphocytes, natural killer cell activity, production of TNF-a, IL-1 a, IFN-g, and the synthesis of thymic hormones like thymulin [12–14]. The recombinant form of this hormone, in addition to being used in the therapy of primary and secondary GH deficiencies, is also used in a broad spectrum of pediatric diseases related with short stature or growth failure: Down- and Turner-syndrome, chronic renal failure (CRF), juvenile rheumatoid arthritis, D-vitamin resistant rickets and osteochondrodystrophies [15]. The objective of this study was to determine whether recombinant human growth hormone (rhGH), already used in growing number of pediatric and adulthood diseases, has any effect on the spontaneous and mitogen induced proliferation of human peripheral blood lymphocytes and on oxidative burst of peripheral blood PMN leukocytes in both healthy and uremic children, in vitro. RhGH treatment in children suffering from growth retardation due to CRF is well regarded. However, there is limited information on how this treatment effects their altered immune system.
2. Patients, materials and methods
2.1. Patients The uremic patients were treated and followed at the haemodialysis center of the 1st Department of Pediatrics, Semmelweis University of Medicine. The uremic group consisted of 11 children (six girls and five boys) with a mean age of 12.18 93.37 years and serum creatinine levels averaging 5879244 mmol/l (normal value B100 mmol/l), white blood cell count 5.75 9 1.44 G/l, absolute number of lymphocytes: 2.139 0.74, monocytes: 0.3990.08, neutrophil granulocytes: 2.82 90.96. Five uremic patients were being treated by haemodialysis performed three times a week for 4 h, using cellulose acetate or Gambrane type of dialysis membranes. These patients were tested just before the
dialysis session. Three patients were being treated by peritoneal dialysis and another three were not on dialysis. The children were treated by recombinant human rhGH 4 U/m2/day, subcutaneously. The patients included in this trial were in a stable state of health without any clinical or laboratory (differential cell count, C-reactive protein) evidence of bacterial infections and were receiving no medication known to interfere with the immune system, such as immunosuppressive drugs. A group of 25 healthy children (10 girls and 15 boys) aged 11.45+ 3.3 years, with an average serum creatinine level of 619 9.1 mmol/l, waiting for same-day surgery, served as a normal control population. White blood cell count: 6.479 1.04, absolute number of lymphocytes: 2.269 0.54, monocytes: 0.419 0.11, granulocytes: 3.4190.97. To be included in the study, the absence of any immunological abnormalities, infections and/or renal dysfunction was required. This study was approved by the Ethical Committee of the Semmelweis Medical University according to the Helsinki Declaration. The parents of each patient gave written informed consent.
2.2. Lymphoblast proliferation test Venous blood samples (9 ml) were collected in heparinized (Vacuette, Greiner) tubes in sterile circumstances. Peripheral blood lymphocytes were separated by centrifugation on Ficoll-Hypaque (Sigma) gradient [16]. Cell viability, as shown by trypan blue exclusion, was over 97%. The cells were suspended in cell culture medium RPMI 1640 (GIBCO) supplemented with 10% heat-inactivated fetal calf serum, glutamine and gentamycine. Five parallel cultures containing 1.5×105 cells/200 ml culture medium in a 96-well plate were established and incubated for 72 h at 37°C in 5% CO2 in the presence of: rhGH 5, 25, 50, 100 and 500 ng/ml (kindly provided by Pharmacia & Upjohn) and PHA 3 mg/ml (Murex Diagnostics) in the last 48 h. Changes in the cell count, reflecting the proliferation were quantified by staining with 3-4,5-dimethylthiazol-2-yl2,5-diphenyltetrazolium bromide (MTT; Sigma). This dye binds to the mitochondrial succinyl dehydrogenase in the cells, producing photometrically measurable extinctions which are proportional to the cell counts [17].
2.3. Separation of granulocytes Polymorphonuclear cells were isolated (95% purity) from 9 ml heparinized sterile venous blood after harvesting the mononuclear cells by Ficoll-Hypaque density-centrifugation (Sigma). Sedimentation on 6% dextran (Dextran T500, Pharmacia & Upjohn) was followed by hypotonic lysis of erythrocytes. Neutrophil cell viability was greater than or equal to 95%, assessed by trypan blue exclusion method.
B. Derfal6i et al. / Immunology Letters 63 (1998) 41–47
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Table 1 Effect of rhGH on spontaneous lymphoproliferation in healthy and uremic children Patient
Control mean 9S.D.
Effect of rhGH over or below mean 9 2 S.D.
The effective rhGH concentration ng/ml
CRF group n =11 Enhancement M.D. V.B. S.L. K.B. Zs.K.
0.133 90.008 0.126 90.007 0.101 90.008 0.07890.012 0.068 90.004
0.152 0.142 0.13 0.157 0.087
100 100 100 50 25
Healthy group n = 25 Enhancement SZ.T. M.K. F.B. L.N. K.B. W.A.
0.32690.023 0.5 90.03 0.03690.003 0.04490.001 0.10690.004 0.03190.003
0.451 0.606 0.047 0.049 0.164 0.039
100 50 50 50 25 25
0.10890.006 0.16690.018 0.13190.003 0.11190.008 0.27690.025
0.093 0.118 0.117 0.088 0.197
500 500 100 100 25
Inhibition B.N. M.SZ. K.B. M.T. S.CS.
CRF: chronic renal failure; GH: growth hormone.
2.4. Measurement of the PMN oxidati6e burst Reactive oxygen intermediers released by PMNs were quantified by measuring luminol (5-amino-2,3-dihydro1,4-phtalazinedione, SIGMA) enhanced chemiluminescence in a Bertold LB 952 type luminometer. Cells were diluted in Hank’s balanced salt solution (HBS) without phenol red to a concentration of 1×105/ml and stimulated with a suboptimal concentration of 2 nM PMA (phorbol-12-myristate-13-acetate, SIGMA) to induce a low chemiluminescence signal. Cells were preincubated with 10, 50 and 300 ng/ml concentration of rhGH (Genotropin, gift from Pharmacia & Upjohn) for 0, 45 and 90 min at 37°C. Chemiluminescence was monitored in the dark at room temperature in 7 min intervals for 45 min. The assay was conducted after the peak chemiluminescence relative light unit (RLU) for each vial had passed. Estimation of the induced chemiluminescence was counted by the integral of the time versus RLU curve. We considered at least 10% change in the integral, in the reactive oxidative metabolite synthesis, to be significant.
2.5. Re6erse transcriptase polymerase chain reaction (RT-PCR) analysis of the GH receptor mRNA RNA was prepared by lysis in guanidinium thiocyanate and extraction in acidic phenolchlorophorm. Complementary DNA was synthesized from total cellu-
lar RNA (1 mg) using cloned murine leukemia virus reverse transcriptase and oligonucleotide primers (Perkin Elmer). cDNA aliquots were amplified using the primers for hGHR: sense-(1594–1617) 5%-CCCGGAAATGGTCTCACTGCCA-3%; antisense-(1902 – 1878), 5%-TCTTTGTCAGGCAAGGGCAAGGCAG-3% (Pharmacia Biotech). The primers for the GHR correspond to a region coding for the intracellular domain of GHR, identifying only the transcript coding specifically for the membrane-associated receptor rather than the soluble GH-binding protein. Amplification with 40 cycles of PCR at 95°C (30 s), 55°C (1 min) and 72°C (1 min) using Taq polymerase (Promega) resulted in PCR products of the expected size. These products were characterized by Hind III restriction enzyme analysis to confirm that the amplification was faithful. PCR products were electrophoresed on 2% agarose gels [3].
2.6. Serum neopterin ELISA-assay The microwell strips as solid phase are coated with anti-neopterin antibodies. Neopterin from the sample competes with the conjugate of neopterin and horseradish peroxidase for binding sites on the solid phase. The substrate of the enzyme is 2,2%-Azino-di-[3ethyl-benzthiazoline sulfonate(6)]. Absorbance at 405 nm is read, results are obtained via a standard calibration curve.
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Table 2 Effect of rhGH on phytohaemagglutinin induced lymphoproliferation in healthy and uremic children Patient
Control mean 9 S.D.
Effect of rhGH over or below mean 9 2 S.D.
The effect of rhGH concentration ng/ml
CRF group n = 11 Enhancement L.T. B.ZS. A.J.
0.0459 0.011 0.069 9 0.01 0.0769 0.01
0.083 0.092 0.139
500 500 25
0.1829 0.013 0.2669 0.013
0.136 0.227
500 50
0.2559 0.073 0.0929 0.021 0.0349 0.003 0.0399 0.013 0.0529 0.012 0.1129 0.003 0.346 9 0.032 0.2629 0.026
0.46 0.172 0.044 0.073 0.152 0.141 0.423 0.395
500 500 500 500 500 50 50 25
0.3449 0.074 0.1429 0.002 0.177 9 0.009 0.3119 0.011
0.107 0.119 0.152 0.253
500 500 100 100
Inhibition M.D. V.B. Healthy group n =25 Enhancement M.K. T.V. F.B. G.D. V.G. S.H. K.E. S.T. Inhibition S.CS. K.Be. B.N. K.Br.
CRF: chronic renal failure; GH: growth hormone.
2.7. Statistics A number of cases where the effect of GH on lymphoblast proliferation exceeded the mean9 2 S.D. range are shown. Multiple variance analyses (MANOVA) of the GH dose/response relationship, and the hormone-PHA interaction was performed. The differences between the healthy and uremic group were studied by x 2 test. Neopterin levels were calculated by Student’s t-test.
3. Results
3.1. Proliferati6e responses of peripheral blood lymphocytes to GH and PHA Tables 1 and 2 show how GH affected the lymphoblast proliferation. In 5/11 uremic and 6/25 healthy children GH had a direct proliferative effect on lymphocytes. In 5/25 cases in the healthy group the cell count decreased below the control mean decreased by 2 S.D. When combined with PHA the hormone increased the proliferation of lymphocytes in further 3/11 patients from the CRF group and 8/25 patients from the healthy group. However, in 2/11 and 4/25 cases GH
had a negative effect on the lectin induced lymphoblast proliferation. No significant differences between the two groups GH sensitivity to lymphoblast proliferation was found by x 2 test. Two-way ANOVA test revealed that in 15/25 (60%) of control children GH alone had a direct stimulatory effect on lymphoid proliferation, and/or further enhanced the effect of PHA significantly. In two cases we observed contradiction while the GH itself increased but when combined with PHA decreased the cell counts. In 6/25 (24%) healthy children the GH alone decreased the blastogenesis, or decreased the proliferative effect of the PHA. In six control children the GH had no effect (while the cells reacted to PHA). In two CRF patients neither the hormone, nor the lectin influenced the lymphocyte cell count.
3.2. PMN oxidati6e burst Fig. 1 shows a representative experiment of PMA induced chemiluminescence enhanced by GH dose dependently, after 45 min incubation time. Other experiments indicated that the GH alone did not stimulate the oxidative burst, but a combination of GH and PMA resulted in a synergistic activation typical for PMN-priming factors.
B. Derfal6i et al. / Immunology Letters 63 (1998) 41–47
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Fig. 1. PMA induced chemiluminescence-time curve of granulocytes separated from a healthy child, after 45 min of preincubation with increasing doses of rhGH. RLU: relative light unit; cpm: counts per minute.
Our results are summarized in Table 3. Granulocytes of healthy and uremic patients were primed by GH in the majority of the children. Six patients (60%) in the CRF group, but only eight individuals (44%) of the healthy group showed a dose- and time-dependent increase of free radical production. On the contrary, inhibition occurs less frequently in the CRF than in the healthy group, since only in 1/10 CRF patients was the oxidative burst slightly inhibited compared to 5/18 in the healthy group. Further, one of the uremic group and three of the healthy subjects the GH increased or decreased the oxidative burst depending on the incubation time. In both groups there were two cases in which granulocyte metabolic burst was not affected by the hormone. The optimal concentrations and incubation times of the hormone in the phorbolester induced metabolic burst differed individually. Day to day variation of the determination was excluded by using the cells of healthy control children each time. The ability of PMNs to produce free radicals to PMA without GH did not show significant difference in the two groups (P= 0.46). Chi-squared-probe did not indicate significant differences between the sensitivity to GH in the two groups.
3.3. Neopterin ELISA Serum neopterin levels were significantly higher in the uremic group, compared to the healthy children (healthy: 5.695.1, CRF: 130.2975, P = 10 − 9).
3.4. GHR gene expression by RT-PCR analysis Fig. 2 demonstrates the gene expression of GHR showing a high inter individual variance in childrens’ lymphocytes. According to our present findings the receptor expression did not correlate with the result on
the lymphoblast proliferation in the presence of GH and did not show any consequent differences in the two groups.
4. Discussion The neuroendocrine and immune system communicate bidirectionally through cytokines, peptide hormones, neurotransmitters, and in this process the receptors can be also shared. In addition to the endocrine actions of pituitary growth hormone, this hormone also appears to be involved in the autocrine/paracrine regulatory actions in various immune cells. Patients with end-stage renal disease and those treated with chronic dialysis have an immunodeficiency clinically manifested by: prolonged skin allograft survival, an increased susceptibility to intracellular pathogens, and about 7–10 times higher incidence of cancer, anergy in type four delayed type hypersensitivity tests, defective responses to T-cell-dependent antigens such as influenza and hepatitis-B virus. They also have abnormal antibody responses, decreased ability to seroconvertion after immunization and an increased incidence of autoantibodies [18]. Malnutrition, anaemia, blood transfusions, iron overload, which enhances bacterial growth and virulence, and the long-term exposure to dialysis membrane might all be involved in altering their immune system [18]. Uremic toxins are also responsible for the decreased immune responses, preincubation of human CD4 + T-cells with sera from haemodialysed and peritoneal dialysed uremic patients inhibited the capacity of the cells to be stimulated by phytohaemagglutinine and by anti-CD3 monoclonal antibody [18,19].
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Table 3 Effect of rhGH on granulocytes oxidative burst in control and uremic children
Healthy (n =18) Chronic renal failure (n =10)
Enhancement (%)
Inhibition (%)
ENH/INH (%)
No effect (%)
8 (44.4) 6 (60)
5 (27.8) 1 (10)
3 (16.7) 1 (10)
2 (11.1) 2 (20)
In the present study we found that in 60% of the control and 72% of the uremic children rhGH had a positive, stimulating effect on lymphoblast proliferation. However the grade of the effect and the range of the optimal concentrations of the hormone varied widely. Conflicting findings about the in vitro and in vivo effect of GH on the PHA induced lymphoproliferative response can be found in the literature [20 – 22], however this action is specific, because anti-hGH-serum completely blocks the modulating capacity of hGH [20]. The proliferation ability of peripheral lymphocytes to polyclonal antigens/mitogens of GH-deficient childrens after 9 months of rhGH treatment showed a transient elevation, however others found no significant changes [23,24]. In Europe, the incidence of end stage renal failure is 3.5 – 10 children per million children under the age of 15 years, and this prepubertal age-group is a candidate for rhGH treatment [25]. Regarding the low incidence of this disease we could involve only 11 children in this study. In our study two chronic renal patients showed blunted lectin induced proliferative responses. These results are consistent with the altered lymphocyte functions reported previously in renal failure patients [18]. These reduced responses can be partially explained by the lower IL-2 receptor density in CD4 + cells compared to those in healthy controls [26]. The present data indicate a preferential enhancement of lymphocytic mitogenesis in the CRF group with relative high individual variance. Despite the only partially understood cellular/molecular mechanisms, this simple blast transformation system used in this study might, in the
future, enhance the predictability of the previously unforeseen in vivo reactivity pattern to GH in individual children. Further, in the case of transplanted children, the results of this in vitro test might shed light on the controversy of whether rhGH treatment has a detrimental effect in acute and chronic rejections. We hypothesised that the differences in the responses to GH can be controled by the quantitative differences in the expression of GH receptors; however, we did not find a correlation between the receptor number and responsiveness by semiquantitative RT-PCR analysis. Therefore, GH mitogen responsiveness appears to be controled by other factors downstream to GH binding, such as at the level of the various steps of signal transduction. These individual differences could be explained by the incomplete maturation of the immune system in children, however we took into consideration that at least quantitatively the immune cells component are homogenous above 7 years. Several authors have investigated the monocytic production of superoxide and other reactive metabolites in response to GH. However, there has been little published regarding the effect of GH on PMN metabolic activity, especially characterized by chemiluminescence. The determination of chemiluminescence is an effective method for detection of the whole spectrum of reactive oxidative intermediers. Our results correlate with those of Spadoni et al., using FACS analysis, that the oxidative burst of PMNs can be significantly enhanced by GH in a time- and dose-dependent manner in vitro, and GH acts as a priming factor [27]. In contrast to PMN stimulators (e.g. phorbolesters) that cause a direct response in PMNs (such as chemotaxis, adhesion, ingestion, induction of metabolic burst, degranulation) PMN
Fig. 2. GH-receptor gene expression detected by reverse transcriptase polymerase chain reaction (RT-PCR). Initials of healthy (KBr, FB) and CRF (VB, VI, KBa, ZsK, SL) patients, (M) molecular weight marker, 308 base pair.
B. Derfal6i et al. / Immunology Letters 63 (1998) 41–47
priming factors enhance the neutrophil response to a stimulus. Patients with CRF have an increased susceptibility to infections due to diminished chemotactic response and impaired superoxide production of PMN cells [28,29]. One reason for that the granulocyte inhibitory proteins in patients sera, which block effective bacterial killing, chemotaxis and oxygen metabolism [30]. Each dialysis session triggers macrophage activation, mainly through the generation of activated complement components, following the contact of blood with dialysis membranes. The increased generation of basal reactive oxygen intermediers of neutrophils and monocytes contrasts with their reduced oxidative response to stimulating agents, such as invading microorganisms. In our results the higher neopterin levels in the CRF group, likely induced by interferon-g of activated T-cells indicate basal monocyte activation as well. In this study we have demonstrated that the GH in vitro could advantageously affect the lymphocyte and granulocyte function of the immunocompromised CRF patients, although it could have dangers in the later transplanted cases. The causes of the different reactivity to GH require further studies as well as the safety of the rhGH treatment in CRF since the pharmacological GH dose is higher than the substitution dose traditionally used as replacement therapy for GH deficient children.
[5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
[15] [16] [17] [18] [19]
[20] [21] [22]
Acknowledgements
[23]
The authors thank the financial support of the ‘Foundation for the Hungarian Science’ of the Hungarian Credit Bank.
[24] [25]
[26]
References
[27] [1] D.A. Weigent, J.E. Blalock, Cell. Immunol. 135 (1991) 55 – 65. [2] N. Maggiano, M. Piantelli, R. Ricci, L.M. Larocca, A. Capelli, F.O. Ranelletti, J. Hystochem. Cytochem. 42 (1994) 1349 – 1354. [3] R. Badolato, H.M. Bond, G. Valerio, A. Petrella, G. Morrone, M.J. Waters, S. Venuta, A. Tenore, J. Clin. Endoc. Metab. 79 (1994) 984 – 990. [4] Y.K. Fu, S. Arkins, G. Fuh, C. Cunningham, J.A. Wells, S.
.
[28] [29] [30]
47
Fong, M.J. Cronin, R. Dantzer, K.W. Welley, J. Clin. Invest. 89 (1992) 451 – 457. J.A. VanderKuur, N. Billestrup, G. Norstedt, S.C. Carter, J. Biol. Chem. 270 (1995) 7587 – 7593. M.C. Postel-Vinay, J. Finidori, Eur. J. Endocrinol. 133 (1995) 654 – 659. C.J. Wiedermann, N. Reinisch, C. Kahler, H. Braunsteiner, J. Ped. Endocrinol. 6 (1993) 85 – 91. W.J. Murphy, H. Rui, D.L. Longo, Life Sci. 57 (1995) 1–14. H. Saito, T. Inoue, K. Fukatsu, L. Ming-Tsan, T. Inaba, R. Fukushima, T. Muto, Horm. Res. 45 (1996) 50 – 54. J. Warwick-Davies, D.B. Lowrie, P.J. Cole, J. Immunol. 154 (1995) 1909 – 1918. C.J. Wiedermann, M. Niedermu¨hlbicher, D. Geissler, H. Beimpold, H. Braunsteiner, Br. J. Haematol. 78 (1991) 19 –22. K.W. Kelley, Ann. New York Acad. Sci. 584 (1990) 95–103. E.C. Snow, T.L. Feldbush, J.A. Oaks, J. Immunol. 121 (1981) 161 – 164. L. Sommese, G. Donnarumma, G. Cipollaro de L’Ero, A. Marcatili, M. Vitiello, M. Galdiero, J. Med. Microbiol. 45 (1996) 40 – 47. B.M. Lippe, J.M. Nakamoto, Rec. Progr. Horm. Res. 48 (1993) 179 – 235. A. Boyum, Scand. J. Clin. Lab. Invest. 97 (1968) 21 – 51. M.V. Berridge, A.S. Tan, K.D. McCoy, R. Wang, Biochemica 4 (1996) 15 – 20. B. Descamps-Latscha, A. Herbelin, Kid. Int. 43 (Suppl. 41) (1993) 135 – 142. R. Hershkovitz, Z. Korzets, M. Rathaus, D. Gilat, I. Drucker, Y.A. Mekori, J. Bernheim, Nephrol. Dial. Transplant. 10 (1995) 2065 – 2069. W. Kiess, H. Holtmann, O. Butenandt, R. Eife, Eur. J. Pediatr. 140 (1983) 47 – 50. R.M. Schimpff, A.M. Repellin, Acta. Endocrinol. Copenh. 120 (1989) 745 – 752. M. Bozzola, A. Valtorta, A. Moretta, D. Montagna, R. Maccario, G.R. Burgio, Thymus 12 (1988) 157 – 165. B.H. Petersen, R. Rapaport, D.P. Henry, C. Huseman, W.V. Moore, J. Clin. Endocrinol. Metab. 70 (1990) 1756 – 1760. G.L. Spadoni, P. Rossi, W. Rango, E. Galli, S. Cianfarani, C. Galasso, B. Boscherini, Acta Paediatr. Scand. 80 (1991) 75–79. Report from the European Dialysis and Transplant Registry 1. EDTA Registry Centre Survey, 1985, Nephrol. Dial. Transplant 2 (1987) 475 – 487. J. Stachowski, M. Pollok, M. Burrichter, C.A. Baldamus, J. Clin. Lab. Immunol. 34 (1991) 171 – 177. G.L. Spadoni, A. Spagnoli, S. Cianfarani, D. Del-Principe, A. Menichelli, S. Di-Giulio, B. Boscherini, Acta Endocrinol. Copenh. 124 (1991) 589 – 594. R.R. Martin, G. Eknoyan, C. Saenz, S.E. Hyde III, J. Olivero, J. Med. 10 (1979) 267 – 278. S.L. Lewis, D.E. Van-Epps, D.E. Chenowth, Clin. Nephrol. 30 (1988) 63 – 72. M. Haag-Weber, W.H. Horl, New-Horiz. 3 (1995) 669–679.