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The differentiation of bone marrow-derived progenitor cells into monocytes, tissue macrophages and some dendritic cell (DC) subtypes requires the growth factor CSF1 and its receptor, CSF1R. Langerhans cells (LCs) and microglia develop from embryonic myeloid precursor cells that populate the epidermis and central nervous system (CNS) before birth. Notably, LCs and microglia are present in CSF1-deficient mice but absent from CSF1R-deficient mice. Here we investigated whether an alternative CSF1R ligand, interleukin 34 (IL-34), is responsible for this discrepancy. Through the use of IL-34-deficient (Il34(LacZ/LacZ)) reporter mice, we found that keratinocytes and neurons were the main sources of IL-34. Il34(LacZ/LacZ) mice selectively lacked LCs and microglia and responded poorly to skin antigens and viral infection of the CNS. Thus, IL-34 specifically directs the differentiation of myeloid cells in the skin epidermis and CNS.
Osteoclasts are generated from monocyte/macrophage-lineage precursors in response to colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). CSF-1-mutated CSF-1(op/op) mice as well as RANKL(-/-) mice exhibit osteopetrosis (OP) caused by osteoclast deficiency. We previously identified RANKL receptor (RANK)/CSF-1 receptor (CSF-1R) double-positive cells as osteoclast precursors (OCPs), which existed in bone in RANKL(-/-) mice. Here we show that OCPs do not exist in bone but in spleen in CSF-1(op/op) mice, and spleen acts as their reservoir. IL-34, a newly discovered CSF-1R ligand, was highly expressed in vascular endothelial cells in spleen in CSF-1(op/op) mice. Vascular endothelial cells in bone also expressed IL-34, but its expression level was much lower than in spleen, suggesting a role of IL-34 in the splenic generation of OCPs. Splenectomy (SPX) blocked CSF-1-induced osteoclastogenesis in CSF-1(op/op) mice. Osteoclasts appeared in aged CSF-1(op/op) mice with up-regulation of IL-34 expression in spleen and bone. Splenectomy blocked the age-associated appearance of osteoclasts. The injection of 2-methylene-19-nor-(20S)-1α,25(OH)(2)D(3) (2MD), a potent analog of 1α,25-dihidroxyvitamin D(3), into CSF-1(op/op) mice induced both hypercalcemia and osteoclastogenesis. Administration of 2MD enhanced IL-34 expression not only in spleen but also in bone through a vitamin D receptor-mediated mechanism. Either splenectomy or siRNA-mediated knockdown of IL-34 suppressed 2MD-induced osteoclastogenesis. These results suggest that IL-34 plays a pivotal role in maintaining the splenic reservoir of OCPs, which are transferred to bone in response to diverse stimuli, in CSF-1(op/op) mice. The present study also suggests that the IL-34 gene in vascular endothelial cells is a unique target of vitamin D.
The CSF-1 receptor (CSF-1R) regulates CNS microglial development. However, the localization and developmental roles of this receptor and its ligands, IL-34 and CSF-1, in the brain are poorly understood. Here we show that compared to wild type mice, CSF-1R-deficient (Csf1r-/-) mice have smaller brains of greater mass. They further exhibit an expansion of lateral ventricle size, an atrophy of the olfactory bulb and a failure of midline crossing of callosal axons. In brain, IL-34 exhibited a broader regional expression than CSF-1, mostly without overlap. Expression of IL-34, CSF-1 and the CSF-1R were maximal during early postnatal development. However, in contrast to the expression of its ligands, CSF-1R expression was very low in adult brain. Postnatal neocortical expression showed that CSF-1 was expressed in layer VI, whereas IL-34 was expressed in the meninges and layers II-V. The broader expression of IL-34 is consistent with its previously implicated role in microglial development. The differential expression of CSF-1R ligands, with respect to CSF-1R expression, could reflect their CSF-1R-independent signaling. Csf1r-/- mice displayed increased proliferation and apoptosis of neocortical progenitors and reduced differentiation of specific excitatory neuronal subtypes. Indeed, addition of CSF-1 or IL-34 to microglia-free, CSF-1R-expressing dorsal forebrain clonal cultures, suppressed progenitor self-renewal and enhanced neuronal differentiation. Consistent with a neural developmental role for the CSF-1R, ablation of the Csf1r gene in Nestin-positive neural progenitors led to a smaller brain size, an expanded neural progenitor pool and elevated cellular apoptosis in cortical forebrain. Thus our results also indicate novel roles for the CSF-1R in the regulation of corticogenesis.
Colony stimulating factor-1 (CSF-1, also known as macrophage-colony stimulating factor, M-CSF) has long been known as the primary growth factor regulating survival, proliferation and differentiation of macrophages and other mononuclear phagocytic (MNP) lineage cells. CSF-1 was subsequently identified as a monocyte/macrophage chemokine, a capacity now recognized to be integral to many of the deleterious as well as positive roles of macrophages in development, homeostasis and disease. The pleiotrophic actions of CSF-1 are all transduced by its high affinity receptor, the CSF-1R, a receptor tyrosine kinase (RTK) and the cellular homologue of the v-fms oncoprotein. While the CSF-1R is the sole receptor for CSF-1, an alternative functional ligand for the receptor, interleukin-34 (IL-34), was recently identified. CSF-1-induced CSF-1R activation triggers autophosphorylation of several intracellular tyrosine residues, leading to initiation of an array of phosphotyrosine-based signaling cascades that mediate the wide variety of cellular responses to CSF-1. Dissecting the contributions of the different phosphorylated tyrosine motifs of the receptor to downstream signaling events in macrophages is not only important for our understanding of CSF-1R function, but also for the development of inhibitors to treat diseases where infiltrating macrophages contribute to their progression. This review will outline our current understanding of the CSF-1/CSF-1R signaling axis and describe how a novel macrophage cell line system, which allows examination of CSF-1R signaling in a mature macrophage context, is helping us to tease apart the diverse signaling pathways initiated by CSF-1R activation.
Interleukin-34 (IL-34) is a recently defined cytokine, showing a functional overlap with macrophage colony stimulating factor (M-CSF). This study was undertaken to address the expression of IL-34 in rheumatoid arthritis (RA) patients and to investigate its regulation and pathogenic role in RA.
IL-34 levels were determined in the RA synovium, synovial fluid (SF) and fibroblast-like synovial cells (FLS) by immunohistochemistry, real-time PCR, enzyme-linked immunosorbent assay and immunoblotting. RA activity was assessed using Disease Activity Score 28 (DAS28) activity in the plasma collected at baseline and one year after treatment. Conditioned media (CM) were prepared from RA FLS culture with tumor necrosis factor alpha (TNFα) for 24 hours and used for functional assay.
IL-34 was expressed in the synovium, SF, and FLS from RA patients. The production of IL-34 in FLS was up-regulated by TNFα in RA samples compared with osteoarthritis (OA) patients. Importantly, the preferential induction of IL-34 rather than M-CSF by TNFα in RAFLS was mediated by the transcription factor nuclear factor kappa B (NF-κB) and activation of c-Jun N-terminal kinase (JNK). IL-34 elevation in plasma from RA patients was decreased after the administration of disease-modifying anti-rheumatic drugs (DMARDs) in accordance with a decrease in DAS28. CM from RAFLS cultured with TNFα promoted chemotactic migration of human peripheral blood mononuclear cells (PBMCs) and subsequent osteoclast (OC) formation, effects that were attenuated by an anti-IL-34 antibody.
These data provide novel information about the production of IL-34 in RA FLS and indicate that IL-34 is an additional osteoclastogenic factor regulated by TNFα in RA, suggesting a discrete role of IL-34 in inflammatory RA diseases.
Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.
It has been widely believed that the cytokines required for osteoclast formation are M-CSF (also known as CSF-1) and RANKL. Recently, a novel cytokine, designated IL-34, has been identified as another ligand of CSF1R. This study was to explore the biological function, specifically osteoclastogenesis and bone metabolism, of the new cytokine. We produced recombinant mouse IL-34 and found that together with RANKL it induces the formation of osteoclasts both from splenocytes as well as dose-dependently from bone marrow cells in mouse and these cells also revealed bone resorption activity. It also promotes osteoclast differentiation from human peripheral blood mononucleated cells. Finally, we show that systemic administration of IL-34 to mice increases the proportion of CD11b+ cells and reduces trabecular bone mass. Our data indicate that IL-34 is another important player in osteoclastogenesis and thus may have a role in bone diseases. Strategies of targeting CSF1/CSF1R have been developed and some of them are already in preclinical and clinical studies for treatment of inflammatory diseases. Our results strongly suggest the need to revisit these strategies as they may provide a new potential pharmaceutical target for the regulation of bone metabolism in addition to their role in the treatment of inflammatory diseases.
Experimental autoimmune encephalomyelitis (EAE), a well-established model of multiple sclerosis, is characterised by microglial activation and lymphocytic infiltration. Lymphocytic activation through the antigen presentation process involves three main signals, the first provided by the engagement of major histocompatibility complex molecules (MHC) with the receptor of T-cells (TCR), the second by the binding of co-stimulatory molecules and the third by the secretion or expression of T-cell polarising molecules in specific populations of antigen presenting cells (APC). Microglial cells are considered to be the main APC population in the central nervous system (CNS). Specifically in EAE an increase in MHCs, co-stimulatory molecules and different T-cell polarising factors have been reported in microglia. However, a growing number of evidences suggest that dendritic cells (DCs), the main APC in the peripheral immune system, may also participate in the regulation of T-cell responses within the CNS. In this review we summarize the principal knowledge regarding microglial/macrophage function in EAE and their role in T-cell modulation, as well as the participation of DCs in the immune response associated to this disease.
Conserved upstream open reading frames (uORFs) are found within many eukaryotic transcripts and are known to regulate protein translation. Evidence from genetic and bioinformatic studies implicates disturbed uORF-mediated translational control in the etiology of human diseases. A genetic mouse model has recently provided proof-of-principle support for the physiological relevance of uORF-mediated translational control in mammals. The targeted disruption of the uORF initiation codon within the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) gene resulted in deregulated C/EBPβ protein isoform expression, associated with defective liver regeneration and impaired osteoclast differentiation. The high prevalence of uORFs in the human transcriptome suggests that intensified search for mutations within 5' RNA leader regions may reveal a multitude of alterations affecting uORFs, causing pathogenic deregulation of protein expression.
CSF-1 and the novel CSF-1 receptor ligand, IL-34, possess similar CSF-1R-mediated activities, but differ in their spatio-temporal expression, permitting complementary CSF-1 receptor activation in vivo.
CSF-1 is broadly expressed and regulates macrophage and osteoclast development. The action and expression of IL-34, a novel CSF-1R ligand, were investigated in the mouse. As expected, huIL-34 stimulated macrophage proliferation via the huCSF-1R, equivalently to huCSF-1, but was much less active at stimulating mouse macrophage proliferation than huCSF-1. Like muCSF-1, muIL-34 and a muIL-34 isoform lacking Q81 stimulated mouse macrophage proliferation, CSF-1R tyrosine phosphorylation, and signaling and synergized with other cytokines to generate macrophages and osteoclasts from cultured progenitors. However, they respectively possessed twofold and fivefold lower affinities for the CSF-1R and correspondingly, lower activities than muCSF-1. Furthermore, muIL-34, when transgenically expressed in a CSF-1-dependent manner in vivo, rescued the bone, osteoclast, tissue macrophage, and fertility defects of Csf1op/op mice, suggesting similar regulation of CSF-1R-expressing cells by IL-34 and CSF-1. Whole-mount IL34 in situ hybridization and CSF-1 reporter expression revealed that IL34 mRNA was strongly expressed in the embryonic brain at E11.5, prior to the expression of Csf1 mRNA. QRT-PCR revealed that compared with Csf1 mRNA, IL34 mRNA levels were lower in pregnant uterus and in cultured osteoblasts, higher in most regions of the brain and heart, and not compensatorily increased in Csf1op/op mouse tissues. Thus, the different spatiotemporal expression of IL-34 and CSF-1 allows for complementary activation of the CSF-1R in developing and adult tissues.
Background
Salmonids are of interest because of their relatively recent genome duplication, and their extensive use in wild fisheries and aquaculture. A comprehensive gene list and a comparison of genes in some of the different species provide valuable genomic information for one of the most widely studied groups of fish.
Results
298,304 expressed sequence tags (ESTs) from Atlantic salmon (69% of the total), 11,664 chinook, 10,813 sockeye, 10,051 brook trout, 10,975 grayling, 8,630 lake whitefish, and 3,624 northern pike ESTs were obtained in this study and have been deposited into the public databases. Contigs were built and putative full-length Atlantic salmon clones have been identified. A database containing ESTs, assemblies, consensus sequences, open reading frames, gene predictions and putative annotation is available. The overall similarity between Atlantic salmon ESTs and those of rainbow trout, chinook, sockeye, brook trout, grayling, lake whitefish, northern pike and rainbow smelt is 93.4, 94.2, 94.6, 94.4, 92.5, 91.7, 89.6, and 86.2% respectively. An analysis of 78 transcript sets show Salmo as a sister group to Oncorhynchus and Salvelinus within Salmoninae, and Thymallinae as a sister group to Salmoninae and Coregoninae within Salmonidae. Extensive gene duplication is consistent with a genome duplication in the common ancestor of salmonids. Using all of the available EST data, a new expanded salmonid cDNA microarray of 32,000 features was created. Cross-species hybridizations to this cDNA microarray indicate that this resource will be useful for studies of all 68 salmonid species.
Conclusion
An extensive collection and analysis of salmonid RNA putative transcripts indicate that Pacific salmon, Atlantic salmon and charr are 94–96% similar while the more distant whitefish, grayling, pike and smelt are 93, 92, 89 and 86% similar to salmon. The salmonid transcriptome reveals a complex history of gene duplication that is consistent with an ancestral salmonid genome duplication hypothesis. Genome resources, including a new 32 K microarray, provide valuable new tools to study salmonids.
Development of Myeloid Immune Cells
As leukocytes develop to maturity, they proceed through an array of phenotypically distinct intermediates. For T and B lymphocyte populations, the different developmental stages, anatomical locations, and cell signals required for progression are well established. However, until recently, much less has been known about how development proceeds in the myeloid lineage, which includes monocytes, macrophages, and dendritic cells. Geissmann et al. (p. 656 ) review our current understanding of myeloid lineage development and describe the developmental pathways and cues that drive differentiation.
A novel IL-1 family member (nIL-1F) has been discovered in fish, adding a further member to this cytokine family. The unique gene organization of nIL-1F, together with its location in the genome and low homology to known family members, suggests that this molecule is not homologous to known IL-1F. Nevertheless, it contains a predicted C-terminal beta-trefoil structure, an IL-1F signature region within the final exon, a potential IL-1 converting enzyme cut site, and its expression level is clearly increased following infection, or stimulation of macrophages with LPS or IL-1beta. A thrombin cut site is also present and may have functional relevance. The C-terminal recombinant protein antagonized the effects of rainbow trout rIL-1beta on inflammatory gene expression in a trout macrophage cell line, suggesting it is an IL-1beta antagonist. Modeling studies confirmed that nIL-1F has the potential to bind to the trout IL-1RI receptor protein, and may be a novel IL-1 receptor antagonist.
Proliferative kidney disease (PKD) of salmonids, caused by Tetracapsuloides bryosalmonae, can lead to high mortalities at elevated water temperature. We evaluated the hypothesis that this mortality is caused by increasing parasite intensity. T. bryosalmonae-infected rainbow trout (Oncorhynchus mykiss) were reared at different water temperatures and changes in parasite concentrations in the kidney were compared to cumulative mortalities. Results of parasite quantification by a newly developed real-time PCR agreed with the number of parasites detected by immunohistochemistry, except for very low or very high parasite loads because of heterogenous distribution of the parasites in the kidney. Two experiments were performed, where fish were exposed to temperatures of 12, 14, 16, 18 or 19 degrees C after an initial exposure to an infectious environment at 12-16 degrees C resulting in 100% prevalence of infected fish after 5 to 14 days of exposure. While mortalities differed significantly between all investigated water temperatures, significant differences in final parasite loads were only found between fish kept at 12 degrees C and all other groups. Differences in parasite load between fish kept at 14 degrees C to 19 degrees C were not significant. These findings provide evidence that there is no direct link between parasite intensity and fish mortality.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
A cell line, RTL-W1, has been developed from the normal liver of an adult rainbow trout by proteolytic dissociation of liver fragments. RTL-W1 can be grown routinely in the basal medium, L-15, supplemented with 5% fetal bovine serum. In this medium, the cells have been passaged approximately 100 times over an 8-year period. The cells do not form colonies or grow in soft agar. The cultures are heteroploid. The cell shape was predominantly polygonal or epithelial-like, but as cultures became confluent, bipolar or fibroblast-like cells appeared. Among the prominent ultrastructural features of RTL-W1 were distended endoplasmic reticulum and desmosomes. Benzo[a]pyrene was cytotoxic to RTL-W1. Activity for the enzyme, 7-ethoxyresorufin O-deethylase (EROD), which is a measure of the cytochrome P4501A1 protein, increased dramatically in RTL-W1 upon their exposure to increasing concentrations of either beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). With these properties, RTL-W1 should be useful for studying the expression of the cytochrome P450 enzymes and as a tool for assessing the toxic potency of environmental contaminants.
Genome sequencing efforts will soon generate hundreds of millions of bases of human genomic DNA containing thousands of novel genes. In the past year, the accuracy of computational gene-finding methods has improved significantly, to the point where a reasonable approximation of the gene structures within an extended genomic region can often be predicted in advance of more detailed experimental studies.
The effects of colony-stimulating factor 1 (CSF-1), the primary regulator of mononuclear phagocyte production, are thought to be mediated by the CSF-1 receptor (CSF-1R), encoded by the c-fms proto-oncogene. To investigate the in vivo specificity of CSF-1 for the CSF-1R, the mouse Csf1r gene was inactivated. The phenotype of Csf1(-)/Csf1r(-) mice closely resembled the phenotype of CSF-1-nullizygous (Csf1(op)/Csf1(op)) mice, including the osteopetrotic, hematopoietic, tissue macrophage, and reproductive phenotypes. Compared with their wild-type littermates, splenic erythroid burst-forming unit and high-proliferative potential colony-forming cell levels in both Csf1(op)/Csf1(op) and Csf1(-)/Csf1r(-) mice were significantly elevated, consistent with a negative regulatory role of CSF-1 in erythropoiesis and the maintenance of primitive hematopoietic progenitor cells. The circulating CSF-1 concentration in Csf1r(-)/Csf1r(-) mice was elevated 20-fold, in agreement with the previously reported clearance of circulating CSF-1 by CSF-1R-mediated endocytosis and intracellular destruction. Despite their overall similarity, several phenotypic characteristics of the Csf1r(-)/Csf1r(-) mice were more severe than those of the Csf1(op)/Csf1(op) mice. The results indicate that all of the effects of CSF-1 are mediated via the CSF-1R, but that subtle effects of the CSF-1R could result from its CSF-1-independent activation.
The Clustal series of programs are widely used in molecular biology for the multiple alignment of both nucleic acid and protein
sequences and for preparing phylogenetic trees. The popularity of the programs depends on a number of factors, including not
only the accuracy of the results, but also the robustness, portability and user-friendliness of the programs. New features
include NEXUS and FASTA format output, printing range numbers and faster tree calculation. Although, Clustal was originally
developed to run on a local computer, numerous Web servers have been set up, notably at the EBI (European Bioinformatics Institute)
(http://www.ebi.ac.uk/clustalw/).
The rapid increase in the amount of protein and DNA sequence information available has become almost overwhelming to researchers. So much information is now accessible that high-quality, functional gene analysis and categorization has become a major goal for many laboratories. To aid in this categorization, there is a need for non-commercial software that is able to both align sequences and also calculate pairwise levels of similarity/identity.
We have developed MatGAT (Matrix Global Alignment Tool), a simple, easy to use computer application that generates similarity/identity matrices for DNA or protein sequences without needing pre-alignment of the data.
The advantages of this program over other software are that it is open-source freeware, can analyze a large number of sequences simultaneously, can visualize both sequence alignment and similarity/identity values concurrently, employs global alignment in calculations, and has been formatted to run under both the Unix and the Microsoft Windows Operating Systems. We are presently completing the Macintosh-based version of the program.
Information on the ontogeny of the fish immune system is largely restricted to a few species of teleosts (e.g., rainbow trout, catfish, zebrafish, sea bass) and has previously focused on morphological features. However, basic questions including the identification of the first lympho-hematopoietic sites, the origin of T- and B-lymphocytes and the acquisition of full immunological capacities remain to be resolved. We review these three main topics with special emphasis on recent results obtained from the zebrafish, a new experimental model particularly suitable for study of the ontogeny of the immune system because of its rapid development and easy manipulation. This species also provides an easy way of creating mutations that can be detected by various types of screens.
A new rainbow trout cell line, RTS11, arose spontaneously from a long-term haemopoietic culture of spleen. For routine subculture, RTS11 requires 20–30% fetal bovine serum, a high cell density, and conditioned medium from previous cultures. Through successive passages, RTS11 cultures maintain a balance between small, non-adherent cells that form the majority of cells (referred to as round cells) and a few larger, granular cells that are either adherent or in suspension surrounded by clusters of round cells. The large granular cells appear to be macrophages, and are referred to as macrophage-like cells, because they are phagocytic, take up DiI-acetylated low density lipoprotein and acridine orange and stain for non-specific esterase, whereas the round cells appear to be at an earlier stage of macrophage development. Medium conditioned by RTS11 also contains lysozyme-like activity, an additional indication that they are of the macrophage lineage. In proliferation assays, the non-adherent cells are stimulated strongly by RTS11 conditioned medium and by lipopolysaccharide. Rainbow trout head kidney leucocytes also show a positive proliferative response to RTS11 conditioned medium. Therefore, RTS11 cells appear to both produce and respond to an autocrine growth factor(s).
Interleukin-34 (IL-34) is a newly discovered regulator of myeloid lineage differentiation, proliferation, and survival, acting via the macrophage-colony stimulating factor receptor (M-CSF receptor, c-fms). M-CSF, the main ligand for c-fms, is required for osteoclastogenesis and has been already identified as a critical contributor of the pathogenesis of giant cell tumours of bone (GCTs), tumours rich in osteoclasts. According to the key role of M-CSF in osteoclastogenesis and GCTs, the expression of IL-34 in human GCTs was first assessed. Quantitative analysis of IL-34 mRNA expression in 14 human GCTs revealed expression of this cytokine in GCTs as well as M-CSF and c-fms. Immunohistochemistry demonstrated that osteoclast-like cells exhibited a huge immunostaining for IL-34 and that mononuclear stromal cells were slightly positive for this protein. In contrast to osteoblasts, bone-resorbing osteoclasts showed very strong staining for IL-34, suggesting its potential role in the pathogenesis of GCTs by facilitating osteoclast formation. The role of IL-34 in osteoclastogenesis was then studied in murine and human models. IL-34 was able to support RANKL-induced osteoclastogenesis in the absence of M-CSF in all models. Multinucleated cells generated in the presence of IL-34 and RANKL expressed specific osteoclastic markers and resorbed dentine. IL-34 induced phosphorylation of ERK 1/2 and Akt through the activation of c-fms, as revealed by the inhibition of signalling by a specific c-fms tyrosine kinase inhibitor. Furthermore, IL-34 stimulated RANKL-induced osteoclastogenesis by promoting the adhesion and proliferation of osteoclast progenitors, and had no effect on osteoclast survival. Overall, these data reveal that IL-34 can be entirely substituted for M-CSF in RANKL-induced osteoclastogenesis, thus identifying a new biological activity for this cytokine and a contribution to the pathogenesis of GCTs.
A histological study of 0+ rainbow trout, exposed to proliferative kidney disease (PKD) on an infected farm, showed evidence of infection from the beginning of June, gross pathological changes from the beginning of July, and clinical signs from mid-July. Evidence of healing was present in some samples from the second week of August. Kidney samples were divided into five grades (0–4) representing increasing severity of pathology. A sixth grade (H) was applied to kidneys showing signs of healing. The most frequent clinical signs were abdominal distension and exophthalmos. Melanosis, corneal cloudiness and ecchymoses were observed occasionally. Apart from renal enlargement, other internal gross pathological changes included ascites, gill and liver pallor, oedema, petechiation, and splenic enlargement. The majority of clinical signs and internal changes were only seen in fish with advanced renal swelling (grades 3 and 4). Histopathology was most marked in the kidney and was of three types: haemopoietic hyperplasia, vascular pathology and diffuse inflammatory change. Vascular changes, including occlusion of blood vessels in several organs and apparent haemoglobin crystallization, and destruction of renal excretory structures within areas of chronic diffuse inflammation were marked features of the histopathology in fish with clinical signs. The pathogenesis of PKD is discussed with reference to the histopathological changes.
Interleukin-34 (IL-34) and colony stimulating factor-1 (CSF-1) both signal through the CSF-1R receptor tyrosine kinase, but they have no sequence homology, and their functions and signaling activities are not identical. We report the crystal structures of mouse IL-34 alone and in complex with the N-terminal three immunoglobulin-like domains (D1-D3) of mouse CSF-1R. IL-34 is structurally related to other helical hematopoietic cytokines, but contains two additional helices integrally associated with the four shared helices. The non-covalently linked IL-34 homodimer recruits two copies of CSF-1R on the sides of the helical bundles, with an overall shape similar to the CSF-1:CSF-1R complex, but the flexible linker between CSF-1R D2 and D3 allows these domains to clamp IL-34 and CSF-1 at different angles. Functional dissection of the IL-34:CSF-1R interface indicates that the hydrophobic interactions, rather than the salt bridge network, dominate the biological activity of IL-34. To degenerately recognize two ligands with completely different surfaces, CSF-1R apparently takes advantage of different subsets of a chemically inert surface that can be tuned to fit different ligand shapes. Differentiated signaling between IL-34 and CSF-1 is likely achieved by the relative thermodynamic independence of IL-34 vs. negative cooperativity of CSF-1 at the receptor-recognition sites, in combination with the difference in hydrophobicity which dictates a more stable IL-34:CSF-1R complex compared to the CSF-1:CSF-1R complex.
Lacking any discernible sequence similarity, interleukin-34 (IL-34) and colony stimulating factor 1 (CSF-1) signal through a common receptor CSF-1R on cells of mononuclear phagocyte lineage. Here, the crystal structure of dimeric IL-34 reveals a helical cytokine fold homologous to CSF-1, and we further show that the complex architecture of IL-34 bound to the N-terminal immunoglobulin domains of CSF-1R is similar to the CSF-1/CSF-1R assembly. However, unique conformational adaptations in the receptor domain geometry and intermolecular interface explain the cross-reactivity of CSF-1R for two such distantly related ligands. The docking adaptations of the IL-34 and CSF-1 quaternary complexes, when compared to the stem cell factor assembly, draw a common evolutionary theme for transmembrane signaling. In addition, the structure of IL-34 engaged by a Fab fragment reveals the mechanism of a neutralizing antibody that can help deconvolute IL-34 from CSF-1 biology, with implications for therapeutic intervention in diseases with myeloid pathogenic mechanisms.
Messenger RNA decay is an essential step in gene expression to set mRNA abundance in the cytoplasm. The binding of proteins and/or noncoding RNAs to specific recognition sequences or secondary structures within mRNAs dictates mRNA decay rates by recruiting specific enzyme complexes that perform the destruction processes. Often, the cell coordinates the degradation or stabilization of functional subsets of mRNAs encoding proteins collectively required for a biological process. As well, extrinsic or intrinsic stimuli activate signal transduction pathways that modify the mRNA decay machinery with consequent effects on decay rates and mRNA abundance. This review is an update to our 2001 Gene review on mRNA stability in mammalian cells, and we survey the enormous progress made over the past decade.
Microglia, macrophage-like resident immune cells in the brain, possess both neurotoxic and neuroprotective properties and have a critical role in the development of Alzheimer's disease (AD). We examined the function of Interleukin-34 (IL-34), a newly discovered cytokine, on microglia because it reportedly induces proliferation of monocytes and macrophages. We observed that the neuronal cells primarily produce IL-34 and that microglia express its receptor, colony-stimulating factor 1 receptor. IL-34 promoted microglial proliferation and clearance of soluble oligomeric amyloid-β (oAβ), which mediates synaptic dysfunction and neuronal damage in AD. IL-34 increased the expression of insulin-degrading enzyme, aiding the clearance of oAβ, and induced the antioxidant enzyme heme oxygenase-1 in microglia to reduce oxidative stress, without producing neurotoxic molecules. Consequently, microglia treated with IL-34 attenuated oAβ neurotoxicity in primary neuron-microglia co-cultures. In vivo, intracerebroventricular administration of IL-34 ameliorated impairment of associative learning and reduced oAβ levels through up-regulation of insulin-degrading enzyme and heme oxygenase-1 in an APP/PS1 transgenic mouse model of AD. These findings support the idea that enhancement of the neuroprotective property of microglia by IL-34 may be an effective approach against oAβ neurotoxicity in AD.
Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates hematopoiesis, inflammation, immune responses and bone homeostasis in mammals. Fish IL-6 has been cloned in recent years but to date no functional studies have been reported. Thus, in this paper we present for the first time in fish the functional characterisation of IL-6, using rainbow trout (Oncorhynchus mykiss) as the fish model and with a focus on macrophage effects. Trout IL-6 (tIL-6) expression in macrophages could be induced by proinflammatory agents (LPS, polyI:C, and IL-1β) and recombinant tIL-6 (rtIL-6) rapidly induced STAT3 phosphorylation and expression of SOCS-1 to -3, CISH and IRF-1, as seen in mammals. However, three findings in this study suggest a novel role of tIL-6 in fish. Firstly, macrophage growth was enhanced by rtIL-6 in vitro, suggesting that IL-6 produced during inflammatory events may promote macrophage proliferation locally. Secondly, rtIL-6 induced the expression of cathelicidin-2, an antimicrobial peptide with immune-modulatory function, but down-regulated the expression of IL-1β and TNF-α, indicating a role of IL-6 in host defence and also in limiting inflammation. Thirdly, rtIL-6 induced the expression of hepcidin in macrophages. In mammals hepcidin is antimicrobial but also regulates iron homeostasis by inhibiting iron absorption, and its expression is induced by IL-6 only in hepatocytes but not macrophages. Thus, in fish if IL-6 is induced in patrolling macrophages during sepsis this may act to reduce iron availability by induction of hepcidin expression and lead to iron deficiency, as a means to limit the spread of infection.
Mammalian interleukin-4 (IL-4) and IL-13 are T helper type 2 (Th2) cytokines with pleiotropic functions in immunity. They signal through receptors containing IL-4Rα and IL-2Rγ or IL-13Rα1. In addition, a decoy receptor, IL-13Rα2, is known to exist and modulates the function of IL-13. The existence of fish orthologues to mammalian IL-4 and IL-13 is still under debate. However, the receptor chains have been predicted in zebrafish, and we have previously cloned IL-2Rγ and IL-13Rα2 in rainbow trout. In this study, we have cloned a further five novel trout IL-4/13 receptors. Thus, each of the IL-4Rα, IL-13Rα1 and IL-13Rα2 chains has two copies. The identities of the receptors is supported by homology analysis, characteristic domain structure, phylogenetic tree analysis and synteny analysis in zebrafish. However, the characteristic WSXWS motif of structural importance in mammalian type I cytokine receptors is missing in all fish IL-4Rα and IL-13Rα1 molecules. All the receptors have a characteristic domain structure that is similar to their mammalian counterparts except for IL-13Rα1b that has the N-terminal Ig domain missing. Since this Ig domain is a specific and critical binding unit for IL-13 but not for IL-4 signalling, its absence potentially converts the IL-13Rα1b into a receptor that can only signal via IL-4 ligation. The existence of duplicated receptor genes perhaps suggests that more ligands still remain to be discovered that will bind these receptors. The duplicated receptors are differentially expressed in most tissues and cell lines examined, and their expression can be modulated by LPS, polyIC and IFN-γ in cell lines. In contrast, the T-cell stimulant phytohaemagglutinin increased the expression of IL-4Rα1 and IL-4Rα2, but not IL-13Rα1/2, suggesting a role of an IL-4-like molecule in T-cell growth/activation in fish.
The aim of this study is to investigate the induction of interleukin-34 (IL-34) and macrophage colony-stimulating factor (M-CSF) mRNA by inflammatory cytokines and the involvement of mitogen-activated protein kinases (MAPKs) in this signaling pathway in human osteoblasts as both IL-34 and M-CSF bind to the same receptor c-FMS. Among four inflammatory cytokines [(IL-1β, IL-6, IL-17, and tumor necrosis factor-α (TNF-α)], IL-34 mRNA expression level was dramatically induced by IL-1β (17-fold) and TNF-α (74-fold). IL-1β and TNF-α activated the intracellular mitogen-activated protein kinases (MAPKs): p44/42 MAPK, p38, and c-Jun N-terminal kinase (JNK) as well as nuclear factor-κB (NF-κB) in osteoblasts. IL-1β- and TNF-α-mediated induction of IL-34 mRNA expression was decreased by JNK inhibitor. Interestingly, although treatment of MEK-1/2 inhibitor showed no reduction in the increase of IL-34 mRNA expression by cytokines, combination of MEK-1/2 inhibitor and JNK inhibitor significantly inhibited IL-1β- and TNF-α-mediated IL-34 mRNA expression level compared to those by each inhibitor alone. On the other hand, M-CSF mRNA expression level was significantly induced by both IL-1β and TNF-α by up to 7- and 11-fold, respectively. IL-1β- and TNF-α-mediated induction of M-CSF mRNA was not affected by p38, JNK, and MEK-1/2 inhibitors. However, NF-κB inhibitor completely inhibited the elevation of M-CSF mRNA expression by these cytokines. These results showed that proinflammatory cytokines, IL-1β and TNF-α, induced the expression of IL-34 mRNA via JNK and p44/42 MAPK but not p38 in human osteoblasts while p38, JNK, and p44/42 MAPK were not involved in the induction of M-CSF mRNA expression by these cytokines.
In mammals, IL-21 is a common γ chain cytokine produced by activated CD4(+) T cells and NKT cells that acts on multiple lineages of cells. Although IL-21 has also been discovered in birds, amphibians, and fish, to date, no functional studies have been reported for any nonmammalian IL-21 molecule. We have sequenced an IL-21 gene (tIL-21) in rainbow trout, which has a six-exon/five-intron structure, is expressed in immune tissues, and is induced by bacterial and viral infection and the T cell stimulant PHA. In contrast to mammals, calcium ionophore and PMA act synergistically to induce tIL-21. Recombinant tIL-21 (rtIL-21) induced a rapid and long-lasting (4-72 h) induction of expression of IFN-γ, IL-10, and IL-22, signature cytokines for Th1-, Th2-, and Th17-type responses, respectively, in head kidney leukocytes. However, rtIL-21 had little effects on the expression of other cytokines studied. rtIL-21 maintained the expression of CD8α, CD8β, and IgM at a late stage of stimulation when their expression was significantly decreased in controls and increased the expression of the Th cell markers CD4, T-bet, and GATA3. Intraperitoneal injection of rtIL-21 confirmed the in vitro bioactivity and increased the expression of IFN-γ, IL-10, IL-21, IL-22, CD8, and IgM. Inhibition experiments revealed that the activation of JAK/STAT3, Akt1/2, and PI3K pathways were responsible for rtIL-21 action. This study helps to clarify the role of IL-21 in lower vertebrates for the first time, to our knowledge, and suggests IL-21 is a likely key regulator of T and B cell function in fish.
The aim of this study is to investigate if macrophage-colony stimulating factor (M-CSF) or interleukin-34 (IL-34) induces cytokines or chemokines using human whole blood (HWB) and if an M-CSF- or IL-34-induced cytokine or chemokine production from HWB is inhibited by soluble M-CSF receptor or c-FMS kinase inhibitors. Among eight cytokines or growth factors tested, only IL-6 level was increased by up to 6-fold by M-CSF or IL-34 in HWB. In contrast, chemokine levels (IP-10/CXCL10, IL-8/CXCL8, and MCP-1/CCL2) were dramatically increased by M-CSF or IL-34 in HWB while exhibiting a large variation among donors. Variability of the MCP-1 signal induced by M-CSF or IL-34 was relatively less among donors compared to the IP-10 and IL-8 signals. The elevation of these chemokine levels was significantly decreased by soluble M-CSF receptor, indicating the elevation of these chemokines was mediated by M-CSF or IL-34. Furthermore, GW2580, a c-FMS kinase inhibitor, inhibited the induction of MCP-1 by M-CSF or IL-34 in a concentration dependent manner. These indicate MCP-1 is the most appropriate chemokine target for a chemokine release assay to evaluate the potency of c-FMS kinase inhibitors and MCP-1 release assay using HWB would be useful, relevant tool for translational pharmacology of c-FMS kinase inhibitors.
The polarization of naïve CD4+ T cells to T helper (Th)1 or Th2 cells is specified by two master transcription factors, T-bet and GATA3, and is an essential feature of mammalian adaptive immune responses to pathogens and the development of long-lasting immunity. We report here the cloning of rainbow trout Oncorhynchus mykiss T-bet and GATA3, to allow the future evaluation of the existence of Th1 and Th2 cells in salmonid fish. The trout T-bet translation shares high amino acid identities to other fish T-bet molecules (71–72%) but low identities to mammalian T-bet genes (41–42%), although the middle T-box DNA binding domain is highly conserved among all the T-bet proteins from fish and mammals. The trout GATA3 has high amino acid sequence identities (73–88%) to all known vertebrate molecules, with two highly conserved zinc finger motifs. The identity of the trout T-bet and GATA3 molecules was confirmed by phylogenetic tree analysis.
Macrophage colony-stimulating factor (M-CSF) regulates the production, survival and function of macrophages through Fms, the receptor tyrosine kinase. Recently, interleukin-34 (IL-34), which shares no sequence homology with M-CSF, was identified as an alternative Fms ligand. Here, we provide the first evidence that these ligands indeed resemble but are not necessarily identical in biological activity and signal activation. In culture systems tested, IL-34 and M-CSF showed an equivalent ability to support cell growth or survival. However, they were different in the ability to induce the production of chemokines such as MCP-1 and eotaxin-2 in primary macrophages, the morphological change in TF-1-fms cells and the migration of J774A.1 cells. Importantly, IL-34 induced a stronger but transient tyrosine phosphorylation of Fms and downstream molecules, and rapidly downregulated Fms. Even in the comparison of active domains, these ligands showed no sequence homology including the position of cysteines. Interestingly, an anti-Fms monoclonal antibody (Mab) blocked both IL-34-Fms and M-CSF-Fms binding, but another MAb blocked only M-CSF-Fms binding. These results suggested that IL-34 and M-CSF differed in their structure and Fms domains that they bound, which caused different bioactivities and signal activation kinetics/strength. Our findings indicate that macrophage phenotype and function are differentially regulated even at the level of the single receptor, Fms.
Interleukin-34 (IL-34) is a newly discovered regulator of myeloid lineage differentiation, proliferation, and survival, acting via the macrophage-colony stimulating factor receptor (M-CSF receptor, c-fms). M-CSF, the main ligand for c-fms, is required for osteoclastogenesis and has been already identified as a critical contributor of the pathogenesis of giant cell tumours of bone (GCTs), tumours rich in osteoclasts. According to the key role of M-CSF in osteoclastogenesis and GCTs, the expression of IL-34 in human GCTs was first assessed. Quantitative analysis of IL-34 mRNA expression in 14 human GCTs revealed expression of this cytokine in GCTs as well as M-CSF and c-fms. Immunohistochemistry demonstrated that osteoclast-like cells exhibited a huge immunostaining for IL-34 and that mononuclear stromal cells were slightly positive for this protein. In contrast to osteoblasts, bone-resorbing osteoclasts showed very strong staining for IL-34, suggesting its potential role in the pathogenesis of GCTs by facilitating osteoclast formation. The role of IL-34 in osteoclastogenesis was then studied in murine and human models. IL-34 was able to support RANKL-induced osteoclastogenesis in the absence of M-CSF in all models. Multinucleated cells generated in the presence of IL-34 and RANKL expressed specific osteoclastic markers and resorbed dentine. IL-34 induced phosphorylation of ERK 1/2 and Akt through the activation of c-fms, as revealed by the inhibition of signalling by a specific c-fms tyrosine kinase inhibitor. Furthermore, IL-34 stimulated RANKL-induced osteoclastogenesis by promoting the adhesion and proliferation of osteoclast progenitors, and had no effect on osteoclast survival. Overall, these data reveal that IL-34 can be entirely substituted for M-CSF in RANKL-induced osteoclastogenesis, thus identifying a new biological activity for this cytokine and a contribution to the pathogenesis of GCTs.
These studies demonstrate that CSF-1 and IL-34 are conserved in birds, and uses evolutionary comparisons to infer structure function relationships among vertebrate animals.
Macrophages are involved in many aspects of development, host defense, pathology, and homeostasis. Their normal differentiation, proliferation, and survival are controlled by CSF-1 via the activation of the CSF1R. A recently discovered cytokine, IL-34, was shown to bind the same receptor in humans. Chicken is a widely used model organism in developmental biology, but the factors that control avian myelopoiesis have not been identified previously. The CSF-1, IL-34, and CSF1R genes in chicken and zebra finch were identified from respective genomic/cDNA sequence resources. Comparative analysis of the avian CSF1R loci revealed likely orthologs of mammalian macrophage-specific promoters and enhancers, and the CSF1R gene is expressed in the developing chick embryo in a pattern consistent with macrophage-specific expression. Chicken CSF-1 and IL-34 were expressed in HEK293 cells and shown to elicit macrophage growth from chicken BM cells in culture. Comparative sequence and co-evolution analysis across all vertebrates suggests that the two ligands interact with distinct regions of the CSF1R. These studies demonstrate that there are two separate ligands for a functional CSF1R across all vertebrates.
Specialized phagocytes are found in the most primitive multicellular organisms. Their roles in homeostasis and in distinguishing self from non-self have evolved with the complexity of organisms and their immune systems. Equally important, but often overlooked, are the roles of macrophages in tissue development. As discussed in this Review, these include functions in branching morphogenesis, neuronal patterning, angiogenesis, bone morphogenesis and the generation of adipose tissue. In each case, macrophage depletion impairs the formation of the tissue and compromises its function. I argue that in several diseases, the unrestrained acquisition of these developmental macrophage functions exacerbates pathology. For example, macrophages enhance tumour progression and metastasis by affecting tumour-cell migration and invasion, as well as angiogenesis.
In addition to being the respiratory organ in fish, the gills form a barrier against the external milieu. Innate and adaptive immune system components have been detected in the gills, but lymphoid cell accumulations similar to that seen in the mammalian mucosa have not been described. The present investigations revealed cell accumulations on the caudal edge of interbranchial septum at the base of the gill filaments in the Atlantic salmon. Cytokeratin immunohistochemical staining and identification of a basal membrane and desmosome cell junctions by electron microscopy showed that the cell accumulation was located intraepithelially. Major histocompatibility complex (MHC) class II+ cells were detected by immunohistochemistry, and laser capture micro-dissection and subsequent RT-PCR analysis revealed expression of T-cell receptor transcripts in the investigated tissue, suggesting the presence of T cells. The intraepithelial tissue reported here may be a suitable location for immune surveillance of gill infections, as well as a target site for new vaccine approaches and investigations of epithelial immunity. This is the first description of a lymphocyte cell aggregation within a teleostian gill epithelium network, illustrating a phylogenetically early form of leukocyte accumulations in a respiratory organ.
We report on the regulation of pro-inflammatory functions of goldfish macrophages and induction of gene expression by recombinant goldfish CSF-1 (rgCSF-1). Recombinant goldfish TNFalpha-2 (rg TNFalpha-2), rgIFNgamma but not rgTGFbeta induced time-dependent increase of CSF-1 expression in macrophages. Treatment of goldfish macrophages with rgCSF-1 increased expression of several immune genes including CXCL-8 (=IL-8), CCL-1, TNFalpha-1, TNFalpha-2, IL-1beta-1, IL-1beta-2, IL-12-p35, IL-12-p40, IFN, IL-10 and iNOS A and B. The rgCSF-1 treatment did not significantly alter the mRNA levels of TGFbeta and NRAMP in macrophages up to 48h post treatment. However, at 72h post treatment, the expression of TGFbeta increased whereas that of NRAMP decreased. The treatment of macrophages with rgCSF-1 enhanced their respiratory burst and nitric oxide responses that were abrogated after addition of soluble CSF-1 receptor (sCSF-1R) to cell cultures. Macrophages exhibited a concentration-dependent chemotactic response toward rgCSF-1 as well as an increase in phagocytic activity that was abrogated after addition of sCSF-1R to cell cultures. Our results indicate that in addition to being an important growth factor of goldfish macrophages, rgCSF-1 also plays a central role in the regulation of their pro-inflammatory responses.
Mammalian macrophage CSF (MCSF; CSF-1) is the primary regulator of the mononuclear phagocyte lineage. We, for the first time, report the complete sequencing of five MCSF cDNAs from three fish species, rainbow trout, zebrafish, and goldfish. Despite the difference in the lengths of the MCSF transcripts, all of the fish MCSF molecules encode a signal peptide, a CSF-1 domain, a transmembrane domain, and an intracellular region. Each fish MCSF gene has a unique exon/intron structure. The primordial MCSF gene may have had a nine exon/eight intron structure. In this model, insertion of an intron in exon 6 in primitive fish created the fish type I MCSF, while the loss of this exon or part of the original exon 6 created the fish type II MCSF. Investigation of alternative splicing variants in trout suggests that no mammalian equivalent splice variants exist. The two trout MCSF genes are differentially expressed in vivo and contributed differently to the high-level expression of MCSF in spleen and head kidney. In contrast to the up-regulation of MCSF by PMA in mammals, in trout MCSF1 expression is down-regulated by PMA treatment. As in mammals, recombinant trout MCSF1 can promote the growth of head kidney leukocytes, and it up-regulates the expression of CXCR3 in head kidney macrophages, with the latter suggesting a role of MCSF in the trafficking of macrophages to sites of inflammation or injury where the CXCR3 ligands are expressed. Thus MCSF has an important role in the immune system of fish as in mammals.
Macrophage colony-stimulating factor (M-CSF) triggers the development of cells of the monocyte-macrophage lineage and has
a variety of stimulatory effects on mature cells of this class. The biologically active form of M-CSF is a disulfide-linked
dimer that activates an intrinsic tyrosine kinase activity on the M-CSF receptor by inducing dimerization of the receptor
molecules. The structure of a recombinant human M-CSF dimer, determined at 2.5 angstroms by x-ray crystallography, contains
two bundles of four alpha helices laid end-to-end, with an interchain disulfide bond. Individual monomers of M-CSF show a
close structural similarity to the cytokines granulocyte-macrophage colony-stimulating factor and human growth hormone. Both
of these cytokines are monomeric in their active form, and their specific receptors lack intrinsic tyrosine kinase activity.
The similarity of these structures suggests that the receptor binding determinants for all three cytokines may be similar.
Bioassay and northern blot analyses revealed that, among several functional murine macrophage (M phi) clones, lipopolysaccharide (LPS) stimulation generated in distinct induction levels of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage colony-stimulating factor (M-CSF). When compared with these induction profiles, the M phi clones could be classified into two types; G type (G-CSF GM-CSF-M-CSF+) and GM type (G-CSF +/- GM-CSF M-CSF+) of M phi clones. Unlike G-CSF and GM-CSF that were inducible factors, M-CSF mRNA was constitutively expressed without stimulation and was differentially controlled between the G and GM types; LPS induction decreased M-CSF mRNA in the former, but increased it in the latter. Further northern blot analysis revealed that interferon-gamma (IFN-gamma) suppressed constitutive expression of M-CSF mRNA, and that costimulation with both LPS and IFN-gamma reduced expression of G-CSF and M-CSF mRNA in the G type of M phi clone, but induced higher expression of GM-CSF and M-CSF mRNAs in the GM type of M phi clone compared with LPS alone. However, in either case, IFN-gamma completely inhibited LPS-induced production of active CSF of the M phi clones, which was observed even in IFN-gamma pretreatment, and also abrogated autoactivation of GM-CSF. Our present results suggested that murine M phi clones had differentially regulated expression of CSFs and that IFN-gamma had a regulatory function of inhibiting CSF production of murine M phi s.
We used a panel of monoclonal antibodies and immunocytochemistry to identify macrophages and dendritic cells in mice that are deficient in macrophage colony stimulating factor (M-CSF or CSF-1) because of the recessive osteopetrotic (op/op) mutation. Prior work had shown that osteopetrosis is associated with a lack of osteoclasts, phagocytic cells required for remodelling in bone. Additional macrophage populations proved to be very M-CSF dependent. op/op mice had few and sometimes no peritoneal cavity phagocytes, splenic marginal zone metallophils, and lymph node subcapsular sinus macrophages. Other populations, however, reached substantial levels in the absence of M-CSF, including phagocytes in the thymic cortex, splenic red pulp, lymph node medulla, intestinal lamina propria, liver (Kupffer cells), lung (alveolar macrophages) and brain (microglia). Dendritic cells, which are specialized accessory cells for T-dependent immune responses and tolerance, were readily identified in skin and in the T-dependent regions of spleen, lymph node and Peyer's patch. The identification of dendritic cells utilized antibodies to MHC class II products and four different antigens that are primarily expressed by these accessory cells. Our findings indicate that only a few macrophage populations are critically dependent upon M-CSF in vivo. With respect to dendritic cells, the data are consistent with prior in vitro work where it was noted that GM-CSF but not M-CSF supported dendritic cell viability, function and growth.
Sixteen polycyclic aromatic hydrocarbons (PAHs) were screened for their ability to be photocytotoxic to a cell line from the rainbow trout gill, RTgill-W1. PAHs could be divided into one of three groups: incapable of being photocytotoxic, able to be both photocytotoxic and directly cytotoxic, or capable of being only photocytotoxic. Photocytotoxicity was distinct from direct cytotoxicity in that EC50 values were lower with the neutral red assay immediately after the PAH/UV treatment than with alamar Blue or CFDA-AM, indicating a more specific action on lysosomes. As well, in photocytotoxicity but not in direct cytotoxicity, the three assays showed increased impairment 24 h after treatment. Most PAHs were found to be strictly photocytotoxic; however, only six compounds were photocytotoxic at concentrations theoretically achievable in water. When photocytotoxic PAHs were ranked relative to fluoranthene to establish fluoranthene equivalent factors (FEFs), benzo[a]pyrene and benzo[g,h,i]perylene were found to be most potent. However, when the water solubility of each compound was taken into account in order to calculate the potential environmental photocytotoxic potency (PEPP), fluoranthene and pyrene appeared to have the most potential to impact fish through photocytotoxicity.
The predicted rainbow trout mature interleukin-1 beta (IL-1 beta) peptide has been produced as a recombinant protein in E. coli. The bioactivity of this molecule has been studied using trout head kidney cell preparations and a trout macrophage cell line (RTS11). Trout rIL-1 beta was shown to increase the expression level of IL-1 beta, cyclooxygenase (COX2) and MHC class II beta chain transcription, as determined by Northern blot analysis. Stimulatory doses of rIL-1 beta were typically > or =10 ng/ml. Induction of IL-1 beta expression occurred within 1h post-stimulation with trout rIL-1 beta and was maximal 3-6h post-stimulation. Trout rIL-1 beta was also able to increase murine D10.G4.1 cell proliferation and trout head kidney leukocyte phagocytic activity, in a dose-dependent manner. However, equivalent D10.G4.1 cell proliferation was induced with approximately 1000-fold lower doses of human rIL-1 beta. That LPS contamination did not contribute to the effects seen was confirmed by determining its concentration in the trout rIL-1 beta preparation, and demonstrating that the rIL-1 beta activity was inhibited by heating or pre-incubation with a polyclonal anti-trout rIL-1 beta antibody.
An established line of fish cells has been propagated in vitro for 21 months and 48 subcultivations. Important characteristics
of the cells are described.
Three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway have been fully sequenced and their expression investigated in rainbow trout (Oncorhynchus mykiss). Trout C1r cDNA encodes a 707-amino-acid (aa) protein with a theoretical M r of 77,200. The trout translation shows highest homology with carp C1r/s, and lower, equal homologies to mammalian C1r and C1s, and MASPs from other vertebrate species. However, phylogenetic analysis and structural features suggest that the trout sequence, together with the two carp sequences, are the orthologues of mammalian C1r. The trout C4 cDNA encodes a 1,724-aa protein with a theoretical M r of 192,600. The trout translation shows higher homologies to the carp C4B and medaka C4, but lower homologies to C4 from other species and the carp C4A. It has a predicted signal peptide of 22 aa, a α-chain of 773 aa, a β-chain of 635 aa and a λ-chain of 288 aa. Trout C1 inhibitor cDNA encodes a 611-aa protein with a theoretical M r of 68,700. The trout translation has a C-terminal serpin domain with high homologies with mammalian counterparts (~37% identities), and a longer N-terminus, with no significant homology to other serpins, which contains two Ig-like domains. A molecule containing two Ig-like domains followed by a serpin domain, has also been found in an EST clone from another bony fish, the Japanese flounder. This suggests a unique structural feature of C1 inhibitor in fish. The functional significance of the Ig domains is discussed. The liver is the major site of expression of the three trout complement components, C1r, C4 and C1 inhibitor, although their expression is also detectable in other tissues. The extra-hepatic expression of complement genes may be important for local protection and inflammatory responses. Low-level constitutive expression of the three components was also detectable in a trout monocyte/macrophage cell line RTS-11, but only the expression of C4 could be upregulated by LPS.
Proliferative kidney disease (PKD) is a parasitic infection of salmonid fish characterized by an apparently abnormal immune response to the presence of the myxozoan parasite, Tetracapsuloides bryosalmonae. In order to examine the nature of the immune response at the molecular level, the expression of a range of immune regulatory genes, including cytokines and cyclooxygenase (COX)-2 was examined in naive unexposed fish and in naive fish exposed to parasite-infected water at three points during the course of a natural outbreak of PKD. Since fish with advanced PKD pathology generally exhibit increased susceptibility to secondary infections which is typical of stress/cortisol-mediated immune suppression, a further aim of this work was to examine in vitro the influence of the glucocorticoid cortisol on the bacterial lipopolysaccharide (LPS)-induced expression of the trout cytokine genes studied. Two weeks after the initial sampling, naive exposed fish showed a specific profile of up-regulated tumor necrosis factor (TNF)-alpha2, COX-2 and, to a lesser extent, transforming growth factor (TGF)-beta1 expression. As the disease pathology increased, TNF-alpha2 and COX-2 expression returned to normal levels. Stress levels of cortisol suppressed the LPS inducibility of pro-inflammatory cytokine genes, although TGF-beta1 and TNF-alpha2 appeared to be refractory. These data demonstrate that specific immune responses at the molecular level are affected during PKD infection, with the cortisol suppression of cytokine expression in vitro providing a possible link to PKD-mediated cytokine down-regulation and immune suppression.