ytology & f C H i o s l t a o n l o r Antosz et al., J Cytol Histol 2014, 5:3 g u y o J Journal of Cytology & Histology DOI: 10.4172/2157-7099.1000228 ISSN: 2157-7099 Research Article Article OpenOpen Access Access Distinct IRAK4 Expression in Normal and B-CLL Lymphocytes Halina Antosz*1, Joanna Sajewicz1, Barbara Marzec-Kotarska1, Dorota Choroszynska1, Iwona Hus2, Malgorzata Jargiello-Baszak3 and Jacek Baszak3 1Department of Clinical Genetics, Medical University of Lublin, Lublin, Poland 2Department of Clinical Immunology, Medical University of Lublin, Lublin, Poland 3Department of Cardiology, Medical University of Lublin, Lublin, Poland Abstract Toll-like receptors (TLR) signaling plays an important role in the B cell biology. It induces the maturation, proliferation and antibody production in response to pathogen recognition. Interleukin-1 Receptor-associated Kinase-4 (IRAK-4) is TLR downstream molecule which is of strategic importance in starting a cascade of NF-kappaB (Nuclear factor-kappaB) and MAPK (Mitogen-activated protein kinase) intracellular signaling pathways. The aim of the study was to identify the differences in IRAK-4 expression in normal and chronic lymphocytic leukemia (CLL) lymphocytes. We have also studied the IRAK-4 expression alteration upon TLR2/4 ligand stimulation in leukemic and normal B cells. We carried out Real-Time PCR and Western blot analysis of IRAK4 mRNA (RQ) and protein expression. The study were performed on the isolated CD19+ normal B lymphocytes and CLL cells (CD5+CD19+) before and 30 min after (Lipopolysaccharide) LPS and (Staphylococcus aureus strain Cowan I) SAC stimulation. IRAK-4 mRNA expression was significantly higher in normal CD19+ lymphocytes as compared to leukemic CD5+CD19+ subpopulation (RQ median 0.90 vs 0.54; p=0.012). In normal lymphocytes, LPS and SAC treatment led to significant IRAK-4 down-regulation both at mRNA (RQ median 0.90 vs 0.53 vs 0.40; p=0.03, p=0.01) and protein level, whereas generally there were no significant changes in IRAK-4 expression neither on mRNA nor protein level. No trend or significant differences in IRAK-4 expression in leukemic lymphocytes after LPS and SAC stimulation may imply the presence of alleged defect of immune tolerance in CLL. Keywords: IRAK-4; TLR; CLL; LPS; SAC IL-1 (interleukin-1), IL-6 (interleukin-6) and TNF (tumor necrosis factor) [11]. Introduction Unlike TIRAP/Mal, MyD88 has an N-terminal death domain The human body defense against pathogens occurs through responsible for interaction with IRAK kinases family members [12]. integration of innate and adaptive immunity. Both types of immune After LPS stimulation, IRAK1 (Interleukin-1 Receptor-associated function are mediated by intrinsic cellular and molecular interactions Kinase-1) is recruited to the TLR4 receptor complex, where it undergoes [1,2]. Various cell types endued with pattern recognition receptors phosphorylation in its threonine key locations by serine/threonine (PRRs), including Toll-like receptors (TLRs), are involved in innate IRAK4 kinases. IRAK4 is of strategic importance in starting a cascade immune system action in mammals. TLRs perform constant supervision of NF-κB and MAPK intracellular signaling pathways. Based on of the environment in search of pathogens, while distinguishing their mutagenesis studies, it was demonstrated that the active kinase IRAK- own antigens from the foreign ones. Adaptive immunity, which is 4 is absolutely essential for the activation of NF-κB [13]. Therefore, evolutionary younger, use of antigen-specific receptors on the surface IRAK-4 is considered to be a central mediator in TIR signaling innate of B and T lymphocytes (BCR, TCR) in response to specific and immunity [14] and has been proposed to be the only true kinase of the changing antigens [3]. B cells play the main role in this context. They IRAK family [15]. Studies on mice and clinical observations of IRAK-4 produce antibodies against these specific antigens, function as antigen deficient patients showed that the absence/deficiency of IRAK-4 results presenting cells for T cells, produce cytokines [4,5]. Lymphocytes T in severe immune deficiency [16,17]. show TLRs receptors expression, which are typical for innate immunity. Severe immune system disorders, i.e. hypogammaglobulinemia, In humans, ten different TLRs have been identified. Some of them impaired immunoglobulin class switching are typical features of are localized on the cell surface (TLR1, 2,4,5,6,10) while the other patients with chronic lymphocytic leukemia (CLL) [18]. This makes ones, on the endosomes (TLR3,7,8,9) They consist of transmembrane domain with leucine-rich repeats (LRRs), and cytoplasmic TIR domain (Toll/interleukin-1 resistance protein) [6,7]. TIR domains *Corresponding author: Halina Antosz, Department of Clinical Genetics, confer specificity to different TLRs enabling each of the receptors (via Medical University of Lublin, 11 Radziwillowska Street, 20-950 Lublin, Poland, Tel/Fax: (0048) 81 5288408; E-mail: [email protected] TIR-TIR homotypic interactions), communication with the signaling adaptor proteins [8-10]. Ligand, binding to the receptor, generally Received December 09, 2013; Accepted March 19, 2014; Published March 21, 2014 induces receptor dimerization, recruitment of adaptor molecules and activation of kinase cascade series. In the case of lipopolysaccharide Citation: Antosz H, Sajewicz J, Marzec-Kotarska B, Choroszynska D, Hus I, et al. (2014) Distinct IRAK4 Expression in Normal and B-CLL Lymphocytes. J Cytol (LPS) receptor TLR4 or the lipoprotein receptor TLR2, the adaptor Histol 5: 228. doi:10.4172/2157-7099.1000228 molecules MyD88 (myeloid differentiation factor 88) and TIRAP/ MAL (TIR-domain-containing adaptor protein, also called MyD88 Copyright: © 2014 Antosz H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted adaptor-like) are recruited to activate nuclear factor-kappaB (NF-κB- use, distribution, and reproduction in any medium, provided the original author and transcription factor) and induce proinflammatory cytokines, such as source are credited. J Cytol Histol ISSN: 2157-7099 JCH, an open access journal Volume 5 • Issue 3 • 1000228 Citation: Antosz H, Sajewicz J, Marzec-Kotarska B, Choroszynska D, Hus I, et al. (2014) Distinct IRAK4 Expression in Normal and B-CLL Lymphocytes. J Cytol Histol 5: 228. doi:10.4172/2157-7099.1000228 Page 2 of 7 patients with CLL very susceptible to infections, which significantly Cell cultures and stimulation contribute to their morbidity and mortality. Normal peripheral blood lymphocytes and CLL cells were cultured Chronic lymphocytic leukemia is a monoclonal disorder in RPMI-1640 medium (Gibco) containing FCS (10%) (Fetal Calf characterized by a progressive accumulation of CD5+CD19+ B cells, Serum), HEPES (10 mM), glutamine 2 mM, streptomycin (100 μg/ml), functionally incompetent, frequently associated by the other immune penicillin (100 U/ml) (Polfa, Poland) on culture plates (106 cells/ml) disturbances. Low expression of BCR (B-cell receptor) is the hallmark of (Corning Inc, USA) at 37ºC in humidified 5% CO2 atmosphere for 30 the CLL lymphocytes [19]. Besides antibody production, normal B cells minutes. CLL cells and normal lymphocytes, at the concentration 1x106 have important antigen presenting functions [20]. It has been shown cells per ml media, were stimulated with purified TLR agonists: 10 μg/ml [21] that the activation of human naive B cells requires a combination LPS (Lipopolysaccharide from Escherichia coli, Sigma) or formalinised of three signals: 1/BCR triggering, 2/cognate T cell help, and 3/ TLR SAC (Staphylococcus aureus strain Cowan I) in the insoluble form, 6 + stimulation. 1.2x10 microorganisms/ml. Normal CD19 subpopulations were isolated from half hour cultured normal lymphocytes using magnetic It seems that there is no harmony between these signals in CLL. columns (Miltenyi Biotech, Bergisch Gladbach, Germany). BCR signaling may be defective, which is related to small numbers of BCR, non-functional BCR assembly [19,22] and mutations in Flow cytometry analysis accessory proteins [23]. Consequently, it abolishes the ability of antigen Immunophenotyping was performed by flow cytometry presentation by CLL cells [24]. In patients with CLL, many T cells (FACSCalibur, Becton Dickinson Immunocytometry System) using acquired defect were also found, such as: increase of T cell number, commercial mouse monoclonal antibodies (anti-human CD19/ reversal of the CD4: CD8 ratio, alterations in T-cell cytoskeleton PE-Cy5, anti-human CD5/FITC, Becton Dickinson) and respective formation and vesicle transportation and production of cytokine immunoglobulin isotype controls. Cells were stained for surface inhibitors, IL-6, IL-10, TNF (Tumor necrosis factor) and TGF-beta markers according to the supplier’s instructions. The cells were gated (Tumor Growth Factor beta) [25-29]. Finally, CLL is associated with according to their FSC (forward scatter) and SSC (side scatter). Analyses the impairment of the innate immune system [30,31]. were carried out using Cell QUEST Software (Becton Dickinson) on gated lymphocytes. TLR stimulation triggers the activation of signals that eventually turn into a transcription of specific genes. Therefore, the defect in any of Real-Time PCR the signaling pathway links concerning activation signal transmission The expressions of IRAK4 in unstimulated, LPS- and SAC-stimulated from TLRs may underlay the cause of immune failure in