Paracrine growth factor role for Interleukin-18 in AIDS associated human B-cell lines.
University of West Florida, Pensacola, FL, United States
AIM:Considering the potential interaction of HIV and IL-18, we propose to study the expression of Interleukin-18 (IL-18), IL-18 Receptor-1 (IL-18 R1), IL-18 accessory protein-like (IL-18AcPL)molecule, and IL-18 protein secretion in AIDS-associated and non-AIDS-associated human B-cell lymphoma cell lines. METHODS:Using RT-PCR and ELISA methods, the expression and secretion of IL-18, IL-18R1 and IL-18 AcPL in 13 tumor derived human B-cell lines were studied for both constitutive and Phorbol 12-Myristate 13-Acetate (PMA) stimulation. CD19+ B-cells isolated from PBMC’s using magnetic beads (Dynabeads, Dynal A.S, Norway)were also used in this investigation. A 1 ug equivalent of total RNA or 0.1 ug of Poly A+ RNA of each cell type (control and PMA) was transcribed into cDNA by reverse transcription (RT) using a Perkin-Elmer 2400 PCR Thermocycler. PCR amplicons of IL-18 (350 bp), IL-18R1 (490 bp), and IL-18AcPL (429 bp) from three representative cell lines were cloned and sequenced to confirm respective genes. Expression patterns were normalized to the house-keeping gene GAPDH. RESULTS:The activity of IL-18 begins with its binding to the IL-18RC. The IL-18RC is a heterodimer composed of an alpha (IL-18R1) and beta subunit (IL-18 AcPL), both members of the interleukin-1 receptor (IL-1R) family. IL-18R1 is the binding portion whereas IL-18AcPL is for inducing signal transduction. Constitutive IL-18, IL-18R1 and IL-18 AcPL transcripts were expressed from 1 to 96 hr with a peak expression at 24 hr. Furthermore, mitogenic stimulation did not significantly change mRNA expression in any of the transcripts compared to untreated control cells, at any time-point. IL-18 secretion was analyzed using the Cytoscreen IL-18 immunoassay (Biosource International, Camarillo, CA), following the manufacturer’s guidelines. This kit recognizes both pro- and mature forms of IL-18. The results of the ELISA show that none of the B-cell lines or the CD19+ B-cells secretes any IL-18 protein either constitutively or upon PMA stimulation. CONCLUSIONS:Our understanding of the role B-cells play in pathogenesis has revealed several important cytokines such as IL-6, IL-7, IL-10, IL-12, IL-16, MIP-1α, and MIP-1β some of which are secreted in autocrine fashion. These findings suggest the possibility that modulation of viral expression may be influenced by some of these regulatory cytokines. In addition, some of these soluble factors secreted by B-cells may in fact have suppressive effects on viral replication. However, with reference to IL-18 and IL-18RC, the presence of EBV, or AIDS association did not influence in the induction of IL-18 protein secretion, thus suggesting possible paracrine role. In a nutshell, the recognition that B-cells express IL-18, IL-18RC, and IL-18AcPL mRNA transcripts adds to the burgeoning list of cytokines and cytokine receptors produced by this class of lymphocytes.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 798 (Growth factors & signalling).