Cicd94-1, an Ascidian Multipurpose C-Type Lectin-Like Receptor Expressed in Ciona Intestinalis Hemocytes and Larval Neural Structures
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r 2007, Copyright the Authors Differentiation (2008) 76:267–282 DOI: 10.1111/j.1432-0436.2007.00214.x Journal compilation r 2007, No claim to US Government Works ORIGINAL ARTICLE Ivana Zucchetti . Rita Marino . Maria Rosaria Pinto . John D. Lambris . Louis Du Pasquier . Rosaria De Santis ciCD94-1, an ascidian multipurpose C-type lectin-like receptor expressed in Ciona intestinalis hemocytes and larval neural structures Received March 8, 2007; accepted in revised form July 4, 2007 Abstract C-type lectins play an important role in the along the evolutionary pathway leading to the NK immune system and are part of a large superfamily that receptors. includes C-type lectin-like domain (CTLD)-containing ciCD94-1 was up-regulated in response to inflamma- proteins. Divergent evolution, acting on the CTLD tion induced by lipopolysaccharide (LPS) acting on a fold, has generated the Ca21-dependent carbohydrate- blood cell type present in both the tunic and circulating binding lectins and molecules, as the lectin-like natural blood. Furthermore, an anti-ciCD94-1 antibody spe- killer (NK) receptors that bind proteins, rather than cifically inhibited the phagocytic activity of these cells. sugars, in a Ca21-independent manner. We have studied ciCD94-1 was also expressed during development in the ciCD94-1, a CTLD-containing protein from the tunic- larva and in the early stages of metamorphosis in struc- ate Ciona intestinalis, which is a homolog of the CD94 tures related to the nervous system, and loss of its vertebrate receptor that is expressed on NK cells and function affected the correct differentiation of these ter- modulates their cytotoxic activity by interacting with ritories. These findings suggest that ciCD94-1 has differ- MHC class I molecules. ciCD94-1 shares structural ent roles in immunity and in development, thus features with the CTLD-containing molecules that rec- strengthening the concept of gene co-option during ognize proteins, suggesting that it could be located evolution and of an evolutionary relationship between the nervous and the immune systems. Ivana Zucchetti Á Maria Rosaria Pinto Á Key words tunicates Á Ciona intestinalis Á nervous Rosaria De Santis (*. ) system Á immune system Á hemocytes Á CD94 Laboratory of Cell Biology Stazione Zoologica ‘‘Anton Dohrn’’ Villa Comunale 80121 Naples, Italy E-mail: [email protected] Introduction Rita Marino C-type lectins, the most diverse family of animal lectins, Gene Expression Service are involved in a broad range of biological processes Stazione Zoologica ‘‘Anton Dohrn’’ that include adhesion, endocytosis, and pathogen rec- Villa Comunale, 80121 Naples, Italy ognition and neutralization (Drickamer and Taylor, John D. Lambris 1993; Weis et al., 1998; Vasta et al., 2004). These pro- Protein Chemistry Laboratory teins were originally identified as carbohydrate-recog- Department of Pathology and Laboratory Medicine nition molecules because of the presence of a C-type School of Medicine, University of Pennsylvania lectin domain, which occurs in Ca21-dependent lectins 401 Stellar Chance in both invertebrates and vertebrates (Drickamer, Philadelphia, PA 19104 1999). These carbohydrate-binding C-type lectin do- mains are part of a larger family of domains known as Louis Du Pasquier Institute of Zoology and Evolutionary Biology C-type lectin-like domains (CTLDs), which seem to University of Basel, Vesalgasse 1 have originated by a process of divergent evolution Basel CH 4051, Switzerland from a common ancestral C-type lectin domain fold 268 (Drickamer, 1999). Many members of the CTLDs bind 0.47 M NaCl, 10 mM KCl, 1 mM Na2SO4, 2.5 mM NaHCO3, to proteins, rather than to sugars, in a Ca21-indepen- 1 mM EDTA, pH 7.5) to prevent cell clotting. After centrifugation dent manner (Vales-Gomez et al., 2000). One such pro- at 500 Â g for 5 min at 41C, hemocytes were resuspended in marine solution (MS: 0.45 M NaCl, 26 mM MgCl , 11 mM KCl, and tein-binding molecule is CD94, which is part of several 2 12 mM CaCl2, pH 7.4), and the cell number was determined in a vertebrate natural killer (NK) lymphocyte receptors Bu¨rker hemocytometer. Samples were used immediately for in vivo that play an important role in innate immunity. CD94 assays or processed for RNA and protein extraction. For in situ forms heterodimers with NKG2 family molecules and hybridization and immunohistochemistry, 100 ml of the cell suspen- sion, containing a final concentration (FC) of 3 Â 106 cells/ml, was either blocks or activates the cytotoxic activity of NK spread on Superfrost Pluss slides (Menzel Glaser GmbH, Bra- cells toward target cells by interacting specifically with unschweig, Germany) and allowed to attach for 30 min. After the MHC class I molecules (Lazetic et al., 1996; Carretero liquid was wiped away, the cells were fixed in 4% paraformalde- et al., 1998). Thus, its activity represents a point of in- hyde in 0.1 M MOPS (pH 7.5) containing 0.5 M NaCl for 30 min. Fragments of tunic isolated from both LT and NLT animals tersection between innate and adaptive immunity. were fixed in 4% paraformaldehyde in 75% SW overnight at 41C Based on homology at the amino acid level, a CD94- for in situ hybridization. For immunohistochemistry experiments, like gene, BsCD94/NKR-P1, has been identified in the the tissue was fixed in Bouin’s fluid for 24 hr. After dehydration, the colonial tunicate Botryllus schlosseri, and its product samples were embedded in paraffin, and 7.5 mm sections were cut and mounted on Superfrost Pluss slides. has been shown to be expressed in a sub-population of Ovary and testis were surgically dissected from the adult animals blood cells that may be involved in colony allorecog- and processed for total RNA extraction. nition (Khalturin et al., 2003). A homolog of BsCD94/ NKR-P1, with features close to those of human CD94 (Du Pasquier et al., 2004), also exists in the Ciona in- Purification of amebocytes testinalis genome (Khalturin et al., 2004) (AY505423) (Ci0100135570 model, Joint Genome Institute C. intes- According to the method of Smith and Peddie (1992), 1 ml of total tinalis v. 1.0). hemocyte suspension from single animals was loaded on an 8 ml C. intestinalis is a member of the Tunicata, a key 0%–60% continuous Percoll gradient in 3.5% NaCl and centri- fuged in a swing-out rotor at 1,900 Â g for 10 min at 41C. The invertebrate group, whose close phylogenetic position fractions containing hyaline and granular amebocytes were collect- to vertebrates has recently been confirmed and strength- ed, checked for cell quality under an Axiophot microscope (Zeiss, ened (Delsuc et al., 2006). This evidence has led to re- Gottingen, Germany) equipped with Nomarsky optics, pooled, and newed interest in this evolutionarily relevant model washed in ASW/EDTA. system. Given this relationship with vertebrates, we reasoned that a more in-depth study of the CD94-like gene in Ciona might provide further information In vitro LPS treatment of whole organisms and tunic regarding the evolution of C-type lectins and their involvement in differentiation and/or immunity. For the treatment of C. intestinalis individuals, 200 ml of 2 mg/ml LPS (Escherichia coli, serotype 055:B5; Sigma-Aldrich, St. Louis, In this paper, we demonstrate that in C. intestinalis MO) in phosphate-buffered saline (PBS) were injected randomly the CD94-1-like gene is expressed in a blood cell type into the tunic between the outer layer and the epidermis. Animals that is involved in the phagocytic activity that occurs were then incubated in buckets containing LPS at 2.6 mg/ml FC in during an immune response. We also show that this SW for 3 hr twice with a 24-hr interval between incubations, during which they were maintained in circulating SW. The animals were molecule plays a role during development and early sacrificed in order to collect blood samples. metamorphosis, confirming several earlier reports of the To induce local inflammatory reactions, 100 ml of 2 mg/ml LPS in involvement of immunity-related molecules in develop- PBS were injected into the tunic of C. intestinalis individuals be- mental processes (Regnier-Vigouroux, 2003; Vasta tween the outer layer and the epidermis in the area between the two siphons, as described previously (Pinto et al., 2003). A fragment et al., 2004). 2 ( 1cm ) of the injured tissue was fixed either in 4% paraform- aldehyde in 75% SW overnight at 41C for in situ hybridization or in Bouin’s fluid for 24 hr for immunohistochemistry experiments. Af- ter dehydration, the samples were embedded in paraffin. For both Methods individuals and local treatment, NLT controls involved injecting equal amounts of PBS instead of LPS. Embryo and tissue preparation C. intestinalis adults were taken from the Gulf of Naples. Eggs and sperm were collected surgically from the gonoducts and used for in LPS treatment of blood cells vitro fertilization. Embryos were raised in Millipore-filtered seawa- ter (SW) at 181C–201C and either fixed in 4% paraformaldehyde in Total cells or Percoll-purified amebocytes were adjusted to 0.1 M MOPS (pH 7.5) containing 0.5 M NaCl for whole-mount in 6 Â 106 cells/ml with ASW/EDTA. Aliquots of the cell suspension situ hybridization and immunohistochemistry, or collected by low- were diluted 1:1 with either a 40 mg/ml LPS suspension or with speed centrifugation for protein solubilization and RNA extraction. ASW/EDTA (for the controls) in a test tube and incubated at 181C. Blood from both lipopolysacchride (LPS)-treated (LT) and non- Samples were collected at the designated intervals, washed by cen- LPS-treated (NLT) control animals was collected from the basal trifugation, and resuspended in ASW/EDTA. One hundred micro- sinus with a syringe in the presence of artificial SW (ASW) con- liters of the cell suspension were spread onto a Superfrost Pluss taining ethylenediaminetetraacetic acid (EDTA) (ASW/EDTA: slide and processed for immunohistochemistry.