Published online before print November 12, 2008
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Hematology and Hemotherapy Center, School of Medicine, UNICAMP, Campinas, Brazil
1 Correspondence: Centro de Hematologia e Hemoterapia, Faculdade de Ciências Medicas, Rua Carlos Chagas, 480, Cidade Universitária, Barão Geraldo, Campinas 13083-970-SP, Brazil. E-mail: ferreira{at}unicamp.br
ABSTRACT
Inflammation, cell adhesion to vascular endothelium, and endothelial injury contribute to sickle cell anemia (SCA) vaso-occlusion. Although alterations in inflammatory cytokines and biomarkers have been related, reports have been conflicting, and a conclusive role for these molecules in the disease remains to be established. Furthermore, the effect of hydroxyurea therapy (HU) on the release of inflammatory mediators is not understood. This study aimed to determine plasma levels and leukocyte gene expressions of inflammatory mediators in healthy controls, steady-state SCA patients, and SCA patients on HU therapy. TNF-
, IL-8, and PGE2 levels were significantly higher in the plasma of SCA individuals when compared with control individuals. HU therapy was associated with a significant reversal of augmented TNF- and, interestingly, increased plasma anti-inflammatory IL-10. IFN-
, IL-10, cyclooxygenase 2 (COX-2), and inducible NO synthase (iNOS) gene expressions were unaltered in SCA mononuclear cells (MC); however, gene expressions of TNF-, IL-8, and the protective enzyme heme oxygenase-1 (HO-1) were significantly higher. HU therapy was not associated with significantly altered SCA MC inflammatory gene expression, although COX-2 mRNA expression was decreased. In SCA neutrophils, gene expressions of IL-8, IFN-, iNOS, and HO-1 were significantly higher than those of control subjects. Patients on HU demonstrated lower iNOS and higher IL-10 neutrophil gene expressions. Taken together, data suggest that alterations in the gene expressions and productions of a number of pro- and anti-inflammatory mediators are present in SCA and importantly, in those patients on HU therapy. Knowledge of these pathways may contribute to further the understanding of the pathophysiology of this disease.
Key Words: neutrophil • mononuclear cell • chemokines • heme oxygenase • gene expression
INTRODUCTION
Growing evidence indicates that inflammatory stresses within the microvasculature may play a significant role in the vaso-occlusion that is characteristic of sickle cell anemia (SCA). Inflammation, leukocyte adhesion to vascular endothelium, and subsequent endothelial injury appear to contribute to the pathogenesis of SCA [1 ]. An elevated baseline leukocyte count is associated with an increased risk of early death, and leukocytes may play a significant role in the initiation of vaso-occlusive events [2 ]. Occlusions of the microcirculation, as well as infections, are important factors that stimulate oxidative stress, production of cytokines, and acute-phase proteins. Cytokines may participate in several mechanisms that contribute to vaso-occlusive pathogenesis in SCA, including vascular endothelial activation, induction of red cell and leukocyte adhesion to vascular endothelium, development of vascular intimal hyperplasia, platelet activation, endothelin-1 production, and dysregulation of endothelial apoptosis [3 ]. Although alterations in a number of inflammatory, anti-inflammatory cytokines, and inflammatory biomarkers have been related previously, reports have been conflicting, and a conclusive role for cytokines in SCA remains to be established. As neutrophils and mononuclear cells (MC) are important producers of cytokines and are activated during infection and inflammation [4 , 5 ], we hypothesize that these cells, in SCA patients, may express altered mRNA levels of cytokines and inflammatory mediators and that the characterizations of the expressions of these genes may further the understanding of the inflammatory mechanisms that contribute to vascular injury.
Treatment of individuals with SCA with hydroxyurea (HU) decreases white blood cell (WBC) counts, myeloperoxidase activity [6 7 8 ], and cell-adhesive properties [9 , 10 ]. Despite the evidence of significant involvements of leukocytes in modulating clinical and pathophysiological aspects of SCA, the mechanism by which leukocyte numbers are increased in patients with SCA remains unclear, although alterations in leukocyte-producing cytokines have been suggested to participate in this phenomenon [11 ]. Furthermore, the effect of HU therapy on the release of inflammatory mediators is not well understood.
An investigation of the production of inflammatory cytokines, anti-inflammatory cytokines, and inflammatory mediators in SCA patients may further elucidate the pathogenesis of the disease and its complications and help in assessing disease severity and prognosis. As such, we determined the plasma levels and leukocyte gene expressions of cytokines and inflammatory mediators in steady-state SCA patients and determined whether HU therapy is associated with alterations in these levels.
MATERIALS AND METHODS
Patients
SCA patients (see Table 1
for clinical details), diagnosed as homozygous for hemoglobin S (HbS; using Hb electrophoresis methods and HPLC), in steady-state and attended at the Hematology and Hemotherapy Center, State University of Campinas (UNICAMP; Brazil), participated in the study. Patients were not in crisis and had not received blood transfusions in the preceding 3 months. None of the patients were taking nonsteroidal anti-inflammatory drugs at the time of the study, and patients on HU therapy had been taking 20–30 mg/kg/day for at least 3 months. Healthy individuals were used as controls (aged 20–50 years). Informed, written consent was obtained from all patients and controls, and the Ethics Committee of UNICAMP approved the study.
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Table 1. Clinical Details of Healthy Controls, Steady-State SCA Patients, and Steady-State SCA Patients on Hydroxyurea Therapy
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, and PGE2 were determined; for this, blood samples were collected in EDTA and centrifuged at 10,000 g for 15 min, and plasma was stored at –20°C. For determination of PGE2, indomethacin was added (10 ng/µl) to plasma samples before storage. Commercially available ELISA kits were used to determine IL-8, IL-10 (BD Biosciences PharMingen, San Diego, CA, USA), TNF- (R&D Systems, Minneapolis, MN, USA), and PGE2 (Cayman Chemical, Ann Arbor, MI, USA) in plasma samples.
Isolation of neutrophils and MC from peripheral blood
Neutrophils and MC were separated from fresh peripheral blood collected in heparin, following a method described previously with adaptations [12
]. Briefly, whole blood was laid over two layers of Ficoll-Hypaque of densities of 1.077 g/L and 1.119 g/L, respectively. After separation of MC and granulocytes by centrifugation at 700 g for 30 min, cells were washed once in PBS (pH 7.4) before lysing contaminating red cells (10 min, 4°C, lysis buffer; 155 mM NH4Cl, 10 mM KHCO3), and cells were washed once again in PBS before resuspending the neutrophil or MC pellets in TRizol reagent (1 ml per 5–10x106 cells; Invitrogen Corp., Carlsbad, CA, USA) for mRNA extraction (according to the manufacturer’s instructions).
RT of mRNA and amplification and quantification of gene expression by real-time PCR
RNA samples (5 µg) were incubated with 1 U DNaseI (Invitrogen Corp., Rockville, MD, USA) for 15 min at room temperature, and EDTA was added to a final concentration of 2 mM to stop the reaction. The DNaseI enzyme was subsequently inactivated by incubation at 65°C for 5 min. DNaseI-treated RNA samples were then reverse-transcribed with Superscript III and RNaseOut (Invitrogen Corp., Carlsbad, CA, USA) for 50 min at 50°C, 15 min at 70°C. cDNA samples were quantified using a Nanodrop spectrophotometer (ND-1000; Nanodrop Technologies Inc., Wilmington, DE, USA). Synthetic oligonucleotide primers were designed to amplify cDNA for the genes encoding the TNF-, IL-6, IL-8, IL-10, IFN-, HO-1 (heme oxygenase 1), cyclooxygenase 2 (COX-2), and inducible NOS (iNOS) (Primer ExpressTM, Applied Biosystems, Foster City, CA, USA). For primer sequences and concentrations, see Table 2
. Primers were synthesized by Invitrogen Corp. (Carlsbad, CA, USA) and IDT (Coralville, IA, USA). All samples were assayed in a 25-µL vol containing 10 ng cDNA (6.25 µL), 12.5 µL SYBR Green Master Mix PCR (Applied Biosystems), and 6.25 µL-specific primers in a MicroAmp Optical 96-well reaction plate (Applied Biosystems) using the 5700 Sequence Detection System (Applied Biosystems), as described previously. Gene expression was quantified using the Gnorm program [13
]. Two replicas were run on the plate for each sample, and each sample was run twice—independently. Results are expressed as mRNA levels of each gene studied, normalized according to β-actin and GAPDH expressions.
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Table 2. Primer Sequences for Performance of Real-Time Quantitative PCR
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RESULTS
Plasma levels of inflammatory mediators in SCA individuals
Plasma levels of the cytokine TNF- and the chemokine IL-8 were elevated significantly in steady-state SCA patients compared with healthy subjects (Fig. 1 A and B
). No significant difference was observed between the levels of the anti-inflammatory cytokine IL-10 in SCA and controls (Fig. 1C)
. Plasma PGE2, a COX-2 eicosanoid product, was also elevated significantly in SCA patients compared with healthy controls (Fig. 1D)
.
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Figure 1. Plasma TNF- (A), IL-8 (B), IL-10 (C), and PGE2 (D) levels in healthy controls, SCA patients, and SCAHU patients. Data are represented as means ± SEM
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when compared with steady-state SCA patients (Fig. 1A)
. Interestingly, IL-10 plasma levels were higher in SCA patients receiving HU than in SCA patients not on HU therapy (Fig. 1C)
. Plasma levels of IL-8 and PGE2 were not significantly different in SCA patients that were taking, or not, HU (Fig. 1 B and D
, respectively).
Gene expression of inflammatory mediators in the MC of SCA patients and healthy controls
Real-time PCR quantification demonstrated that the expressions of TNF- (TNF) mRNA and IL-8 (IL8) were significantly higher in the MC of SCA individuals than in control individuals (Fig. 2 A and B
). The mRNA gene expressions of the cytokines IL-10 (IL10) and IFN- (IFNG) were not significantly different in the MC of SCA patients when compared with control MC (Fig. 2 C and D
, respectively). The gene expressions of the inflammatory mediator enzymes, HO-1 (HMOX1), iNOS (NOS2A), and COX-2 (PTGS2), were also determined in these cells (see Fig. 4 A-C
). iNOS and COX-2 gene expressions were not altered significantly; in contrast, HO-1 gene expression was increased significantly in SCA MC.
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Figure 2. Expressions of TNF (A), IL8 (B), IL10 (C), and IFNG (D) mRNAs in the MC of steady-state SCA patients; steady-state SCAHU patients (20–30 mg/kg/day), and healthy controls. Values recorded were normalized according to the quantity of ACTB and GAPDH mRNA in each sample measured in the same RT-PCR run. *, P < 0.02, compared with controls.
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Figure 4. Expressions of PTGS2 (A and D), NOS2A (B and E), and HMOX1 (C and F) mRNAs in the MC (A–C) and neutrophils (D–F) of steady-state SCA patients, steady-state SCAHU patients (20–30 mg/kg/day), and healthy controls. Values recorded were normalized according to the quantity of ACTB and GAPDH mRNA in each sample measured in the same RT-PCR run. *, P < 0.05; **, P < 0.01; ***, P< 0.001, compared with normal controls; #, P < 0.05, compared with SCA.
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, IL-10, and COX-2 were not significantly different in the neutrophils of SCA patients when compared with those of healthy subjects (Figs. 3 A and C, and 4D
, respectively); however, the gene expressions of IL-8, IFN-, iNOS, and HO-1 were significantly higher in the neutrophils of SCA patients, compared with those of healthy controls (Figs. 3 B and D
, and 4 E and F
, respectively).
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Figure 3. Expressions of TNF (A), IL8 (B), IL10 (C), and IFNG (D) mRNAs in neutrophils of steady-state SCA patients; steady-state SCAHU patients (20–30 mg/kg/day), and healthy controls. Values recorded were normalized according to the quantity of ACTB and GAPDH mRNA in each sample measured in the same RT-PCR run. *, P < 0.02, compared with healthy controls; #, P < 0.05, compared with SCA.
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, and iNOS were found in the MC of SCAHU patients, compared with SCA individuals not on HU (Figs. 2 C and D
, and 4B
, respectively). Conversely, expressions of the genes encoding TNF-, IL-8, COX-2, and HO-1 (Figs. 2 A and B
, and 4 A and C
) were lower in the MC of SCAHU, compared with those in the MC of SCA patients not on HU therapy; however, a significant difference was observed only in COX-2 expression.
The gene expressions of TNF-, COX-2, and HO-1 were not significantly different in the neutrophils of SCAHU, compared with SCA patients not on HU (Figs. 3A
and 4 E and F
, respectively). However, significantly lower expressions of IL-8, IFN-, and iNOS mRNA were observed in SCAHU neutrophils, compared with SCA neutrophils (Figs. 3 B and D
, and 4E
, respectively). In contrast, IL-10 mRNA levels were significantly higher in neutrophils from SCAHU, compared with neutrophils of SCA patients not on HU therapy (Fig. 3C)
.
DISCUSSION
Although sickle cell disease (SCD) is now recognized to characterize a chronic inflammatory state [14 ], the mechanisms of induction of inflammatory mediator production by inflammatory cells and the consequent effect of these molecules on the inflammatory response are little understood in the disease. Indeed, data from studies to date have been conflicting, possibly as a result of the different study populations under investigation and the use of different methods for the detection of cytokines and other inflammatory mediators in individuals with SCD.
Data presented herein demonstrate significantly higher circulating levels of the cyto/chemokines TNF- and IL-8 in our population of SCA patients. Previous studies have demonstrated increased levels of circulating TNF- in SCD individuals in steady-state [15
] and during crisis events [16
]. Circulating IL-8, on the other hand, has previously been detected only in SCD patients when in crisis or presenting acute syndrome [16
17
18
19
]; the current availability of more sensitive detection tools, however, probably explains this discrepancy. A role for TNF-, an important proinflammatory cytokine, has been appointed in a number of inflammatory diseases that include sepsis, rheumatoid arthritis, and Chrohn’s disease, where this molecule appears to participate in up-regulating the cytokine cascades responsible for inflammation [20
, 21
]. IL-8 (CXCL8) is a key mediator in neutrophil-mediated acute inflammation, with a crucial role in a number of chronic inflammatory diseases and lung pathologies [22
]. Production of IL-8 is regulated at the level of gene transcription and mRNA stability [23
] and can be induced by proinflammatory cytokines, such as TNF- and IL-1 [24
], and other inflammatory molecules including bacterial products [22
]. As such, we monitored the production at a transcriptional level of these and other inflammatory mediators from MC (cells include monocytes, lymphocytes, and macrophages) and neutrophils in an attempt to determine which white cell types contribute to inflammatory mediator production in SCA. The gene expressions of TNF- and IL-8 genes were found to be significantly higher in SCA MC, and levels of IL-8 mRNA were higher in the neutrophils of SCA individuals. Results indicate that MC and neutrophils are important for inflammatory molecule production in SCA, supporting previous reports showing that monocytes appear to circulate in an activated state in SCD patients and probably serve as a source of the increased plasma levels of TNF- [25
, 26
]. Notably, a substantial variation in cytokine levels is observed in our population and in other populations of SCA individuals; for example, those patients that presented the most elevated levels of TNF- were not the same individuals as those that demonstrated the highest levels of plasma IL-8. These findings reflect an interesting feature of SCA, a disease that is characterized by a high variability in clinical symptoms and severity despite the fact that all individuals suffer from a single gene mutation. Importantly, no correlation was observed between proinflammatory mediator protein/mRNA levels and fetal HbF levels in patients (see Supplemental Table 1).
IFN- levels have previously been determined in our population of SCD patients; however, no significant increase in serum levels was found in patients during steady-state [27
]. Others, however, have found a small but significant elevation in circulating IFN- levels in steady-state SCD individuals [16
]. In contrast to the lack of alteration in circulating IFN- in our population of steady-state patients, we found IFN- mRNA expression to be increased significantly in the neutrophils of these individuals compared with control individuals; previous studies have also demonstrated that SCD lymphocytes, when cultured in the presence of activating phytohemagglutinin, produce significantly higher levels of IFN- than control lymphocytes [28
]. IFN- is reported to be a major trigger for the formation and release of reactive oxygen species (ROS) [28
, 29
], and it may be that at sites of infiltration and particularly, at vaso-occlusive sites, where leukocytes are known to play a central role [30
], local production of this proinflammatory cytokine may participate in the oxidative stress that aggravates the chronic inflammatory state.
Plasma levels of the principal inflammatory PGE2 were found to be significantly higher in steady-state SCA compared with controls; similarly, PGE1 has been demonstrated to be increased in the same population of SCA individuals [31 ]. PGE2 has been reported previously to be elevated in SCD patients during crisis and post-crisis [32 ]; however, to our knowledge, this is the first report of elevated PGE2 in SCD individuals during steady-state. PGs are lipid mediators that are synthesized by nearly all cell types from arachidonic acid via the actions of COX enzymes constitutively or in response to trauma or signaling molecules [33 ]. Although PGE2 is known principally as an inflammatory mediator, particularly important in diseases such as rheumatoid arthritis and osteoarthritis [33 ], in SCD, this molecule may have beneficial and detrimental effects. PGE2 mediates some of its effects through up-regulation of cAMP-dependent protein kinase A activity [34 ] and has been found to induce the synthesis of fetal Hb in peripheral, blood-derived erythroid burst colonies [35 ], a mechanism that may be regulated by cAMP-mediated signaling [36 , 37 ]. However, increased intracellular cAMP may increase leukocyte survival under some circumstances and increase the adhesive properties of neutrophils and RBC, all of which contribute to the pathophysiology of SCD [10 , 31 , 38 ].
Inducible COX-2 enzyme is the principal PGE2-synthesizing COX under inflammatory conditions [33
]. The production and activity of this enzyme, in turn, are induced by proinflammatory cytokines, such as IL-1 or IFN-, NO, iNOS, bacterial LPS signaling, and ROS [39
]. COX-2 gene expression, however, was not altered significantly in MC or neutrophils of SCA individuals; however, the possibility that COX-2 activity may be induced should not be disregarded. iNOS enzyme, for example, can bind directly to COX-2, s-nitrosylating it and increasing its enzyme activity [40
]. iNOS is expressed in numerous cell types including endothelial cells and inflammatory cells in the presence of inflammatory stimuli such as cytokine signaling (e.g., TNF-, IL-1β), bacterial LPS, and ROS [41
]. iNOS gene expression was increased significantly in SCA neutrophils, possibly reflecting the inflammatory and oxidative stress present in these individuals [42
]; indeed, iNOS can be up-regulated in blood vessels to compensate for loss of functional endothelium and endothelial NOS during endothelial dysfunction as a result of injury; however, it is possible that this production of NO, in association with ROS production, may lead to additional tissue damage [43
].
The inducible cytoprotective enzyme HO-1 breaks down heme released during hemolysis, protecting tissues and cells from ischemia/reperfusion injury, oxidative stress, inflammation, and apoptosis [44
, 45
]. This enzyme is induced at the transcriptional level by heme, hypoxia, and cytokines amongst other stimuli. HO-1 expression is elevated in the organs of transgenic sickle mice [46
], and we found the gene expression of this enzyme to be induced significantly in the MC and neutrophils of our SCA patient population, as also reported in another population of SCD individuals [47
], suggesting this to be one of the anti-inflammatory mechanisms used by the organism in SCA. Interestingly, HU therapy slightly, but not significantly, increased the expression of this enzyme in these inflammatory cells, indicating this to be one of the beneficial effects of HU therapy. Similarly, IL-10, an anti-inflammatory cytokine, found previously to be elevated in the plasma of a group of SCD patients [48
], was not found to be altered significantly in our population of SCA individuals; however, levels were elevated significantly in those patients on HU therapy. IL-10 limits the production of proinflammatory cytokines (including IL- and -β, IL-6, IL-12, IL-18, and TNF-) and chemokines (MCP-1, MCP-5, RANTES, IL-8, IFN-inducible protein 10, and MIP-2) [49
], and PGE2 has been reported to be a potent inducer of IL-10 production in bone marrow-derived dendritic cells (DC) [50
]. No significant associations between detectable IL-10 plasma levels and decreased proinflammatory cytokine levels were observed in SCAHU patients (data not shown). The IL-10 cytokine is produced by various cell types including macrophages, DC, lymphocytes, and other leukocytes [51
]; IL-10 mRNA production was found to be elevated significantly in the neutrophils of patients on HU therapy, and importantly, a significant positive correlation (P=0.03) between neutrophil IL10 gene expression and levels of HbF was found in SCA and SCAHU patients when grouped together (see Supplemental Table 1), indicating that HbF may have a direct role in increasing anti-inflammatory IL-10. HU therapy also decreased plasma TNF- levels significantly, associated with a nonsignificant decrease in TNF- gene expression in MC cells, but did not alter plasma IL-8 or PGE2 concentrations. HU therapy diminished IFN- and iNOS gene expression in neutrophils and COX-2 gene expression in MC. The exact mechanisms by which HU alters the expressions of these genes is not clear; however, as PGE2 levels are not decreased in the plasma of SCAHU individuals, the decrease in COX-2 gene expression observed may not be high enough to significantly alter PG production. Alterations in IFN-, iNOS, and COX-2 expressions, however, may alleviate oxidative stress at sites of inflammation, and it would seem that neutrophils may play an important role in the reversal of the inflammatory state in patients on HU.
In conclusion, we report alterations in the gene expressions and productions of a number of inflammatory mediators in SCA individuals, all of which may contribute to the pathophysiology of this disease. A number of anti-inflammatory mechanisms were also found up-regulated in these individuals, indicating efforts to counteract these alterations. HU therapy diminished the production of some inflammatory mediators and up-regulated protective mechanisms, demonstrating further beneficial effects of this therapy in SCD. Knowledge of these pathways may contribute to the understanding of the pathophysiology of SCD.
ACKNOWLEDGEMENTS
This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and The State of São Paulo Research Foundation (FAPESP).
Received July 28, 2008; revised October 2, 2008; accepted October 20, 2008.
REFERENCES
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