In vitro substance P-dependent induction of bone marrow cells in common (CD10) acute lymphoblastic leukaemia

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Leukemia Research 32 (2008) 97–102

In vitro substance P-dependent induction of bone marrow cells in common (CD10) acute lymphoblastic leukaemia M. Nowicki a,∗ , D. Ostalska-Nowicka b , B. Miskowiak c a Department of Histology and Embryology, University of Medical Sciences in Poznan, Swiecickiego 6, 60-781 Poznan, Poland Department of Pediatric Cardiology and Nephrology, University of Medical Sciences in Poznan, Szpitalna 27/33, 60-572 Poznan, Poland Department of Optometry and Biology of the Visual System, University of Medical Sciences in Poznan, Rokietnicka 5D, 60-806 Poznan, Poland b

c

Received 27 January 2007; received in revised form 10 May 2007; accepted 14 May 2007 Available online 22 June 2007

Abstract The aim of the present research was to investigate the possible in vitro stimulatory effect of substance P (SP) on blasts induction in childhood common acute lymphoblastic leukaemia (ALL). Bone marrow aspirates were incubated with SP receptor agonist or antagonist (spantide) and subsequently assayed for the presence of human interleukin (IL)-1b using ELISA kit. Blast cells incubated with SP receptor agonist were found to result in a significant increase of IL-1b concentration while incubated with spantide resulted in control levels of IL-1b. These findings suggest the novel possible role of SP in blasts proliferation in childhood ALL of common (CD10) origin. © 2007 Elsevier Ltd. All rights reserved. Keywords: Cell proliferation; Children; Interleukin-1b; Leukaemia; Substance P

1. Introduction Substance P (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-GlyLeu-Met-NH2 ), widely distributed in the nervous system, plays an essential role as a neuromediator [1,2]. First of all, it is antidromally released from sensory nerve endings of C type nerve fibers [3,4] in response to mechanical, chemical, and thermal insults as well as under effect of factors released at sites of tissue injury [4–8]. According to several authors, the presence of peptidergic nerve endings in the closest vicinity of immuno-competent cells in organs most exposed to contact with foreign antigens represents an anatomical exponent of functional links between the mentioned fibers and cells and, more generally, between nervous system and immune system [9–11]. In humans, receptor for SP, known as the neurokinin1 receptor (NK-1R) can be demonstrated on around 40% peripheral blood lymphocytes [7]. As compared to mature ∗

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lymphocytes, lymphoblasts carry around 3–4-fold higher amounts of receptors for SP [12]. Apart from lymphocytes, receptors for SP also can be noted on monocytes [13], endothelial cells [14], fibroblasts [15] and hematopoietic stem cells [16]. Substance P augments proliferative activity of human and mouse lymphocytes T [17,18], human smooth muscle cells [19], mouse fibroblasts [20], fibroblasts of human skin [15], smooth muscle fibers of arterial walls [21], human synovial cells [22] and human cells forming colonies of granulocytes and monocytes or of erythrocytes [23]. SP stimulates also production of cytokines such as interleukin-1 (IL) [24,25], IL-2 [7,26], IL-3, IL-6, tumour necrosis factor-␣ (TNF) [22], interferon-␥ [27], granulocyte–monocyte colony stimulating factor (GM-CSF) and stem cell factor (SCF) [26]. It may intensify expression of adhesion molecules, i.e. ICAM-1, which promote implantation of grafted hematopoietic cells [28]. Due to the present in bone marrow (BM) peptidergic nerve endings, SP has an easy access both to hematopoietic cells and to cells forming sublayer of BM [29]. Earlier conducted studies demonstrated that

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SP is also released in BM from macrophages [30], eosinophils [31,32] and from cells of vascular endothelium [33]. Most of cells present in BM, i.e. both hematopoietic cells [23], and cells forming BM stroma [14,34], as well as lymphocytes present there, particularly lymphocytes T [35], are equipped with the SP-specific receptor, NK-1R. Cytokines linked to the hematopoietic functions of SP include IL-1, IL-3, GM-CSF and SCF [36]. SP induces the production of these cytokines, which exhibit stimulatory effects on hematopoiesis. Alternatively, the cytokines induced by SP could activate BM cells through an autocrine and/or paracrine mechanism to produce other cytokines with hematopoiesis-stimulatory effects [37]. For example, SP induces the production of IL-1, which stimulates the induction of hematopoietic factors with direct and indirect effects on haematopoietic stem cells (HSCs) [38]. In contrast to SP, the hematopoietic effects of NK-A could be stimulatory or inhibitory, depending on the particular hematopoietic lineage [23,39]. NK-A inhibits the proliferation of granulocyte–monocyte progenitors, but stimulates erythrocyte progenitors [29]. The negative functions of NK-A can be explained by the production of hematopoietic suppressors and transforming growth factor ␤ [36]. In acute lymphoblastic leukaemia (ALL), which forms the major malignant disease in children, as well as the most common type of childhood leukaemia, the plasma level of SP are not detectable. Although in our previous study we demonstrated that blast cells in ALL of T-cell and common (CD10) origin are able to produce SP [40], it is still not yet clear whether, in vivo, ALL cells stimulate themselves in a SP-dependent autocrine manner. On the other hand, SP expression in blast cells in BM biopsies in childhood ALL have been found to be associated with increased risk of unfavourable leukaemia course and have been found to be significantly associated with recurrent ALL [41]. In line with the above, the aim of the present research was to investigate the possible in vitro stimulatory effect of SP on blasts proliferation in childhood common acute lymphoblastic leukaemia.

2. Materials and methods 2.1. Patients BM samples were taken from children treated in the Department of Paediatric Oncology, Haematology and Transplantation, Poznan University of Medical Sciences, between 1 January 1998 and 13 December 2002. The research protocol was approved by the Ethics Commission of the University. Thirty children designated to the study protocol were diagnosed with common acute lymphoblastic leukaemia (CD10-positive). Half of them (SP(+) group) were additionally diagnosed with the expression of SP in blast cells as previously described [40,41, see also SP detection paragraph below]. The rest of them were SP-negative (SP(−), n = 15). The bone marrow from all the leukemic children was sampled at the moment of diagnosis and before administration of any chemotherapy regimen. Ten age- and sex-matched children, all of whom presented with only one enlarged lymph node, served as the control group. Here, histopathological examination of the enlarged node indicated an inflammatory response only. Subsequent observation of these control children in the Out-patient Clinic for Hyperplastic Diseases for around 12 months detected no clinical traits of neoplastic disease. To participate in the investigation, children had to meet the following eligibility criteria: age 4–18 years, CD10-positive phenotype (as described below), non-lymphocytes cell count (monocytes and granulocytes) <0.1 G/l, percentage of blasts in BM > 98.5% and sterile blood cultures (obtained at least three times) at the time of the first hospitalisation. The relevant data are presented in Table 1. 2.2. SP detection In order to demonstrate the presence of SP in bone marrow cells, an indirect immunocytochemical procedure was performed with rabbit antibodies against human SP (Serotec, PEP A40) and the StreptABComplex/HRP method modified

Table 1 Clinical pre-treatment characteristics, selected laboratory data and results of substance P determination in children with ALL Characteristics

Number of patients Age (years) Sex (male/female) ALL phenotype Risk group WBC, G/L Monocytes and granulocytes cell count (G/l) %Blasts in BM SP %blast Blood culture Treatment protocol

Study group

Control group

SP(+)

SP(−)

15 8 (4–18) 7/8 Common Standard 12.3 ± 4.8 <0.1 99.5 ± 0.5 84.3 ± 12.2 Sterile ALL-BFM-95

15 7 (5–17) 8/7 Common Standard 14.6 ± 6.7 <0.1 99.3 ± 0.7 – Sterile ALL-BFM-95

10 8 (6–18) 5/5 – – 6.7 ± 2.2 4.5 ± 1.8 – – Sterile –

Values represent the mean ± S.D., except for age which is expressed as the median (range); SP = substance P; ALL = acute lymphoblastic leukemia; WBC = white blood cells count at the moment of diagnosis; common = common acute lymphoblastic leukaemia antigen (CALLA, CD10) immunophenotype; SP %blast = the percentage of SP-positive blast cells to the total number of nucleated cells in SP-positive children.

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by the application of biotinylated tyramine (Dako Catalysed Signal Amplification System, Peroxidase, K 1500). Heat-induced antigen demasking pre-treatment was also carried out (Target Retrieval Solution, Dako S 1699.15). The endogenous activity of peroxidase was blocked by 10 min preincubation in 10% hydrogen peroxide. The smears were then incubated with anti-SP antibodies diluted 1:500–1:2000 for 12 h at +41 ◦ C. Incubation with a second antibody (biotinylated goat anti-rabbit, Dako E 0432, diluted 1:300) was performed at room temperature for 60 min followed by incubation with diaminobenzidine (DAB, Dako S 3000). For detection of mRNA encoding the amino acid sequence of human substance P, a 5 -biotinylated probe of the nucleotide sequence 5 -TCT GGG TTC GGA GTC GTC AAG AAA CCT AAT TAC-3 , was used. The probe was originally synthesised by the DNAGdansk (Gdansk, Poland) and was complementary to the nucleotide sequence of human substance P as available in the GenBank (locus = 7q21–q22, gene = “TAC1”, product = “substance P”, bp = 68–100; http:// www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=M68907). The smears were incubated with the probe (concentration: 200 ng/1 ml) for 18 h at +37 ◦ C in a hybridisation chamber. This was followed by incubation with a streptavidin–biotin complex (Dako, K 1500, diluted 1:100) at room temperature for 60 min and incubation with DAB for 5 min. Both techniques complied with the principles of positive and negative controls and were performed blind on coded samples. Results of the immunocytochemical reactions and of the in situ hybridisation were examined under a light microscope (Eclipse 600, Nikon), at 1:200–1:400 magnification. Based on the results of the haematological staining, which were analysed through the use of Microimage (Olympus) morphometric software, the content of reaction-positive cells was determined by comparing the number of cells with a positive reaction for SP or mRNA SP with the total number of blasts. A percentage of SP-positive cells less than 5% was deemed to reflect technical errors and was classified as a negative result. 2.3. Reagents SP receptor agonist—identical to natural sequence of amino acids; supplied by Bachem, Bubendorf, Switzerland (H-1890.0005). SP receptor antagonist—spantide, H-DLys(nicotinol)-Pro-␤-(3-pyridyl)-Ala-Pro-3,4-dichloro-DPhe-Asn-D-Trp-Phe-D-Trp-Leu-Nle-NH2 (H-8310.0001, provided by the same manufacturer). ELISA kit—high sensitivity interleukin-1 beta [(h)IL—1␤] human ELISA system; RPN 2781; Biotrak; Amersham Pharmacia Biotech UK Ltd., Amersham Place Little Chalfont Buckinghamshire, UK. 2.4. BM cell identification The samples of BM were taken in the typical way from the posterior superior iliac spine as previously described [40]. The leukaemia samples were classified according to

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bright field microscopic identification (Giemsa staining) and subsequent phenotyping by flow cytometry assay. All the children in whom the percentage of blast cells was lower than 98.5% were excluded from the analysis. Samples were stained with the four-colour combination CD34-FITC/CD10PE/CD19-PerCP/CD22-APC (Becton Dickinson, San Jose, CA). Erythrocytes were lysed (FACSLyse, BD), and the samples were resuspended and fixed in paraformaldehyde before running on a FACSCalibur flow cytometer (Becton Dickinson). Presented analysis enabled recognition of common (CD10-positive) ALL as well as estimation of the total number of cells used in the subsequent incubation (an average number of BM cells in 100 ␮l was rated for 4 × 104 ). 2.5. Cell preparation Aspirates were placed into preservative-free 0.5% heparin phosphate buffer saline (PBS) solution and quartered into aseptic test-tubes. All of them were placed in 37 ◦ C for 2 h following an addition of SP receptor agonist (concentration of 10−9 mol/L) or spantide (concentration of 10−6 mol/L) into two of them. The third test-tube was equipped with both SP receptor agonist and spantide at the same concentrations as stated above. The last test-tube was free of any reagents. After the incubation, all the aspirates were centrifuged (4000 rpm for 5 min), supernatants were collected and kept in −80 ◦ C until the assay for the presence of biologically active IL-1b was performed. 2.6. Biological assay for IL-1b Defrosted supernatants were again clarified by centrifugation (4000 rpm for 2 min) and than assayed for the presence of biologically active human IL-1b. The following steps of ELISA were performed according to the instructions as provided by the manufacturer. The estimation of an average IL-1b concentration in each patient from both study and control groups was founded on double cytokine evaluation by reading in spectrophotometer at 450 nm. 2.7. Statistical analysis The statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test at p < 0.05. These were tracked by employing Statistica 6.1 software. 3. Results In the control incubations, bone marrow was free of any reagents except of heparin. The average IL-1b concentration in SP(+) patients was 7.3 ± 0.9 pg, in SP(−) group—2.2 ± 0.9 pg (p = 0.001) and finally 3.8 ± 1.4 pg in control children (p = 0.02). There was no significant difference between IL-1b concentration between SP(−) and control subjects.

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Fig. 1. Diagrammatic representation of the sample IL-1b concentrations in the bone marrow. IL = interleukin; BM = bone marrow; ALL = acute lymphoblastic leukemia; SP = substance P; SP(+) = leukemic children diagnosed with the expression of SP in blast cells of BM; SP(−) = leukemic children with no expression of SP in blast cells of BM; *p = 0.01; **p = 0.001.

The average IL-1b concentration in the bone marrow incubated with SP receptor agonist was as follow: SP(+)—4.2 ± 0.8 pg (p = 0.01 as compared to SP(+) control incubation), SP(−)—9.1 ± 0.8 pg (p = 0.001 as compared to SP(−) control incubation) and control children—4.0 ± 0.8 pg (ns). Moreover, IL-1b concentration in SP(−) group was significantly higher as compare to both SP(+) and control patients (p = 0.01). Following IL-1b concentrations in the bone marrow incubated with spantide were found: SP(+)—1.9 ± 0.3 pg (p = 0.001 as compared to both previous SP(+) incubations), SP(−)—2.7 ± 0.2 pg (p = 0.001 as compared to both previous SP(−) incubations) and control children—1.5 ± 0.3 pg (p = 0.001 as compared to both previous incubations). IL-1b concentrations in all the bone marrow samples incubated with spantide did not differ significantly. Finally, the last bone marrow samples incubated together with SP receptor agonist and spantide, revealed not significantly different IL-1b concentrations as presented in results of incubation only with spantide (SP(+)—2.0 ± 0.7 pg, SP(−)—2.5 ± 0.4 pg and control children—1.4 ± 0.3 pg. The relevant data were summarized in Fig. 1.

4. Discussion Tested in short term cultures of human BM in methylcellulose, SP alone was demonstrated to support hematopoiesis in vitro [23]. The authors showed that SP, at the concentration of 10−11 to 10−8 mol/l could substitute for IL-3, G-CSF and GM-CSF, the presence of which was indispensable for growth of colonies [23]. On the other hand, substance P could not substitute for erythropoietin even if, added together, it augmented activity of the latter. Specificity of this stimulatory action of SP was confirmed by administering it together with blockers of the known subtypes of SP receptor. Such a parallel administration of SP and a blocker for a subtype of NK-1R receptor yielded results at the control level. On the other hand, blocker of NK-2R receptor exerted no effect

on SP activity [16]. SP was also found to affect hematopoietic cells in an indirect manner, i.e. through the stromal cells, stimulating their production of cytokines. Supplementation of SP-stimulated cultures with antibodies specific for IL-1, IL-3, IL-6 and GM-CSF resulted in partial inhibition of the cell growth proving that SP can act through the induction of the cytokine synthesis. SP induces also synthesis of IL-1 and SCF in BM stromal cells [37]. In line with the above, IL-1b concentration, regarded as the most specific inductive element of haematopoietic cells proliferation [23], was chosen in these experimental studies involving bone marrow samples obtained from ALL children. As already mentioned, these subjects were preliminary selected according to the age, phenotype (CD10), percentage of blast cells (>98.5%) and SP expression in blast cells. Finally, bone marrow samples prior to 2-h incubations were adjusted to the number of cells (an average of 4 × 104 cells per 100 ␮l). In that way aspirate sampling cellularity did not significantly vary from patient to patient. Although blasts were not primarily sorted, the percentage of other than blast cells in samples was lower than 1.5%, which certainly did not affect decisive results. It must be also emphasized that bone marrow samples were not initially treated and because of relatively short period of incubation, the medium did not contain RPMI which could interfere obtained results. The experimental 2-h incubation of blast cells with SP receptor agonist was found to result in a significant increase of IL-1b concentration in BM sampled from children with ALL who showed no SP expression in leukemic cells. Interestingly, ALL children with original expression of SP in blast cells incubated with the same agonist decreased by half the original production of IL-1b. Such a discrepancy could be explained by the presence of NK-1R on the surface of all CD10-positive blasts [12]. The cells which produce SP by themselves are forced for secretion of IL-1b in an autocrine manner but within relatively narrow range of SP concentrations (10−10 to 10−8 mol/L) [23]. Both higher and lower concentrations result in reduction of IL-1b synthesis. On the other hand, immunohistochemically SP-negative blasts could act the same when modulated by SP released from BM nerve endings. Increased concentration of IL-1b in SP(−) samples cannot be then explained by the presence of other cells (neutrophils or macrophages) capable for production of SP, since control BM samples (reach in such cells) resulted with significantly lower IL-1b concentrations. All the incubations of the cells with SP receptor antagonist (spantide) with or without SP receptor agonist resulted in control levels of IL-1b. This observation needs a separate discussion. Why does spantide decrease IL-1b production in control cells? Do they produce SP? It can be stated that control cells are capable to produce IL-1 in NK receptor dependent manner. Decreased secretion of IL-1b production after spantide administration suggests that these cells produce tachykinins but another than SP. No acceleration of IL-1b production after administration of SP receptor agonist was observed which indicates that IL-1b secretion is

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not dependent upon NK-1R. Spantide is pan-NK receptor antagonist (involving also NK-2R and NK-3R) and probably depletion of IL-1b production is dependent upon another than SP tachykinin. As previously reported, SP-positive blast immunophenotype resulted with unfavourable leukaemia course. On the one hand, it could be explained by extraordinary tachykinins expressions in normal and leukemic hematopoietic cells. The preprotachykinin gene (TAC) encodes neuropeptides that belong to the tachykinin family [42]. TAC-1 encodes both SP and neurokinin (NK)-A [43]. It comprises 7 exons, which can be alternately spliced and modified to form 4 transcripts: ␣-, ␤-, ␥-, and ␦-TAC-1. SP is encoded by exon 3, present in each transcript, and NK-A is encoded by exon 6, present in two transcripts, ␤ and ␥ [43]. The hematopoietic effects mediated by SP and NK-A correlate with the cytokines that each peptide produced in BM cells [44]. The negative effects of NK-A on the proliferation of BM progenitors suggest that NK-A might be protective to HSCs [37]. A protective role for NK-A is construed based on the predominant types of TAC-1 transcripts found in normal BM cells and in leukemic cells. Normal BM stromal cells express ␤-TAC-1, while leukemic cells express only ␣-TAC-1 [45]. While ␤-TAC-1 is capable of producing both NK-A and SP, ␣-TAC-1 can only produce SP. In normal haematopoiesis, SP and NK-A, through the production of distinct cytokines, exert opposite effects with respect to the proliferation of haematopoietic progenitors. This suggests that SP and NK-A might be able to regulate the proliferation of HSC through autocrine and/or paracrine mechanism. Such a regulatory mechanism might not be possible in leukemic cells, which produce only SP [40,41,45–47]. Moreover, SP is believed to play an essential role as a modulator of synaptic transmission in sympathetic nerve fibers [48]. On the other hand, sympathetic nervous system regulates the egress of stem and progenitor cells from their niche in BM [49]. It was found out in the model study employing UDP-galactose ceramide galactosyltransferasedeficient mice which exhibited aberrant nerve conduction and displayed no stem and progenitor cells escape from BM following G-CSF administration. These results raise also an interesting possibility, i.e. SP-derived alterations in the sympathetic tone may explain why sometimes blasts infiltration in the bone marrow does not refer to the blast leukocytosis observed in the peripheral blood. NK-1R (SP) and NK-2R (NK-A) exhibiting a yin-yang relationship with respect to their expression in BM stroma are also expressed on mesenchymal stem cells (MSC) [36,50]. These are reported to provide information to exiting immune cells of the barrier between the periphery and the BM understood as a “gatekeeper” function, in which the MSC regulate the movement of cells in and out of the BM [51]. A relevant scenario would be that the over-expressed SP in the BM environment might inhibit to some degree the blasts passage to peripheral blood. These expectations, involving also widely understood IL-1b

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dependent SP pleiotrophy, must be, however, supported by further investigations. In conclusion, these findings suggest the novel role of substance P in blasts induction in childhood acute lymphoblastic leukaemia of common (CD10) origin as well as the consideration of SP receptor antagonists employed as anti-neoplastic drugs, e.g. by direct or indirect blocking of tumour cell proliferation through inhibition of growth factor production, including also IL-1b synthesis.

Competing interest statement The corresponding author, on behalf of all the authors, declares that there are no competing interests. Acknowledgments The authors are grateful to Prof. Geoffrey Shaw for his assistance in the English text editing and Prof. Elzbieta Kaczmarek for her input in the statistical analysis. This study was supported by the State Committee for Scientific Research (KBN, grant no. 2PO5E 07130). Contributions. Michal Nowicki (corresponding author) contributed in study design, sample collection, and manuscript writing. Danuta Ostalska-Nowicka and Bogdan Miskowiak participated in manuscript writing.

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