Characterization of granulocyte colony stimulating factor receptor of the goldfish (Carassius auratus L.)

Abstract

Granulocyte colony stimulating factor receptor (GCSFR) is a member of the class I cytokine receptor superfamily and signaling through this receptor is important for the proliferation, differentiation and activation of neutrophils and their precursors. In this study we report on the cloning and molecular characterization of goldfish GCSFR. The identified goldfish GCSFR sequence possesses the conserved Ig-like domain, the cytokine receptor homology domain (CRH), three fibronectin domains as well as several intracellular signaling motifs characteristic of other vertebrate GCSFRs. Goldfish gcsfr mRNA was highly expressed in kidney and spleen, and in primary kidney neutrophils. The neutrophils have significantly higher mRNA levels of the transcription factors pu.1 and cebpα, and down-regulated levels of transcription factors important for macrophage development such as egr1 and cjun, compared to progenitor cells from the kidney. The gcsfr mRNA was present in the kidney progenitor cells, albeit at much lower levels compared to the neutrophils, and the expression of gcsfr in progenitor cells was not affected by duration of cultivation. Furthermore, gcsfr mRNA levels were up-regulated in neutrophils after treatment with heat-killed Aeromonas salmonicida A449 or with mitogens. Our results indicate that GCSFR may be a useful marker for fish neutrophils.

Introduction

Neutrophils are one of the first cells at the site of injury and possess an arsenal of potent antimicrobial responses for the elimination of infectious agents such as phagocytosis, respiratory burst, degranulation and release of toxic compounds and the ability to send out neutrophil extracellular traps (NETs) (Brinkmann et al., 2004, Hallett and Dewitt, 2007, Martinelli et al., 2004, Nathan, 2006, Pham, 2006). The potential toxic threat that neutrophils pose to host tissue should they undergo a spontaneous response is limited due to the short lifespan of neutrophils in vivo and in vitro (estimated to be ∼90 h and ∼8 h, respectively) by pre-programmed apoptosis (Witko-Sarsat et al., 2011). Thus, the continuous production of neutrophils, and the necessity of the immune system to respond to pathogens by increasing neutrophil numbers, must be tightly regulated. It is primarily through the action of granulocyte colony stimulating factor (GCSF) through its receptor (GCSFR) that is required for the development of neutrophils from bone marrow progenitors to committed neutrophil precursors (reviewed in (Panopoulos and Watowich, 2008)).

GCSFR is a member of the class I cytokine receptor superfamily which includes other receptors such as GP130. The structure of GCSFR is comprised of a signal peptide, an immunoglobulin-like domain, a cytokine receptor homology (CRH) domain, containing the class I cytokine receptor superfamily motif, W-S-X-W-S, three fibronectin domains, a transmembrane domain, and an intracellular cytoplasmic signaling domain containing three motifs termed Box 1, Box 2, and Box 3, important for signal transduction (Panopoulos and Watowich, 2008). Binding of a homodimeric GCSF complex to the Ig and CRH domains of two GCSF receptors triggers intracellular signaling through the Jak/STAT, Ras/Raf/Erk, or PI3-kinase pathways (Avalos, 1996, Liongue et al., 2009a, Richards et al., 2003). These signaling pathways ultimately lead to the migration, survival, proliferation, and differentiation of neutrophils. While there are reports of GCSFR expression on other hematopoietic cells such as monocytes (Shimoda et al., 1992) and lymphocytes, as well as some non-hematopoietic cells, GCSFR is primarily expressed on neutrophils and their precursors (Liongue et al., 2009a, Panopoulos and Watowich, 2008).

Recently, we identified and characterized goldfish neutrophil-like cells that are cytochemically and functionally similar to other vertebrate neutrophils (Katzenback and Belosevic, 2009a). While a number of studies have examined the antimicrobial arsenal of teleost neutrophils such as chemotaxis, phagocytosis, respiratory burst, degranulation and NETs (Harun et al., 2008, Mayumi et al., 2008, Overland et al., 2010, Palic et al., 2005, Palic et al., 2007a, Palic et al., 2007b), the process of neutrophil development, including the receptors and growth factors that are involved have remained largely unexamined in teleosts. To our knowledge, there is only one study that directly examined GCSFR in zebrafish myeloid cell development. Liongue et al. (2009a), demonstrated the presence of gcsf and gcsfr mRNA in zebrafish, and showed that gcsfr morpholino zebrafish had a reduction in the number of myeloid cells as well as neutropenia. Interestingly, neutrophils were still present in morpholino-injected fish presumably due to a GCSF/GCSFR-independent developmental pathway (Liongue et al., 2009a).

In this study, we report on the identification and in silico and molecular analysis of a goldfish gcsfr. Goldfish gcsfr mRNA showed highest expression in the kidney, and mRNA levels of gcsfr were higher in neutrophils isolated from the goldfish kidney. These neutrophils showed significantly higher mRNA levels of the transcription factors pu.1 and cebpα compared to kidney progenitor cells. We also demonstrate that gcsfr mRNA levels were up-regulated in neutrophils after treatment with mitogens or heat-killed Aeromonas salmonicida A449.