RESEARCH REPORT ISJ 4: 95-100, 2007 ISSN 1824-307X RESEARCH REPORT Vertebrate interleukins originated in invertebrates? S Gerber, P Cadet, M Sheehan, GB Stefano, KJ Mantione Neuroscience Research Institute, State University of New York - College at Old Westbury, Old Westbury, NY 11568-0210, USA Accepted October 30, 2007 Abstract Previous studies have demonstrated that invertebrate immune and neural tissues contain mammalian-like cytokines, which activate specific cellular functions. Therefore, it was of interest to attempt to identify these molecules via Applied Biosystems Human Genome Survey Arrays. The array was used to analyze the transcriptional profiles of Mytilus edulis RNA samples. The Applied Biosystems Human Genome Survey Array contains 31,700 60-mer oligonucleotides probes representing a set of 27, 868 individual human genes and more than 1,000 control probes. We show interleukin-like and tumor necrosis factor-like genes among other cytokine-like genes significantly expressed in this invertebrate tissue with a signal to noise value greater than 2. In morphine treated tissue additional cytokine genes were expressed. These cytokine-like genes are directly related to previously discovered molecules in invertebrates, suggesting that they first appeared earlier in evolution. Key words: mussel; Mytilus edulis; cytokines; microarray Introduction Mytilus edulis neural tissues contain both immune- and neural-like signaling molecules found in mammals (see Stefano, 1982, 1990a, 1992). In regard to catecholamines, the neural tissues of Mytilus contain both dopamine and norepinephrine (Stefano, 1982, 1990b). In reference to cytokine-like molecules, interleukin (IL)-1-, IL-6- and tumor necrosis factor (TNF)-like molecules have been identified via radio-immune assay in Mytilus ganglia and immune tissues (Hughes et al., 1990, 1991a; Hughes and Chin, 1994; Scharrer et al., 1996). Thus, invertebrate ganglia and immune tissues appear to have the potential to respond like mammalian tissues to these signaling molecules. Concerning the signaling of these molecules, invertebrate immune tissues, i.e., immunocytes, ___________________________________________________________________________ Corresponding author: Kirk J Mantione Neuroscience Research Institute State University of New York - College at Old Westbury Old Westbury, NY 11568-0210, USA E-mail: kjmantione@sunynri.org respond to IL-1 and IL-6 by undergoing conformational changes indicative of becoming activated, i.e., amoeboid, including stimulating mobility (Hughes et al., 1990, 1991a,b; Hughes and Chin, 1994; Scharrer et al., 1996). Thus, given their presence and action in invertebrate physiological systems it was of great interest to determine if human microarray chips would also show that they are present given the many biochemical and pharmacological similarities. Materials and Methods Mytilus edulis were harvested from the shores of Long Island Sound at Mattituck, New York during the month of March. Animals were then transported to the laboratory in chilled seawater (4-10 °C). In the laboratory, they were maintained as previously described in detail (Stefano et al., 1994). M. edulis pedal ganglia (20 per array) were dissected and kept on ice until needed. In order to determine the presence of 95 Table 1 Interleukin-like, tumor necrosis factor-like, and other cytokine-like genes that were significantly expressed as analyzed by the Human Genome Survey Microarray (Applied Biosystems) with a signal to noise value greater than 2 in the untreated Mytilus edulis pedal ganglia tissue. Tumor necrosis factor (TNF)-like molecules present TNFRSF11A Tumor necrosis factor receptor superfamily, member 11a, activator of NFKB TNFRSF25; KIAA0720 Tumor necrosis factor receptor superfamily, member 25; putative NFKB activating protein Interleukins-like molecules present IL16 Interleukin 16 (lymphocyte chemoattractant factor) IL7 Interleukin 7 IL31RA Interleukin 31 receptor A IL15 Interleukin 15 IL11RA Interleukin 11 receptor, alpha IL18BP Interleukin 18 binding protein IL23R Interleukin-23 receptor Additional cytokine-like molecules present Chemokine and chemokine receptor activity CCL23 Chemokine (C-C motif) ligand 23 CCRL2 Chemokine (C-C motif) receptor-like 2 CCL1 Chemokine (C-C motif) ligand 1 CCR9 Chemokine (C-C motif) receptor 9 CXCR3 Chemokine (C-X-C motif) receptor 3 CCL13 Chemokine (C-C motif) ligand 13 CCL15; CCL14 Chemokine (C-C motif) ligand 15; chemokine (C-C motif) ligand 14 CCL19 Chemokine (C-C motif) ligand 19 CCL24 Chemokine (C-C motif) ligand 24 MGC12815; CCL3L1; CCL3 Chemokine (C-C motif) ligand 3-like, centromeric; Chemokine (C-C motif) ligand 3-like 1; Chemokine (C-C motif) ligand 3 CCR6 Chemokine (C-C motif) receptor 6 CXCL12 Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) Cell growth and growth factor activity GDF8 Growth differentiation factor 8 GDF2 Growth differentiation factor 2 EBAF Endometrial bleeding associated factor (left-right determination, factor A; transforming growth factor beta superfamily) BMP1 Bone morphogenetic protein 1 MYH11 Myosin, heavy polypeptide 11, smooth muscle Cytokinesis and other cell-cycle activity CDK6 Cyclin-dependent kinase 6 CDC23 CDC23 (cell division cycle 23, yeast, homolog) CCND3 Cyclin D3 CDC25A Cell division cycle 25A CCNC Cyclin C CCNL1 Cyclin L1 CDC14A CDC14 cell division cycle 14 homolog A (S. cerevisiae) PARD6A Par-6 partitioning defective 6 homolog alpha (C.elegans) PARD3 Par-3 partitioning defective 3 homolog (C. elegans) PRC1 Protein regulator of cytokinesis 1 STAT1 Signal transducer and activator of transcription 1, 91kDa NEDD5 Neural precursor cell expressed, developmentally down-regulated 5 3-Sep Septin 3 DOCK1 Dedicator of cytokinesis 1 Immune activity LIF Leukemia inhibitory factor (cholinergic differentiation factor) OSM Oncostatin M PF4 Platelet factor 4 (chemokine (C-X-C motif) ligand 4) 96 TLR2 Toll-like receptor 2 IFNK Interferon, kappa Other DAPK1 Death-associated protein kinase 1 LATS1 LATS, large tumor suppressor, homolog 1 (Drosophila) PIN1 Protein (peptidyl-prolyl cis/trans isomerase) NIMA-interacting 1 PIN1L Protein (peptidyl-prolyl cis/trans isomerase) NIMA-interacting 1-like MOBK1B MOB1, Mps One Binder kinase activator-like 1B (yeast) SDFR1 Stromal cell derived factor receptor 1 C17 Cytokine-like protein C17 EPOR Erythropoietin receptor CSF2RB Colony stimulating factor 2 receptor, beta, low-affinity (granulocyte-macrophage) ACVR2B Activin A receptor, type IIB NRP1 Neuropilin 1 PBEF1 Pre-B-cell colony enhancing factor 1 OBRGRP; LEPR Leptin receptor gene-related protein; Leptin receptor TLT4 TREM-like transcript 4 LOC392255 Similar to growth differentiation factor 16 aforementioned molecules upon stimulation with a neuroimmune effecter using microarray, ganglia were incubated at 4 °C in filtered seawater or treated with 1 μM morphine for 18 h. Applied Biosystems expression array analysis Applied Biosystems Human Genome Survey Arrays were used to analyze the transcriptional profiles of RNA samples. The Applied Biosystems Human Genome Survey Array contains 31,700 60- mer oligonucleotides probes representing a set of 27, 868 individual human genes and more than 1,000 control probes. Sequences used for microarray probe design are from curated transcripts from the Celera Genomics Human Genome Database (www.celeradiscoverysystem.com), RefSeq transcripts that have been structurally curated from the LocusLink (http://ncbi.nlm.nih.bov/LocusLink/refseq.html) public database, high-quality cDNA sequences from the Mammalian Gene Collection (MGC) (http://mgc.nci.nih.gov) and transcripts that were experimentally validated at Applied Biosystems. Total RNA from 20 M. edulis pedal ganglia was isolated with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The tissue was lysed in 600 µl buffer RLT and homogenized by passing the lysate 5 times through a 20-gauge needle fitted to a 3 ml syringe. The samples were then processed following the manufacturer's detailed instructions. In the final step, the RNA was eluted with 50 µl of RNase-free water by centrifugation for 1 min at 10,000 rpm. Quality of the RNA was analyzed using Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) using the total RNA nanochip according to manufacturer’s protocol. Digoxigenin-UTP labeled cRNA was generated and linearly amplified from 1 µg of total RNA using Applied Biosystems Chemiluminescent RT-IVT Labeling Kit v 2.0 and manufacturer's protocol. Array hybridization, chemiluminescence detection, image acquisition and analysis were performed using Applied Biosystems Chemiluminescence Detection Kit and Applied Biosystems 1700 Chemiluminescent Microarray Analyzer following manufacturer's protocol. To each chip, 15 µg of labeled cRNA targets were hybridized at 55 °C for 19 h. AB1700 Expression System software was used to extract assay signal, and assay signal to noise ratio values from the microarray images. To select expressed genes, the gene list was further filtered by removing genes with a signal to noise value less than two. Results The previously discovered invertebrate cytokine-like molecules include tumor necrosis factor-like molecules as well as IL-1-, IL-2-, IL-4-, IL- 6- and IL-10-like molecules. Table 1 demonstrates interleukin-like and tumor necrosis factor-like genes among other cytokine-like genes that were significantly expressed as analyzed by the Human Gene Survey microarray (Applied Biosystems) with a signal to noise value greater than 2 in the untreated M. edulis pedal ganglia tissue. With a signal to noise value greater than 2, all genes expressed are thus considered to have a strong significant presence. Tumor necrosis-like factors were present in the untreated tissue. Additionally, several interleukin-like molecules also were present. In the morphine treated tissue, however, several additional genes were expressed (Table 2). Among these genes expressed was IL-10, an interleukin-like molecule previously demonstrated in 97 http://www.celeradiscoverysystem.com/ http://ncbi.nlm.nih.bov/LocusLink/refseq.html http://mgc.nci.nih.gov/ Table 2 Interleukin-like, tumor necrosis factor-like, and other cytokine-like genes that were significantly expressed in Mytilus edulis pedal ganglia after morphine treatment. Genes with a signal to noise ratio greater than 2 as analyzed by the Human Genome Survey Microarray (Applied Biosystems) were listed. Tumor necrosis factor (TNF)-like molecules present TNFRSF10A Tumor necrosis factor receptor superfamily, member 10a TNFSF12- TNFSF13; TNFSF12; TNFSF13 Tumor necrosis factor (ligand) superfamily, member 12-member 13; tumor necrosis factor (ligand) superfamily, member 12; tumor necrosis factor (ligand) superfamily, member 13 Interleukin-like molecules present IL17RB Interleukin 17 receptor B IL10 Interleukin 10 IL23A Interleukin 23, alpha subunit p19 IL5 Interleukin 5 (colony-stimulating factor, eosinophil) IL31RA Interleukin 31 receptor A IL18 Interleukin 18 (interferon-gamma-inducing factor) IL15RA Interleukin 15 receptor, alpha Additional cytokine-like molecules present Cytokinesis and other cell-cycle activity CDC25B Cell division cycle 25B ANAPC5 Anaphase promoting complex subunit 5 CDK7 Cyclin-dependent kinase 7 (MO15 homolog, Xenopus laevis, cdk-activating kinase) CDK4 Cyclin-dependent kinase 4 ROPN1 Ropporin, rhophilin associated protein 1 SOCS2 Suppressor of cytokine signaling 2 PNUTL1; GP1BB Peanut-like 1 (Drosophila); glycoprotein Ib (platelet), beta polypeptide SPAG5 Sperm associated antigen 5 ANAPC4 Anaphase promoting complex subunit 4 CDC6 CDC6 cell division cycle 6 homolog (S. cerevisiae) UBE2C Ubiquitin-conjugating enzyme E2C DOCK3 Dedicator of cytokinesis 3 Other CNTFR Ciliary neurotrophic factor receptor ASB9 Ankyrin repeat and SOCS box-containing 9 PREI3 Preimplantation protein 3 STAT2 Signal transducer and activator of transcription 2, 113kDa GAB3 GRB2-associated binding protein 3 OBRGRP; LEPR Leptin receptor gene-related protein; leptin receptor ASB10 Ankyrin repeat and SOCS box-containing 10 CRLF3 Cytokine receptor-like factor 3 11-Sep Septin 11 ASB1 Ankyrin repeat and SOCS box-containing 1 IKBKB Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta 98 Table 3 Additional interleukin-like genes expressed in presence of morphine as analyzed by the Human Genome Survey Microarray (Applied Biosystems) with a signal to noise ratio between 1 and 2. Gene Name Gene Ontology IL1F10 Interleukin 1 family member 10 (theta) Immune response, interleukin-1 receptor antagonist activity, extracellular space IL1F5 Interleukin 1 family member 5, delta Immune response; interleukin-1 receptor antagonist activity; extracellular space IL1RL2 Interleukin 1 receptor-like 2 Interleukin-1, Type I, activating receptor activity, transmembrane receptor activity, integral to membrane IL1RL1 Interleukin 1 receptor-like 1 Signal transduction, transmembrane receptor activity; receptor signaling protein activity;interleukin-1 receptor activity IL1F9 Interleukin 1 family member 9 Cell-cell signaling; immune response; response to pest/pathogen/parasite; interleukin-1 receptor antagonist activity; extracellular space IL6ST Interleukin 6 signal transducer (gp 130, oncostatin M receptor) Extracellular space; integral to membrane; protein binding; glycogen metabolic process; positive regulation of cell proliferation; regulation of Notch signaling pathway; signal transduction IL4 Interleukin 4 Cholesterol metabolism; regulation of isotype switching; cell proliferation; B-cell differentiation; cellular defense response; T-helper 2 type immune response; connective tissue growth factor biosynthesis; chemotaxis, interleukin- 4 receptor binding; extracellular space invertebrates (Stefano et al., 1999) and TNF-like molecules different from those expressed in the untreated tissue. Proinflammatory cytokines and TNF play a major role in inflammation response. The immunosuppressive effect of morphine treatment is demonstrated by a significant presence in expression of the anti-inflammatory IL-10-like molecule. Additionally, the significant presence of TNF receptor-like molecules indicates the down regulation of proinflammatory TNF-like molecules. The additional newly discovered cytokine-like molecules detected by microarray in both the untreated and morphine treated tissue provide researchers with a multitude of possible subjects for future investigation. Given the logarithmic analysis supplied by the SpotFire for functional genomics program (SpotFire, Somerville, Maine), any positive signal to noise value indicates gene presence is in greater amounts than background noise. Furthermore, the gene sequence of the human transcript on the microarray chip is not identical to the gene sequence of the corresponding M. edulis transcript. However, the array hybridization, as well as the washes, used the same stringency as human nucleic acid assays and we were still able to detect approximately 5000 genes. It is thus important to note the presence of any additional neuro-immune significant interleukin- like signal molecules that were detected upon morphine treatment of M. edulis pedal ganglia tissue (Table 3). These interleukin-like genes are directly related to previously discovered interleukin-like molecules in invertebrates. Discussion As noted earlier, invertebrate ganglia, immunocytes, and microglia contain IL-1- and IL-6- like signaling molecules (Beck and Habicht, 1986; Hughes et al, 1990, 1991a; Paemen et al, 1992; Stefano, 1992; Stefano et al, 1992; Hughes and Chin, 1994; Scharrer et al, 1996). Based on these findings, one can surmise that an interleukin-like molecule secreted from these invertebrate cells may have the ability to release dopamine from neurons. Recently, Sawada and colleagues, as well as others, demonstrated that mammalian IL-1 and IL-2 and -4 have the ability to alter invertebrate neural ion channels in a stereoselective manner, further strengthening the hypothesis that these immunocyte-derived molecules can alter neural activities as well as stimulate them ( Sawada et al., 1991; Szucs et al., 1992; Franchini et al., 1996; Rozsa et al., 1997; Kletsas et al., 1998). In previous and current research, measures are taken to confirm gene expression including TaqMan Probes (Applied Biosystems) and molecular methods including Western blotting. The ability of microarray to corroborate with and/or confirm an expanse of previous research is demonstrated in this study of cytokine-like molecules found in M. edulis. 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