Evolution of class I cytokine receptors

Background
The Class I cytokine receptors have a wide range of actions, including a major role in the development and function of immune and blood cells. However, the evolution of the genes encoding them remains poorly understood. To address this we have used bioinformatics to analyze the Class I receptor repertoire in sea squirt (Ciona intestinalis) and zebrafish (Danio rerio).
Results
Only two Class I receptors were identified in sea squirt, one with homology to the archetypal GP130 receptor, and the other with high conservation with the divergent orphan receptor CLF-3. In contrast, 36 Class I cytokine receptors were present in zebrafish, including representative members for each of the five structural groups found in mammals. This allowed the identification of 27 core receptors belonging to the last common ancestor of teleosts and mammals.
Conclusion
This study suggests that the majority of diversification of this receptor family occurred after the divergence of urochordates and vertebrates approximately 794 million years ago (MYA), but before the divergence of ray-finned from lobe-finned fishes around 476 MYA. Since then, only relatively limited lineage-specific diversification within the different Class I receptor structural groups has occurred.

Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression

Background: RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express short-hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter.

Results: We identified and characterised the ch7SK promoter sequence upstream of the full-length 7SK small nuclear RNA (snRNA) sequence in the chicken genome and used this to construct vectors to express shRNAs targeting enhanced green fluorescent protein (EGFP). We transfected chicken DF-1 cells with these constructs and found that anti-EGFP-shRNAs (shEGFP) expressed from the ch7SK promoter could induce efficient knockdown of EGFP expression. We further compared the efficiency of ch7SK-directed knockdown to that of chicken U6 (cU6) promoters and found that the efficiency of the ch7SK promoter was not greater than, but comparable to the efficiency of cU6 promoters.

Conclusion: In this study we have demonstrated that the ch7SK promoter can express shRNAs capable of mediating efficient RNAi in a chicken cell line. However, our finding that RNAi driven by the ch7SK promoter is not more efficient than cU6 promoters contrasts previous comparisons of mammalian U6 and 7SK promoters. Since the ch7SK promoter is the first non-mammalian vertebrate 7SK promoter to be characterised, this finding may be helpful in understanding the divergence of pol III promoter activities between mammalian and non-mammalian vertebrates. This aside, our results clearly indicate that the ch7SK promoter is an efficient alternative to U6-based shRNA expression systems for inducing efficient RNAi activity in chicken cells.

Blood-stage Plasmodium infection induces CD8+ T lymphocytes to parasite-expressed antigens, largely regulated by CD8α+ dendritic cells

Although CD8+ T cells do not contribute to protection against the blood stage of Plasmodium infection, there is mounting evidence that they are principal mediators of murine experimental cerebral malaria (ECM). At present, there is no direct evidence that the CD8+ T cells mediating ECM are parasite-specific or, for that matter, whether parasite-specific CD8+ T cells are generated in response to blood-stage infection. To resolve this and to define the cellular requirements for such priming, we generated transgenic P. berghei parasites expressing model T cell epitopes. This approach was necessary as MHC class I-restricted antigens to blood-stage infection have not been defined. Here, we show that blood-stage infection leads to parasite-specific CD8+ and CD4+ T cell responses. Furthermore, we show that P. berghei-expressed antigens are cross-presented by the CD8α+ subset of dendritic cells (DC), and that this induces pathogen-specific cytotoxic T lymphocytes (CTL) capable of lysing cells presenting antigens expressed by blood-stage parasites. Finally, using three different experimental approaches, we provide evidence that CTL specific for parasite-expressed antigens contribute to ECM.

Identification of novel genes differentially expressed in haemopoietic progenitor cells

The biochemical and molecular processes that maintain the stem cell pool, and govern the proliferation and differentiation of haemopoietic stem/progenitor cells (HSPCs) have been widely investigated but are incompletely understood. The purpose of this study was to identify and characterise novel genes that may play a part in regulating the mechanisms that control the proliferation, differentiation and self-renewal of human HSPCs. Reverse transcription differential display polymerase chain reaction (dd-PCR) was used to identify differences in gene expression between a HSPC population defined by expression of the CD34 phenotype, and the more mature CD34 depleted populations. A total of 6 differentially expressed complementary deoxyribonucleic acid (cDNA) sequences were identified. Four of these transcripts were homologous to well characterised genes, while two (band 1 and band 20) were homologous to unknown and uncharacterised partial gene sequences on the GenBank database and were thus chosen for further investigation. The partial cDNA sequences for band 1 and band 20 were designated ORP-3 and MERP-1 (respectively) due to homologies with other well-characterised gene families. Differential expression of the ORP-3 and MERP-1 genes was confirmed using Taqman™ real-time polymerase chain reaction (PCR) with 3 - 4-fold and 4-10 -fold higher levels in the CD34+ fractions of haemopoietic cells compared to CD34- populations respectively. Additionally, expression of both these genes was down regulated with proliferation and differentiation of CD34+ cells further confirming higher expression in a less differentiated subset of haemopoietic cells. The full coding sequences of ORP-3 and MERP-1 were elucidated using bioinformatics, rapid amplification of cDNA ends (RACE) and PCR amplification. The MERP-1 cDNA is 2600 nucleotides (nt) long, and localizes by bioinformatics to chromosome 7.. It consists of three exons and 2 introns spanning an entire length of 31.4 kilobases (kb). The MERP-1 open reading frame (ORF) codes for a putative 344 amino acid (aa) type II transmembrane protein with an extracellular C-terminal ependymin like-domain and an intracellular N-terminal sequence with significant homology to the cytoplasmic domains of members of the protocadherin family of transmembrane glycoproteins. Ependymins and protocadherins are well-characterised calcium-dependant cell adhesion glycoproteins. Although the function of MERP-1 remains to be elucidated, it is possible that MERP-1 like its homologues plays a role in calcium dependent cell adhesion. Differential expression of the MERP-1 gene in haemopoietic cells suggests a role in haemopoietic stem cell proliferation and differentiation, however, its broad tissue distribution implies that it may also play a role in many cell types. Characterization of the MERP-1 protein is required to elucidate these possible roles. The ORP-3 cDNA is 6631nt long, and localizes by bioinformatics to chromosome 7pl5-p21. It consists of 23 exons and 22 introns spanning an entire length of 183.5kb. The ORP-3 ORF codes for a putative 887aa protein which displays the consensus sequence for a highly conserved oxysterol-binding domain. Other well-characterised proteins expressing these domains have been demonstrated to bind oxysterols (OS) in a dose dependant fashion. OS are hydroxylated derivatives of cholesterol Their biological activities include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, including haemopoietic cells. Differential expression of the ORP-3 gene in haemopoietic cells suggests a possible role in the transduction of OS effects on haemopoietic cells, however, its broad tissue distribution implies that it may also play a role in many cell types. Further investigation of ORP-3 gene expression demonstrates a significant correlation with CD34+ sample purity, and 2-fold higher expression in a population of haemopoietic cells defined by the CD34+38- phenotype compared to more mature CD34+38+ cells. This finding, taken together with the previous observation of down-regulation of ORP-3 expression with proliferation and differentiation of CD34+ cells, indicates that ORP-3 expression may be higher in a less differentiated subset of cells with a higher proliferative capacity. This hypothesis is supported by the observation that expression of the ORP-3 gene is approximately 2-fold lower in differentiated HL60 promyelocytic cells compared to control, undifferentiated cells. ORP-3 expression in HL60 cells during normal culture conditions was also found to vary with expression positively correlated with cell number. This indicates a possible cell cycle effect on ORP-3 gene expression with levels highest when cell density, and therefore the percentage of cells in G(0)/G(1) phase of the cell cycle is highest. This observation also correlates with the observation of higher ORP-3 expression in CD34+38-cells, and in CD34+ and HL60 cells undergoing OS induced and camptothecin induced apoptosis that is preceded by cell cycle arrest at G(0)/G(1). Expression of the ORP-3 gene in CD34+ HSPCs from UCB was significantly decreased to approximately half the levels observed in control cells after 24 hours incubation in transforming growth factor beta-1 (TGFâl). As ≥90% of these cells are stimulated into cell cycle entry by TGFâl, this observation further supports the hypothesis that ORP-3 expression is highest when cells reside in the G(0)/G(1) phase of the cell cycle. Data obtained from investigation of ORP-3 gene expression in synchronised HL60 cells however does not support nor disprove this hypothesis. Culture of CD34+ enriched HSPCs and HL60 cells with 25-OHC significantly increased ORP-3 gene expression to approximately 1.5 times control levels. However, as 25-OHC treatment also increased the percentage of apoptotic cells in these experiments, it is not valid to make any conclusions regarding the regulation of ORP-3 gene expression by OS. Indeed, the observation that camptothecin induced apoptosis also increased ORP-3 gene expression in HL60 cells raises the possibility that up-regulation of ORP-3 gene expression is also associated with apoptosis, Taken together, expression of the ORP-3 gene appears to be regulated by differentiation and apoptosis of haemopoietic progenitors, and may also be positively associated with proliferative and G(0)/G(1) cell cycle status indicating a possible role in all of these processes. Given the important regulatory role of apoptosis in haemopoiesis and differential expression of the ORP-3 gene in haemopoietic progenitors, final investigations were conducted to examine the effects OS on human HSPCs. Granulocyte/macrophage colony forming units (CFU-GM) generated from human bone marrow (ABM) and umbilical cord blood (UCB) were grown in the presence of varying concentrations of three different OS - 7keto-cholesterol (7K-C), 7beta-hydroxycholesterol (7p-OHC) and 25-hydroxycholesterol (25-OHC). Similarly, the effect of OS on HL60 and CD34+ cells was investigated using annexin-V staining and flow cytometry to measure apoptosis. Reduction of nitroblue tetrazolium (NBT) was used to assess differentiative status of HL60 cells. CFU-GM from ABM and HL60 growth was inhibited by all three OS tested, with 25-OHC being the most potent. 25-OHC inhibited ≥50% of bone marrow CFU-GM and ≥95% of HL60 cell growth at a level of 1 ug/ml. Compared to UCB, CFU-GM derived from ABM were more sensitive to the effects of all OS tested. Only 25-OHC and 7(5-OHC significantly inhibited growth of UCB derived CFU-GM. OS treatment increased the number of annexin-V CD34+ cells and NBT positive HL60 cells indicating that OS inhibition of CFU-GM and HL60 cell growth can be attributed to induction of apoptosis and differentiation. From these studies, it can be concluded that dd-PCR is an excellent tool for the discovery of novel genes expressed in human HSPCs. Characterisation of the proteins encoded by the novel genes ORP-3 and MERP-1 may reveal a regulatory role for these genes in haemopoiesis. Finally, investigations into the effects of OS on haemopoietic progenitor cells has revealed that OS are a new class of inhibitors of HSPC proliferation of potential relevance in vivo and in vitro.

Targeting Hsp90 with small molecule inhibitors induces the over-expression of the anti-apoptotic molecule, survivin, in human A549, HONE-1 and HT-29 cancer cells

Survivin is a dual functioning protein. It inhibits the apoptosis of cancer cells by inhibiting caspases, and also promotes cancer cell growth by stabilizing microtubules during mitosis. Since the molecular chaperone Hsp90 binds and stabilizes survivin, it is widely believed that down-regulation of survivin is one of the important therapeutic functions of Hsp90 inhibitors such as the phase III clinically trialed compound 17-AAG. However, Hsp90 interferes with a number of molecules that up-regulate the intracellular level of survivin, raising the question that clinical use of Hsp90 inhibitors may indirectly induce survivin expression and subsequently enhance cancer anti-drug responses. The purpose of this study is to determine whether targeting Hsp90 can alter survivin expression differently in different cancer cell lines and to explore possible mechanisms that cause the alteration in survivin expression.

Evolution of class I cytokine receptor signalling

Cytokines are proteins that provide essential signals to blood and immune cells. The evolution of this system was determined from primitive organisms to humans and zebrafish. Analysis of zebrafish granulocyte colony-stimulating factor (GCSF) signalling revealed broad conservation of function with mammals and a novel role in white blood cell migration.

CD1d-restricted NKT cells contribute to malarial splenomegaly and enhance parasite-specific antibody responses

CD1d-restricted NKT cells are a novel T cell lineage with unusual features. They co-express some NK cell receptors and recognize glycolipid antigens through an invariant T cell receptor (TCR) in the context of CD1d molecules. Upon activation through the TCR, NKT cells produce large amounts of IFN- and IL-4. It has been proposed that rapid cytokine output by activated NKT cells may induce bystander activation of other lymphoid lineages. The impact of CD1d-restricted NKT cell activation in the induction of B cell-mediated immune responses to infection is still unclear. We show here that CD1-restricted NKT cells contribute to malarial splenomegaly associated with expansion of the splenic B cell pool and enhance parasite-specific antibody formation in response to Plasmodium berghei infection. The increased B cell-mediated response correlates with the ability of NKT cells to promote Th2 immune responses. Additionally, antibody responses against the glycosylphosphatidylinositol (GPI)-anchored protein merozoite surface protein 1 (MSP-1) were found to be significantly lower in CD1-/- mice compared to wild-type animals. P. berghei-infected MHC class II (MHCII)-/- mice also generated antibodies against MSP-1, suggesting that antibody production against GPI-anchored antigens in response to malaria infection can arisefrom both MHCII-dependent and independent pathways.

Systemic activation of dendritic cells by toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity

The mechanisms responsible for the immunosuppression associated with sepsis or some chronic blood infections remain poorly understood. Here we show that infection with a malaria parasite (Plasmodium berghei) or simple systemic exposure to bacterial or viral Toll-like receptor ligands inhibited cross-priming. Reduced cross-priming was a consequence of downregulation of cross-presentation by activated dendritic cells due to systemic activation that did not otherwise globally inhibit T cell proliferation. Although activated dendritic cells retained their capacity to present viral antigens via the endogenous major histocompatibility complex class I processing pathway, antiviral responses were greatly impaired in mice exposed to Toll-like receptor ligands. This is consistent with a key function for cross-presentation in antiviral immunity and helps explain the immunosuppressive effects of systemic infection. Moreover, inhibition of cross-presentation was overcome by injection of dendritic cells bearing antigen, which provides a new strategy for generating immunity during immunosuppressive blood infections.

Differential tumor necrosis factor receptor 2-mediated editing of virus-specific CD8+ effector T cells

Much of the CD8+ T cell response in H2b mice with influenza pneumonia is directed at the nucleoprotein366-374 (NP366) and acid polymerase224-233 (PA224) peptides presented by the H2Db MHC class I glycoprotein. These DbNP366- and DbPA224-specific T cell populations are readily analyzed by staining with tetrameric complexes of MHC+ peptide (tetramers) or by cytokine production subsequent to in vitro stimulation with the cognate peptides. The DbPA224-specific CD8+ effector T cells make more tumor necrosis factor (TNF) α than the comparable CD8+DbNP366+ set, a difference reflected in the greater sensitivity of the CD8+DbPA224+ population to TNF receptor (TNFR) 2-mediated apoptosis under conditions of in vitro culture. Freshly isolated CD8+DbNP366+ and CD8+DbPA224+ T cells from influenza-infected TNFR2-/- mice produce higher levels of IFN-γ and TNF-α after in vitro stimulation with peptide, although the avidity of the T cell receptor-epitope interaction does not change. Increased numbers of both CD8+DbPA224+ and CD8+DbNP366+ T cells were recovered from the lungs (but not the spleens) of secondarily challenged TNFR2-/- mice, a pattern that correlates with the profiles of TNFR expression in the TNFR2+/+ controls. Thus, it seems that TNFR2-mediated editing of influenza-specific CD8+ T cells functions to limit the numbers of effectors that have localized to the site of pathology in the lung but does not modify the size of the less activated responder T cell populations in the spleen. Therefore, the massive difference in magnitude for the secondary, although not the primary, response to these DbNP366 and DbPA224 epitopes cannot be considered to reflect differential TNFR2-mediated T cell editing.

Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins : Detection probes and affinity standards

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four CuI ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their CuI affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with CuI to yield distinct 1:2 chromatophoric complexes [CuIL2] 3- with formation constants β2 = 1017.2 and 1019.8 M-2, respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu+ concentrations from 10-12 to 10-19 M. Dtt binds CuI with KD∼10-15 M at pH 7, but it is air-sensitive, and its CuI affinity varies with pH. The CuI binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity CuI binding and the individual quantitative affinities (KD values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind CuI with sub-femtomolar affinities, consistent with tight control of labile Cu+ concentrations in living cells.

Genetic basis for the increased expression of vacuolar H+ translocating ATPase genes upon imatinib treatment in human lymphoblastoid cells

Purpose The role of v-ATPases in cancer biology is being increasingly recognized. Yeast studies indicate that the tyrosine kinase inhibitor imatinib may interact with the v-ATPase genes and alter the course of cancer progression. Data from humans in this regard are lacking.

Methods We constructed 55 lymphoblastoid cell lines from pedigreed, cancer-free human subjects and treated them with IC20 concentration of imatinib mesylate. Using these cell lines, we (i) estimated the heritability and differential expression of 19 genes encoding several subunits of the v-ATPase protein in response to imatinib treatment; (ii) estimated the genetic similarity among these genes; and (iii) conducted a high-density scan to find cis-regulating genetic variation associated with differential expression of these genes.

Results We found that the imatinib response of the genes encoding v-ATPase subunits is significantly heritable and can be clustered to identify novel drug targets in imatinib therapy. Further, five of these genes were significantly cis-regulated and together represented nearly half-log fold change in response to imatinib (p = 0.0107) that was homogenous (p = 0.2598).

Conclusions Our results proffer support to the growing view that personalized regimens using proton pump inhibitors or v-ATPase inhibitors may improve outcomes of imatinib therapy in various cancers.

Turnover of bone marrow-derived cells in the irradiated mouse cornea

In light of an increasing awareness of the presence of bone marrow (BM)-derived macrophages in the normal cornea and their uncertain role in corneal diseases, it is important that the turnover rate of these resident immune cells be established. The baseline density and distribution of macrophages in the corneal stroma was investigated in Cx3cr1gfp transgenic mice in which all monocyte-derived cells express enhanced green fluorescent protein (eGFP). To quantify turnover, BM-derived cells from transgenic eGFP mice were transplanted into whole-body irradiated wild-type recipients. Additionally, wild-type BM-derived cells were injected into irradiated Cx3cr1+/gfp recipients, creating reverse chimeras. At 2, 4 and 8 weeks post-reconstitution, the number of eGFP+ cells in each corneal whole mount was calculated using epifluorescence microscopy, immunofluorescence staining and confocal microscopy. The total density of myeloid-derived cells in the normal Cx3cr1+/gfp cornea was 366 cells/mm2. In BM chimeras 2 weeks post-reconstitution, 24% of the myeloid-derived cells had been replenished and were predominantly located in the anterior stroma. By 8 weeks post-reconstitution 75% of the myeloid-derived cells had been replaced and these cells were distributed uniformly throughout the stroma. All donor eGFP+ cells expressed low to moderate levels of CD45 and CD11b, with approximately 25% coexpressing major histocompatibility complex class II, a phenotype characteristic of previous descriptions of corneal stromal macrophages. In conclusion, 75% of the myeloid-derived cells in the mouse corneal stroma are replenished after 8 weeks. These data provide a strong basis for functional investigations of the role of resident stromal macrophages versus non-haematopoietic cells using BM chimeric mice in models of corneal inflammation.

Airway clearance of apoptotic cells in COPD

Apoptosis of bronchial epithelial cells and the phagocytic clearance of these cells by alveolar macrophages (a process termed efferocytosis) are integral processes leading to repair of airway epithelial injury. Efferocytosis allows for the removal of apoptotic material with minimal inflammation and prevents the development of secondary necrosis and ongoing inflammation. Defective efferocytosis and the increased presence of apoptotic cells have been identified in the airways of subjects with chronic obstructive pulmonary disease (COPD). There are three major potential causes for this accumulation of apoptotic cells: (i) increased apoptosis per se as a result of an increase in apoptotic mediators, (ii) defects in the recognition of apoptotic cells by AM and (iii) failure to clear the unwanted cells by the process of efferocytosis. The implications of these processes in COPD and novel treatment strategies aimed at improving clearance of apoptotic cells form the focus of the present review.

Characterization of zebrafish polymerase iii promoters for the expression of short hairpin RNA interference molecules

RNA interference (RNAi) is a powerful, sequence specific, and long-lasting method of gene knockdown, and can be elicited by the expression of short-hairpin RNA (shRNA) molecules driven via polymerase III type 3 promoters from a DNA vector or transgene. To further develop RNAi as a tool in zebrafish, we have characterized the zebrafish U6 and H1 snRNA promoters and compared the efficiency of each of the promoters to express an shRNA and silence a reporter gene, relative to previously characterized U6 promoters from pufferfish, chicken, and mouse. Our results show that the zebrafish polymerase III promoters were capable of effective gene silencing in the zebrafish ZF4 cell line, but were ineffective in mammalian Vero cells. In contrast, mouse and chicken promoters were active in Vero but not ZF4 cells, highlighting the importance of homologous promoters to achieve effective silencing.