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Up-regulation of CXCL8/interleukin-8 production in response to CXCL12 in chronic lymphocytic leukemia a
a
bc
d
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Omar Perbellini , Federica Cioffi , Giorgio Malpeli , Elisabetta Zanolin , Ornella Lovato , bc
a
Aldo Scarpa , Giovanni Pizzolo & Maria T. Scupoli
ace
a
Department of Medicine – Section of Hematology
b
Department of Pathology and Diagnostics – Section of Pathology
c
Applied Research on Cancer-Network (ARC-NET)
d
Department of Medicine and Public Health – Section of Epidemiology and Medical Statistics
e
Click for updates
Interdepartmental Laboratory of Medical Research (LURM),University of Verona, Verona, Italy Published online: 24 Jun 2015.
To cite this article: Omar Perbellini, Federica Cioffi, Giorgio Malpeli, Elisabetta Zanolin, Ornella Lovato, Aldo Scarpa, Giovanni Pizzolo & Maria T. Scupoli (2015) Up-regulation of CXCL8/interleukin-8 production in response to CXCL12 in chronic lymphocytic leukemia, Leukemia & Lymphoma, 56:6, 1897-1900 To link to this article: http://dx.doi.org/10.3109/10428194.2014.977889
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Leukemia & Lymphoma, June 2015; 56(6): 1897–1900 © 2014 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2014.977889
LETTER TO THE EDITOR
Up-regulation of CXCL8/interleukin-8 production in response to CXCL12 in chronic lymphocytic leukemia Omar Perbellini1, Federica Cioffi1, Giorgio Malpeli2,3, Elisabetta Zanolin4, Ornella Lovato5, Aldo Scarpa2,3, Giovanni Pizzolo1 & Maria T. Scupoli1,3,5 1Department of Medicine – Section of Hematology, 2Department of Pathology and Diagnostics – Section of Pathology, 3Applied Research on Cancer-Network (ARC-NET), 4Department of Medicine and Public Health – Section of Epidemiology
Downloaded by [Emory University] at 11:26 05 August 2015
and Medical Statistics and 5Interdepartmental Laboratory of Medical Research (LURM),University of Verona, Verona, Italy
Within the tissue microenvironment, chronic lymphocytic leukemia (CLL) growth largely depends on a complex crosstalk between leukemic and stromal cells that involves antigens, cytokines, adhesion molecules and surface receptors [1]. The heterogeneous clinical course of patients with CLL reflects, at least in part, cell responses to these microenvironmental interactions that affect CLL cell proliferation, survival, migration and resistance to drug-induced apoptosis [1]. Consequently, several cytokines and chemokines have been reported to be elevated in the plasma of patients with CLL and to correlate with the clinical course [2–5]. CXCL8/interleukin-8 (IL8), a proinflammatory chemokine that has been shown to contribute to the progression of several human cancers [6], is one of these chemokines [3,5]. In patients with CLL, increased levels of CXCL8 mRNA expression have been associated in vitro with prolonged cell survival [7], which may represent the mechanistic link between increased CXCL8 plasma levels and CLL progression. Expression of CXCL8 is regulated by a number of different stimuli [6]. In several cell types comprising human mast cells, endothelial cells, fibroblast-like synoviocytes and T-cell lymphoblastic leukemia cells, CXCL8 expression has been shown to be up-regulated by the CXCR4/CXCL12 axis [8–10], known to play an important role in homing of CLL cells into the bone marrow and in CLL cell survival through cell-to-cell contacts with marrow stromal and/or nurse-like cells [1]. In this study, we analyzed in vitro CXCL8 production in CLL cells, either in the basal condition or following stimulation with microenvironmental stimuli, i.e. bone marrow stromal cells (BMSCs) or exogenous CXCL12. Peripheral blood mononuclear cell (PBMC) samples with high leukemia involvement (75–99%, median 90%) from patients with B-CLL (n ⫽ 24) were collected and cryopreserved at the Hematology Unit, Azienda Ospedaliera Universitaria Integrata (AOUI) in Verona (Italy), under a protocol approved by the local Ethics Committee (Comitato
Etico per la Sperimentazione – AOUI). All patients provided written informed consent. Clinical and biological characteristics at diagnosis of patients with CLL are summarized in Table I. The murine BMSC line M2-10B4 was used for co-culture experiments based on the following features: (i) M2-10B4 cells produce murine CXCL12 chemokine (data not shown), known to share 99% sequence identity with the human protein and to be able to signal through human CXCR4; (ii) expression of human CXCL8 in mouse cells is absent, thus M2-10B4 cells lack a direct homolog of human CXCL8, avoiding possible cross-reactions with anti-human CXCL8 antibodies used in cytokine production analysis. CLL cells were cultured with confluent BMSCs or exogenous, recombinant CXCL12 for the indicated time. Control CLL cells were cultured in the same condition but in the absence of BMSCs or CXCL12. For blocking experiments, monoclonal antibodies recognizing functional epitopes of CXCL12 or CXCR4 were added to the CLL–BMSC co-cultures at the start of the assay. Unrelated, isotype-matched monoclonal antibody was added in the co-cultures as a control. CXCL8 and CXCL12 concentrations were measured by enzymelinked immunosorbent assay (ELISA). Total RNA extraction was performed using TRIzol reagent and retrotranscribed to cDNA by a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Forward and reverse 5’–3’ primer sequences and PCR product lengths were as follows: F-GGGCCAAGAGAATATCCGAACT, R-GCAGACTAGGGTTGCCAGATTT, 166 bp for CXCL8; F-CGCGAGAAGATGACCCAGAT, R-GTCACCGGAGTCCATCACG, 125 bp, for ACTB. A cycle threshold (Ct) was taken above the background fluorescence. The Ct value, used for subsequent calculation, was the average of three replicates. The relative expression level of CXCL8 was calculated using the transcript level of ACTB as an endogenous reference. Data analysis was performed according to the comparative method as indicated in the User Bulletin #2 (Applied Biosystems).
Correspondence: Maria Teresa Scupoli, PhD, Laboratorio Universitario di Ricerca Medica (LURM), Policlinico G.B. Rossi, Ple. L. A. Scuro, 10, 37134 Verona, Italy. Tel: ⫹ 39-045-812 8425. Fax: ⫹ 39-045-802 7403. E-mail: [email protected] Received 4 February 2014; accepted 12 October 2014
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Table I. Characteristics of patients with CLL. All patients (n ⫽ 24) Median age, years (range) Female gender Median lymphocyte count, ⫻ 103/mm3 (range) Binet A stage ZAP-70 (⬎ 20%) CD38 (⬎ 30%)
61.45 (37.2–81.6) 8 18.9 (6–190) 17 13 11
Downloaded by [Emory University] at 11:26 05 August 2015
CLL, chronic lymphocytic leukemia.
Relative frequencies or median values and range were calculated for each categorical or continuous variable, respectively. For categorical variables, the differences were assessed by Fisher exact test. The Mann–Whitney U-test or the Wilcoxon test for paired samples was performed to compare continuous variables not normally distributed. Non-parametric Spearman rank correlation coefficient (r) was used to assess the presence of correlation between variables of interest. p-Values ⬍ 0.05 were considered statistically significant. CLL cells express CXCL8 mRNA and secrete biologically active CXCL8 protein at higher levels than healthy B cells [11]. To investigate whether the bone marrow microenvironment could modulate CXCL8 in CLL, first we measured CXCL8 protein levels in supernatants from CLL–BMSC transwell co-cultures. CLL cells plated onto BMSCs showed a
significant increase of CXCL8 supernatant concentration with respect to CLL alone [mean increase ⫾ standard deviation (SD) ⫽ 7.0 ⫾ 7.0; range ⫽ 1.3–20-fold; n ⫽ 6; Figure 1(A)]. Since the mouse BMSCs used in this study lack a direct homolog of human CXCL8, the CXCL8 amounts in the supernatants were attributable to CLL cells. Thus, these results show that BMSCs induced a statistically significant increased secretion of CXCL8 in CLL. Among the soluble factors secreted by BMSCs possibly involved in CXCL8 up-regulation, we focused on CXCL12, since it has been previously shown to enhance CXCL8 expression in several cell types [8–10]. Therefore, we co-cultured CLL cells with BMSCs in the presence of antiCXCL12 or anti-CXCR4 neutralizing antibodies. Blockage of CXCL12 or CXCR4 molecules significantly inhibited BMSCinduced CXCL8 secretion in CLL cells [Figure 1(B)]. CXCL8 levels were undetectable in the BMSC culture supernatant (data not shown). Next, we investigated the direct effects of CXCL12 on CXCL8 expression in CLL. In preliminary experiments, CLL cells were stimulated with different doses of recombinant CXCL12 (0.01, 0.1 and 1 μg/mL) at different time points (4 and 18 h). The maximal CXCL8 secretion was observed in the presence of 1 μg/mL CXCL12 at 18 h (data not shown), and therefore all experiments were performed under these experimental conditions. In the basal condition, CLL cells secreted heterogeneous levels of CXCL8, ranging from
(A)
(B)
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Figure 1. BMSCs and CXCL12 induce increased CXCL8 production in CLL cells. (A) CLL cells (n ⫽ 6 samples) were cultured with BMSCs for 18 h in a trans-well 24-well plate. After co-culture, CXCL8 concentration in cell-free supernatant was quantified by ELISA. Graph shows CXCL8 concentration in supernatant of each sample before (CLL) and after co-culture with BMSCs (CLL ⫹ BMSC). Values are expressed as pg/mL in log10. Wilcoxon test was performed to compare differences in CXCL8 production from CLL samples before and after co-culture with BMSCs. (B) CLL cells were cultured with BMSCs for 18 h in the presence of anti-CXCL12 or anti-CXCR4 neutralizing antibodies or an irrelevant antibody as control. After co-culture, CXCL8 concentration in cell-free supernatant was quantified by ELISA. Values represent average percentage ⫾ standard error of the mean (SEM) (n ⫽ 3) of CXCL8 concentration in CLL–BMSC co-cultures in presence of anti-CXCL12 or anti-CXCR4 neutralizing antibodies versus CLL–BMSC co-cultures in presence of irrelevant antibody (CTRL, referred as 100%). (C) CLL cells from 24 patients were stimulated with CXCL12 (1 μg/mL) for 18 h. After incubation, CXCL8 concentration in cell-free supernatant was quantified by ELISA. Values are expressed in pg/ mL; error bars indicate SEM. Wilcoxon test was performed to compare differences in CXCL8 production from CLL cells in presence or absence of CXCL12. (D) Time course of relative CXCL8 mRNA expression in a representative case (n ⫽ 3). Values represent average fold difference in secreted CXCL8 from CXCL12-stimulated divided unstimulated cells.
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Letter to the Editor 1899
(A)
(B)
(C)
(D)
Figure 2. Correlation between CXCL8 production and CD38 or ZAP-70 expression. CLL cells from 24 samples were cultured for 18 h in absence (A, C) or presence (B, D) of exogenous, recombinant CXCL12. After incubation, CXCL8 concentration in cell-free supernatant was quantified by ELISA and correlated with expression level of CD38 (A, B) or ZAP-70 (C, D) in CLL cells. Values of CXCL8 concentration are expressed as pg/mL. ZAP-70 and CD38 were determined using a cut-off of 20% and 30%, respectively. Spearman rank correlation coefficients and p-values are shown for each graph.
82 to 25 ⫻ 103 pg/mL (referred to 5 ⫻ 106 cells cultured for 18 h, n ⫽ 24) [Figure 1(C)]. CXCL12 stimulation induced a statistically significant increase of CXCL8 secretion in CLL cells [Figure 1(C)]. Real-time polymerase chain reaction (PCR) analysis of CXCL8 mRNA confirmed the protein expression data, showing that CXCL12 induced CXCL8 mRNA expression that was maximal at 1.5 and 4 h after stimulation [Figure 1(D)]. CXCL8 amounts secreted by CLL cells in response to CXCL12 stimulation were then considered in relation to CD38 and ZAP-70 expression, which correlate with a more aggressive form of the disease [12]. Spearman rank correlation showed a positive concordance between CXCL8 amounts secreted in basal conditions and CD38 expression levels, although it was not statistically significant [Figure 2(A)]. Remarkably, correlation between CXCL8 amounts secreted in response to CXCL12 and CD38 expression was statistically significant [Figure 2(B)]. A positive association, although not statistically significant in this small series, was also detected between CXCL8 levels, measured in either the basal or CXCL12-stimulated condition, and ZAP-70 expression [Figures 2(C) and 2(D)]. Chemokines secreted by CLL cells play a key role in creating a supportive microenvironment by recruiting accessory cells [1]. Among these chemokines, CXCL8 has been showed to provide additional survival signals to malignant cells by partially protecting CLL cells from spontaneous and drug-induced apoptosis [1,7,13]. Our study shows that BMSCs can increase CXCL8 secretion in CLL cells in vitro and identifies CXCL12 as a novel stimulus for CXCL8 up-regulation.
CXCL12 is the best studied among the chemokines secreted by stromal cells in the bone marrow and lymphnode microenvironments that, through CXCR4, lead to CLL cell chemotaxis into the tissue and, independently and directly, provide survival signals to CLL cells [1]. Our in vitro findings indicate that CXCR4 can also regulate CXCL8 secretion in vivo, thus amplifying its functions in recruiting accessory cells and in providing direct pro-survival stimuli to CLL cells. The amounts of CXCL8 produced by CLL cells in response to CXCL12 stimulation are positively associated with CD38 expression. CD38, a marker of cellular activation and aggressiveness, has been shown to have a pivotal role in initiating and modulating a series of input signals from the microenvironment [14]. Correlation between CXCL8 levels in response to stimulation with CXCL12 and CD38 expression suggests that CD38 marks CLL clones that are functionally active and more responsive to stimuli from the microenvironment. Together with previous data from the literature, these results support the idea of a molecular network in the CLL microenvironment whereby molecules may have pleiotropic roles in conferring the ability to migrate to privileged sites, survive and modulate the expression of other molecules. Strategies designed to disrupt this molecular circuit, for instance by inhibiting the CXCR4/CXL12 axis or the secretion of CXCL8, hold attractive and promising therapeutic choices, especially in progressive disease. Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.
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References
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[1] Burger JA , Gribben JG. The microenvironment in chronic lymphocytic leukemia (CLL) and other B cell malignancies: insight into disease biology and new targeted therapies. Semin Cancer Biol 2014;24:71–81. [2] Fayad L, Keating MJ, Reuben JM, et al. Interleukin-6 and interleukin10 levels in chronic lymphocytic leukemia: correlation with phenotypic characteristics and outcome. Blood 2001;97:256–263. [3] Wierda WG, Johnson MM, Do KA , et al. Plasma interleukin8 level predicts for survival in chronic lymphocytic leukaemia. Br J Haematol 2003;120:452–456. [4] Ferrajoli A , Keating MJ, Manshouri T, et al. The clinical significance of tumor necrosis factor-alpha plasma level in patients having chronic lymphocytic leukemia. Blood 2002;100:1215–1219. [5] Yoon JY, Lafarge S, Dawe D, et al. Association of interleukin-6 and interleukin-8 with poor prognosis in elderly patients with chronic lymphocytic leukemia. Leuk Lymphoma 2012;53:1735–1742. [6] Waugh DJ, Wilson C. The interleukin-8 pathway in cancer. Clin Cancer Res 2008;14:6735–6741. [7] Francia di Celle P, Mariani S, Riera L, et al. Interleukin-8 induces the accumulation of B-cell chronic lymphocytic leukemia cells by prolonging survival in an autocrine fashion. Blood 1996;87:4382–4389.
[8] Lin TJ, Issekutz TB, Marshall JS. Human mast cells transmigrate through human umbilical vein endothelial monolayers and selectively produce IL-8 in response to stromal cell-derived factor-1 alpha. J Immunol 2000;165:211–220. [9] Nanki T, Nagasaka K, Hayashida K, et al. Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis. J Immunol 2001;167:5381–5385. [10] Scupoli MT, Donadelli M, Cioffi F, et al. Bone marrow stromal cells and the upregulation of interleukin-8 production in human T-cell acute lymphoblastic leukemia through the CXCL12/CXCR4 axis and the NFkappaB and JNK/AP-1 pathways. Haematologica 2008;93:524–532. [11] Di Celle PF, Carbone A , Marchis D, et al. Cytokine gene expression in B-cell chronic lymphocytic leukemia: evidence of constitutive interleukin-8 (IL-8) mRNA expression and secretion of biologically active IL-8 protein. Blood 1994;84:220–228. [12] Cramer P, Hallek M. Prognostic factors in chronic lymphocytic leukemia-what do we need to know? Nat Rev Clin Oncol 2011;8:38–47. [13] Binsky I, Haran M, Starlets D, et al. IL-8 secreted in a macrophage migration-inhibitory factor- and CD74-dependent manner regulates B cell chronic lymphocytic leukemia survival. Proc Natl Acad Sci USA 2007;104:13408–13413. [14] Malavasi F, Deaglio S, Damle R, et al. CD38 and chronic lymphocytic leukemia: a decade later. Blood 2011;118:3470–3478.
Leukemia & Lymphoma Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ilal20
Up-regulation of CXCL8/interleukin-8 production in response to CXCL12 in chronic lymphocytic leukemia a
a
bc
d
e
Omar Perbellini , Federica Cioffi , Giorgio Malpeli , Elisabetta Zanolin , Ornella Lovato , bc
a
Aldo Scarpa , Giovanni Pizzolo & Maria T. Scupoli
ace
a
Department of Medicine – Section of Hematology
b
Department of Pathology and Diagnostics – Section of Pathology
c
Applied Research on Cancer-Network (ARC-NET)
d
Department of Medicine and Public Health – Section of Epidemiology and Medical Statistics
e
Click for updates
Interdepartmental Laboratory of Medical Research (LURM),University of Verona, Verona, Italy Published online: 24 Jun 2015.
To cite this article: Omar Perbellini, Federica Cioffi, Giorgio Malpeli, Elisabetta Zanolin, Ornella Lovato, Aldo Scarpa, Giovanni Pizzolo & Maria T. Scupoli (2015) Up-regulation of CXCL8/interleukin-8 production in response to CXCL12 in chronic lymphocytic leukemia, Leukemia & Lymphoma, 56:6, 1897-1900 To link to this article: http://dx.doi.org/10.3109/10428194.2014.977889
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Leukemia & Lymphoma, June 2015; 56(6): 1897–1900 © 2014 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2014.977889
LETTER TO THE EDITOR
Up-regulation of CXCL8/interleukin-8 production in response to CXCL12 in chronic lymphocytic leukemia Omar Perbellini1, Federica Cioffi1, Giorgio Malpeli2,3, Elisabetta Zanolin4, Ornella Lovato5, Aldo Scarpa2,3, Giovanni Pizzolo1 & Maria T. Scupoli1,3,5 1Department of Medicine – Section of Hematology, 2Department of Pathology and Diagnostics – Section of Pathology, 3Applied Research on Cancer-Network (ARC-NET), 4Department of Medicine and Public Health – Section of Epidemiology
Downloaded by [Emory University] at 11:26 05 August 2015
and Medical Statistics and 5Interdepartmental Laboratory of Medical Research (LURM),University of Verona, Verona, Italy
Within the tissue microenvironment, chronic lymphocytic leukemia (CLL) growth largely depends on a complex crosstalk between leukemic and stromal cells that involves antigens, cytokines, adhesion molecules and surface receptors [1]. The heterogeneous clinical course of patients with CLL reflects, at least in part, cell responses to these microenvironmental interactions that affect CLL cell proliferation, survival, migration and resistance to drug-induced apoptosis [1]. Consequently, several cytokines and chemokines have been reported to be elevated in the plasma of patients with CLL and to correlate with the clinical course [2–5]. CXCL8/interleukin-8 (IL8), a proinflammatory chemokine that has been shown to contribute to the progression of several human cancers [6], is one of these chemokines [3,5]. In patients with CLL, increased levels of CXCL8 mRNA expression have been associated in vitro with prolonged cell survival [7], which may represent the mechanistic link between increased CXCL8 plasma levels and CLL progression. Expression of CXCL8 is regulated by a number of different stimuli [6]. In several cell types comprising human mast cells, endothelial cells, fibroblast-like synoviocytes and T-cell lymphoblastic leukemia cells, CXCL8 expression has been shown to be up-regulated by the CXCR4/CXCL12 axis [8–10], known to play an important role in homing of CLL cells into the bone marrow and in CLL cell survival through cell-to-cell contacts with marrow stromal and/or nurse-like cells [1]. In this study, we analyzed in vitro CXCL8 production in CLL cells, either in the basal condition or following stimulation with microenvironmental stimuli, i.e. bone marrow stromal cells (BMSCs) or exogenous CXCL12. Peripheral blood mononuclear cell (PBMC) samples with high leukemia involvement (75–99%, median 90%) from patients with B-CLL (n ⫽ 24) were collected and cryopreserved at the Hematology Unit, Azienda Ospedaliera Universitaria Integrata (AOUI) in Verona (Italy), under a protocol approved by the local Ethics Committee (Comitato
Etico per la Sperimentazione – AOUI). All patients provided written informed consent. Clinical and biological characteristics at diagnosis of patients with CLL are summarized in Table I. The murine BMSC line M2-10B4 was used for co-culture experiments based on the following features: (i) M2-10B4 cells produce murine CXCL12 chemokine (data not shown), known to share 99% sequence identity with the human protein and to be able to signal through human CXCR4; (ii) expression of human CXCL8 in mouse cells is absent, thus M2-10B4 cells lack a direct homolog of human CXCL8, avoiding possible cross-reactions with anti-human CXCL8 antibodies used in cytokine production analysis. CLL cells were cultured with confluent BMSCs or exogenous, recombinant CXCL12 for the indicated time. Control CLL cells were cultured in the same condition but in the absence of BMSCs or CXCL12. For blocking experiments, monoclonal antibodies recognizing functional epitopes of CXCL12 or CXCR4 were added to the CLL–BMSC co-cultures at the start of the assay. Unrelated, isotype-matched monoclonal antibody was added in the co-cultures as a control. CXCL8 and CXCL12 concentrations were measured by enzymelinked immunosorbent assay (ELISA). Total RNA extraction was performed using TRIzol reagent and retrotranscribed to cDNA by a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Forward and reverse 5’–3’ primer sequences and PCR product lengths were as follows: F-GGGCCAAGAGAATATCCGAACT, R-GCAGACTAGGGTTGCCAGATTT, 166 bp for CXCL8; F-CGCGAGAAGATGACCCAGAT, R-GTCACCGGAGTCCATCACG, 125 bp, for ACTB. A cycle threshold (Ct) was taken above the background fluorescence. The Ct value, used for subsequent calculation, was the average of three replicates. The relative expression level of CXCL8 was calculated using the transcript level of ACTB as an endogenous reference. Data analysis was performed according to the comparative method as indicated in the User Bulletin #2 (Applied Biosystems).
Correspondence: Maria Teresa Scupoli, PhD, Laboratorio Universitario di Ricerca Medica (LURM), Policlinico G.B. Rossi, Ple. L. A. Scuro, 10, 37134 Verona, Italy. Tel: ⫹ 39-045-812 8425. Fax: ⫹ 39-045-802 7403. E-mail: [email protected] Received 4 February 2014; accepted 12 October 2014
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Table I. Characteristics of patients with CLL. All patients (n ⫽ 24) Median age, years (range) Female gender Median lymphocyte count, ⫻ 103/mm3 (range) Binet A stage ZAP-70 (⬎ 20%) CD38 (⬎ 30%)
61.45 (37.2–81.6) 8 18.9 (6–190) 17 13 11
Downloaded by [Emory University] at 11:26 05 August 2015
CLL, chronic lymphocytic leukemia.
Relative frequencies or median values and range were calculated for each categorical or continuous variable, respectively. For categorical variables, the differences were assessed by Fisher exact test. The Mann–Whitney U-test or the Wilcoxon test for paired samples was performed to compare continuous variables not normally distributed. Non-parametric Spearman rank correlation coefficient (r) was used to assess the presence of correlation between variables of interest. p-Values ⬍ 0.05 were considered statistically significant. CLL cells express CXCL8 mRNA and secrete biologically active CXCL8 protein at higher levels than healthy B cells [11]. To investigate whether the bone marrow microenvironment could modulate CXCL8 in CLL, first we measured CXCL8 protein levels in supernatants from CLL–BMSC transwell co-cultures. CLL cells plated onto BMSCs showed a
significant increase of CXCL8 supernatant concentration with respect to CLL alone [mean increase ⫾ standard deviation (SD) ⫽ 7.0 ⫾ 7.0; range ⫽ 1.3–20-fold; n ⫽ 6; Figure 1(A)]. Since the mouse BMSCs used in this study lack a direct homolog of human CXCL8, the CXCL8 amounts in the supernatants were attributable to CLL cells. Thus, these results show that BMSCs induced a statistically significant increased secretion of CXCL8 in CLL. Among the soluble factors secreted by BMSCs possibly involved in CXCL8 up-regulation, we focused on CXCL12, since it has been previously shown to enhance CXCL8 expression in several cell types [8–10]. Therefore, we co-cultured CLL cells with BMSCs in the presence of antiCXCL12 or anti-CXCR4 neutralizing antibodies. Blockage of CXCL12 or CXCR4 molecules significantly inhibited BMSCinduced CXCL8 secretion in CLL cells [Figure 1(B)]. CXCL8 levels were undetectable in the BMSC culture supernatant (data not shown). Next, we investigated the direct effects of CXCL12 on CXCL8 expression in CLL. In preliminary experiments, CLL cells were stimulated with different doses of recombinant CXCL12 (0.01, 0.1 and 1 μg/mL) at different time points (4 and 18 h). The maximal CXCL8 secretion was observed in the presence of 1 μg/mL CXCL12 at 18 h (data not shown), and therefore all experiments were performed under these experimental conditions. In the basal condition, CLL cells secreted heterogeneous levels of CXCL8, ranging from
(A)
(B)
(C)
(D)
Figure 1. BMSCs and CXCL12 induce increased CXCL8 production in CLL cells. (A) CLL cells (n ⫽ 6 samples) were cultured with BMSCs for 18 h in a trans-well 24-well plate. After co-culture, CXCL8 concentration in cell-free supernatant was quantified by ELISA. Graph shows CXCL8 concentration in supernatant of each sample before (CLL) and after co-culture with BMSCs (CLL ⫹ BMSC). Values are expressed as pg/mL in log10. Wilcoxon test was performed to compare differences in CXCL8 production from CLL samples before and after co-culture with BMSCs. (B) CLL cells were cultured with BMSCs for 18 h in the presence of anti-CXCL12 or anti-CXCR4 neutralizing antibodies or an irrelevant antibody as control. After co-culture, CXCL8 concentration in cell-free supernatant was quantified by ELISA. Values represent average percentage ⫾ standard error of the mean (SEM) (n ⫽ 3) of CXCL8 concentration in CLL–BMSC co-cultures in presence of anti-CXCL12 or anti-CXCR4 neutralizing antibodies versus CLL–BMSC co-cultures in presence of irrelevant antibody (CTRL, referred as 100%). (C) CLL cells from 24 patients were stimulated with CXCL12 (1 μg/mL) for 18 h. After incubation, CXCL8 concentration in cell-free supernatant was quantified by ELISA. Values are expressed in pg/ mL; error bars indicate SEM. Wilcoxon test was performed to compare differences in CXCL8 production from CLL cells in presence or absence of CXCL12. (D) Time course of relative CXCL8 mRNA expression in a representative case (n ⫽ 3). Values represent average fold difference in secreted CXCL8 from CXCL12-stimulated divided unstimulated cells.
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Letter to the Editor 1899
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(B)
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(D)
Figure 2. Correlation between CXCL8 production and CD38 or ZAP-70 expression. CLL cells from 24 samples were cultured for 18 h in absence (A, C) or presence (B, D) of exogenous, recombinant CXCL12. After incubation, CXCL8 concentration in cell-free supernatant was quantified by ELISA and correlated with expression level of CD38 (A, B) or ZAP-70 (C, D) in CLL cells. Values of CXCL8 concentration are expressed as pg/mL. ZAP-70 and CD38 were determined using a cut-off of 20% and 30%, respectively. Spearman rank correlation coefficients and p-values are shown for each graph.
82 to 25 ⫻ 103 pg/mL (referred to 5 ⫻ 106 cells cultured for 18 h, n ⫽ 24) [Figure 1(C)]. CXCL12 stimulation induced a statistically significant increase of CXCL8 secretion in CLL cells [Figure 1(C)]. Real-time polymerase chain reaction (PCR) analysis of CXCL8 mRNA confirmed the protein expression data, showing that CXCL12 induced CXCL8 mRNA expression that was maximal at 1.5 and 4 h after stimulation [Figure 1(D)]. CXCL8 amounts secreted by CLL cells in response to CXCL12 stimulation were then considered in relation to CD38 and ZAP-70 expression, which correlate with a more aggressive form of the disease [12]. Spearman rank correlation showed a positive concordance between CXCL8 amounts secreted in basal conditions and CD38 expression levels, although it was not statistically significant [Figure 2(A)]. Remarkably, correlation between CXCL8 amounts secreted in response to CXCL12 and CD38 expression was statistically significant [Figure 2(B)]. A positive association, although not statistically significant in this small series, was also detected between CXCL8 levels, measured in either the basal or CXCL12-stimulated condition, and ZAP-70 expression [Figures 2(C) and 2(D)]. Chemokines secreted by CLL cells play a key role in creating a supportive microenvironment by recruiting accessory cells [1]. Among these chemokines, CXCL8 has been showed to provide additional survival signals to malignant cells by partially protecting CLL cells from spontaneous and drug-induced apoptosis [1,7,13]. Our study shows that BMSCs can increase CXCL8 secretion in CLL cells in vitro and identifies CXCL12 as a novel stimulus for CXCL8 up-regulation.
CXCL12 is the best studied among the chemokines secreted by stromal cells in the bone marrow and lymphnode microenvironments that, through CXCR4, lead to CLL cell chemotaxis into the tissue and, independently and directly, provide survival signals to CLL cells [1]. Our in vitro findings indicate that CXCR4 can also regulate CXCL8 secretion in vivo, thus amplifying its functions in recruiting accessory cells and in providing direct pro-survival stimuli to CLL cells. The amounts of CXCL8 produced by CLL cells in response to CXCL12 stimulation are positively associated with CD38 expression. CD38, a marker of cellular activation and aggressiveness, has been shown to have a pivotal role in initiating and modulating a series of input signals from the microenvironment [14]. Correlation between CXCL8 levels in response to stimulation with CXCL12 and CD38 expression suggests that CD38 marks CLL clones that are functionally active and more responsive to stimuli from the microenvironment. Together with previous data from the literature, these results support the idea of a molecular network in the CLL microenvironment whereby molecules may have pleiotropic roles in conferring the ability to migrate to privileged sites, survive and modulate the expression of other molecules. Strategies designed to disrupt this molecular circuit, for instance by inhibiting the CXCR4/CXL12 axis or the secretion of CXCL8, hold attractive and promising therapeutic choices, especially in progressive disease. Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.
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References
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