Choline Transporter-like Protein Research Articles

Functional analysis of choline transporters in rheumatoid arthritis synovial fibroblasts

Objectives: In this study, we examined the functional characteristics of choline uptake and sought to identify the transporters in rheumatoid arthritis synovial fibroblasts (RASFs).Methods: The expression of choline transporters was evaluated by quantitative real-time PCR, western blotting, and immunocytochemistry. Time course, Na+-dependency, and kinetics of [3H]choline uptake were investigated. Effects of cationic drugs on the uptake of [3H]choline, cell viability, and caspase-3/7 activity were also examined. Finally, we investigated the influence of choline uptake inhibitor, hemicholinium-3 (HC-3), and choline deficiency on cell viability and caspase-3/7 activity.Results: Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in RASFs and were localized to the plasma membrane. [3H]Choline uptake occurred via a Na+-independent and pH-dependent transport system. The cells have two different [3H]choline transport systems, high- and low-affinity. Various organic cations, HC-3 and choline deficiency inhibited both [3H]choline uptake and cell viability, and enhanced the caspase-3/7 activity. The functional inhibition of choline transporters could promote apoptotic cell death. In RASFs, [3H]choline uptake was significantly increased compared with that in OASFs without a change in gene expression.Conclusions: These results suggest that CTL1 (high-affinity) and CTL2 (low-affinity) are highly expressed in RASFs and choline may be transported by a choline/H+ antiport system. Identification of this CTL1- and CTL2-mediated choline transport system should provide a potential new target for RA therapy.

Interaction of leucocyte antibodies with endothelial cells in transfusion‐related acute lung injury

Transfusion‐related acute lung injury (TRALI) is a severe pulmonary reaction characterized by pulmonary oedema, breathing distress and consequently hypoxia temporally associated with a blood transfusion event. In antibody‐mediated TRALI, alloantibodies reactive with recipient's neutrophils are mainly considered as responsible for lung injury. These antibodies target either human leucocyte antigens (HLAs) class I or II or human neutrophil antigens (HNAs). Antibody interaction with the cognate antigen is believed to induce neutrophil activation and sequestration, which consequently leads to endothelial damage and lung injury. Analysing the effects of these antibodies on neutrophil‐depleted animals however has challenged this dogma. Previous studies have documented that among anti‐HNA antibodies, anti‐HNA‐3a are implicated in severe and fatal TRALI. Epitopes for anti‐HNA‐3 antibodies reside on choline transporter‐like protein 2 (CTL2) which has a broad expression pattern on different cells including neutrophils and endothelial cells. In vitro as well as in vivo research has identified the binding of anti‐HNA‐3a to endothelial cells as initiator of lung injury in anti‐HNA‐3a‐mediated TRALI. Studies have also demonstrated that binding of HLA antibodies to cognate antigens expressed on endothelial cells, by triggering multiple signalling pathways, influences endothelial functions. This presentation will summarize findings on the effects of HNA and HLA antibodies binding to endothelial cells and highlight the contribution of endothelial‐reactive antibodies in the mechanism of antibody‐mediated TRALI.

An invertebrate-specific miRNA targeted the ancient cholinergic neuroendocrine system of oyster

Acetylcholine (ACh) is the main neurotransmitter in the cholinergic neuroendocrine system and plays an indispensable role in modulating diverse immune responses. As important transporters in choline uptake, choline transporter-like proteins (CTLs) can control ACh synthesis and release indirectly in multiple organisms. In this study, cgi-miR-2d, an invertebrate-specific miRNA in oyster Crassostrea gigas, is proved to repress the synthesis/release of ACh by targeting CgCTL1 and choline uptake in haemocytes during the early stage of pathogen infection. In short, an opposite expression pattern between CgCTL1 and cgi-miR-2d is observed during Vibrio splendidus infection, accompanied by changes in haemolymph ACh. In addition, the expression level of CgCTL1 is found to be significantly repressed after cgi-miR-2d overexpression in vivo, while both haemocyte choline and haemolymph ACh are also decreased simultaneously, similar to the finding in CgCTL1 knock-down assay. As a result, the expression of two tumour necrosis factor-like proteins and the bacteriostatic activity of oyster haemocytes are found to be altered significantly by either gain-of-function cgi-miR-2d or knock-down of CgCTL1. To our knowledge, this is the first miRNA identified in invertebrates that can target the ancient cholinergic system and augment immune response during infection.

PT712. Functional expression of choline transporter like-protein 1 (CTL1) and CTL2 in human brain microvascular endothelial cells

PT712. Functional expression of choline transporter like-protein 1 (CTL1) and CTL2 in human brain microvascular endothelial cells

Identification and functional analysis of choline transporter in tongue cancer: A novel molecular target for tongue cancer therapy

We examined the functional characteristics of choline uptake in human tongue carcinoma using the cell line HSC-3. Furthermore, we explored the possible correlation between the inhibition of choline uptake and apoptotic cell death. Both choline transporter-like protein 1 (CTL1) and CTL2 mRNAs and proteins were expressed, and were located in plasma membrane and mitochondria, respectively. Choline uptake was saturable and mediated by a single transport system, which is pH-dependent. Several cationic drugs inhibited cell viability and [3H]choline uptake. Choline uptake inhibitors and choline deficiency inhibited cell viability and increased caspase-3/7 activity. We conclude that extracellular choline is mainly transported via a CTL1 that relies on a directed H+ gradient as a driving force. The functional inhibition of CTL1 by cationic drugs could promote apoptotic cell death. Furthermore, CTL2 may be the major site for the control of choline oxidation in mitochondria and hence for the supply of endogenous betaine and S-adenosyl methionine, which serves as a major methyl donor. Identification of this CTL1- and CTL2-mediated choline transport system provides a potential new target for tongue cancer therapy.

Functional expression of choline transporter like-protein 1 (CTL1) and CTL2 in human brain microvascular endothelial cells

In this study, we examined the molecular and functional characterization of choline transporter in human brain microvascular endothelial cells (hBMECs). Choline uptake into hBMECs was a saturable process that was mediated by a Na+-independent, membrane potential and pH-dependent transport system. The cells have two different [3H]choline transport systems with Km values of 35.0 ± 4.9 μM and 54.1 ± 8.1 μM, respectively. Choline uptake was inhibited by choline, acetylcholine (ACh) and the choline analog hemicholinium-3 (HC-3). Various organic cations also interacted with the choline transport system. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA were highly expressed, while mRNA for high-affinity choline transporter 1 (CHT1) and organic cation transporters (OCTs) were not expressed in hBMECs. CTL1 and CTL2 proteins were localized to brain microvascular endothelial cells in human brain cortical sections. Both CTL1 and CTL2 proteins were expressed on the plasma membrane and mitochondria. CTL1 and CTL2 proteins are mainly expressed in plasma membrane and mitochondria, respectively. We conclude that choline is mainly transported via an intermediate-affinity choline transport system, CTL1 and CTL2, in hBMECs. These transporters are responsible for the uptake of extracellular choline and organic cations. CTL2 participate in choline transport mainly in mitochondria, and may be the major site for the control of choline oxidation.

Genotype frequency of human neutrophil antigen-3 polymorphisms in the Yi, Han, and Tibetan populations of China.

Human neutrophil antigen-3 (HNA-3) alloantibodies can cause fatal transfusion-related acute lung injury (TRALI). Most frequencies of SLC44A2 alleles encoding the HNA-3a/b antigens have been established in Han individuals by polymerase chain reaction with sequence-specific priming (PCR-SSP). We sequenced SLC44A2 gene fragments and determined allele frequencies in three ethnicities of China. Genomic DNA was extracted from 448 samples of 100 blood donors of Yi ethnicity in Xichang, Liangshan; 248 Han in Nanjing, Jiangsu; and 100 Tibetan in Lhasa, Tibet. A PCR-SSP was applied to determine the phase of two single-nucleotide polymorphisms (SNPs); SLC44A2 haplotypes were constructed. In the 567 nucleotides of the SLC44A2 gene covered by our sequencing approach in Han individuals, we detected the known 331-44G>A (rs12972963) and 461G>A (rs2288904) polymorphisms. In the 243 nucleotides sequenced in Yi and Tibetan populations, we detected the known 461G>A and 503-15T>C (rs1560711) polymorphisms. A PCR-SSP for the common HNA-3a/b SNP was 100% concordant. The frequencies of the HNA-3a allele were 0.58, 0.66, and 0.69 in Yi, Han (Nanjing), and Tibetan, respectively (0.42, 0.34, and 0.31 for HNA-3b). The Yi population of China had the highest frequency of blood donors at risk of harboring anti-HNA-3a compared to any population studied so far. We confirmed that the underlying SLC44A2*2 allele is more common in China than in any European or African populations.

Hair Cell Loss, Spiral Ganglion Degeneration, and Progressive Sensorineural Hearing Loss in Mice with Targeted Deletion of Slc44a2/Ctl2.

SLC44A2 (solute carrier 44a2), also known as CTL2 (choline transporter-like protein 2), is expressed in many supporting cell types in the cochlea and is implicated in hair cell survival and antibody-induced hearing loss. In mice with the mixed C57BL/6-129 background, homozygous deletion of Slc44a2 exons 3–10 (Slc44a2Δ/Δ) resulted in high-frequency hearing loss and hair cell death. To reduce effects associated with age-related hearing loss (ARHL) in these strains, mice carrying the Slc44a2Δ allele were backcrossed to the ARHL-resistant FVB/NJ strain and evaluated after backcross seven (N7) (99 % FVB). Slc44a2Δ/Δ mice produced abnormally spliced Slc44a2 transcripts that contain a frameshift and premature stop codons. Neither full-length SLC44A2 nor a putative truncated protein could be detected in Slc44a2Δ/Δ mice, suggesting a likely null allele. Auditory brain stem responses (ABRs) of mice carrying the Slc44a2Δ allele on an FVB/NJ genetic background were tested longitudinally between the ages of 2 and 10 months. By 6 months of age, Slc44a2Δ/Δ mice exhibited hearing loss at 32 kHz, but at 12 and 24 kHz had sound thresholds similar to those of wild-type Slc44a2+/+ and heterozygous +/Slc44a2Δ mice. After 6 months of age, Slc44a2Δ/Δ mutants exhibited progressive hearing loss at all frequencies and +/Slc44a2Δ mice exhibited moderate threshold elevations at high frequency. Histologic evaluation of Slc44a2Δ/Δ mice revealed extensive hair cell and spiral ganglion cell loss, especially in the basal turn of the cochlea. We conclude that Slc44a2 function is required for long-term hair cell survival and maintenance of hearing.

Full-length nucleotide sequences of 30 common SLC44A2 alleles encoding human neutrophil antigen-3.

Human neutrophil antigen-3a (HNA-3a) alloantibodies can cause severe transfusion-related acute lung injury. The frequencies of the single-nucleotide polymorphisms (SNPs) indicative of the two clinically relevant HNA-3a/b antigens are known in many populations. In this study, we determined the full-length nucleotide sequence of common SLC44A2 alleles encoding the choline transporter-like protein-2 that harbors HNA-3a/b antigens. A method was devised to determine the full-length coding sequence (CDS) and adjacent intron sequences from genomic DNA by eight polymerase chain reaction amplifications covering all 22 SLC44A2 exons. Samples from 200 African American, 96 Caucasian, two Hispanic, and four Asian blood donors were analyzed. We developed a decision tree to determine alleles (confirmed haplotypes) from the genotype data. A total of 10 SNPs were detected in the SLC44A2 CDS. The noncoding sequences harbored an additional 28 SNPs (one in the 5'-untranslated region [UTR]; 23 in the introns; and four in the 3'-UTR). No SNP indicative of a nonfunctional allele was detected. The nucleotide sequences for 30 SLC44A2 alleles (haplotypes) were confirmed. There may be 66 haplotypes among the 604 chromosomes screened. We found 38 SNPs, including one novel SNP, in 8192 nucleotides covering the CDS of the SLC44A2 gene among 302 blood donors. Population frequencies of these SNPs were established for African Americans and Caucasians. Because alleles encoding HNA-3b are more common than non-functional SLC44A2 alleles, we confirmed our previous postulate that African American donors are less likely to form HNA-3a antibodies compared to Caucasians.

Choline Transporter-Like Protein-2: New von Willebrand Factor-Binding Partner Involved in Antibody-Mediated Neutrophil Activation and Transfusion-Related Acute Lung Injury.

In contrast to other antibodies involved in transfusion-related acute lung injury, anti-HNA-3a antibodies are incapable of inducing direct neutrophil activation and seem to interact with endothelial cells (ECs) primarily. In animal studies, anti-HNA-3a-mediated transfusion-related acute lung injury could be precipitated in the absence of neutrophils, but was stronger when neutrophils were present. In a different context the target protein of these antibodies, choline transporter-like protein-2 (CTL-2), was reported to interact with a protein of the inner ear carrying 2 von Willebrand factor (VWF) A-domains. These observations prompted us to investigate whether VWF might be involved in anti-HNA-3a-mediated neutrophil activation, and whether signaling via CD11b/CD18 is involved, as in various other experimental settings. Cell adhesion demonstrated specific binding of CTL-2 to VWF. Immunoprecipitation analysis of CTL-2/CD11b/CD18 coexpressing cells indicated that anti-HNA-3a colocalizes CTL-2 and CD11b/CD18 when VWF is present. Functional studies revealed that anti-HNA-3a-mediated neutrophil agglutination is an active, protein kinase C-dependent and partially Fc-dependent process. Agglutination and the production of reactive oxygen species seem to require the formation of a trimolecular complex between the target antigen (CTL-2), CD11b/CD18 and VWF. In line with these observations, anti-HNA-3a induced less severe transfusion-related acute lung injury and less neutrophil recruitment to the alveolar space in VWF knockout mice. We introduce CTL-2 as a new binding partner for VWF. Interaction of neutrophils with VWF via CTL-2 allows anti-HNA-3a to induce signal transduction via CD11b/CD18, which leads to neutrophil activation and agglutination. In transfusion-related acute lung injury, this mechanism may further aggravate endothelial leakage.

Molecular and functional characterization of choline transporter in the human trophoblastic cell line JEG-3 cells

IntroductionCholine is essential for the synthesis of the major membrane phospholipid phosphatidylcholine (PC), the methyl donor betaine and the neurotransmitter acetylcholine (ACh), which is involved in several vital biological functions that play key roles in fetal development. In this study, we examined the molecular and functional characteristics of choline uptake in the human trophoblastic cell line JEG-3. MethodsWe examined [3H]choline uptake in the human trophoblastic cell line JEG-3. The expression of CTL1 and CTL2 was evaluated by quantitative real-time PCR, western blotting and immunocytochemistry. ResultsWe demonstrated that JEG-3 cells take up [3H] choline by a saturable process that is mediated by a Na+-independent and pH-dependent transport system. The cells have two different [3H] choline transport systems, high- and low-affinity, with Km values of 28.4 ± 5.0 μM and 210.6 ± 55.1 μM, respectively. Cationic compounds and hemicholinium-3 (HC-3) inhibited choline uptake. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in JEG-3 cells and were localized to the plasma membrane. DiscussionThe present results suggest that choline is mainly transported via a high-affinity choline transport system (CTL1) and a low-affinity choline transport system (CTL2) in human trophoblastic JEG-3 cells. These transporters play an important role in the growth of the fetus.

Human neutrophil antigens: a nomenclature update based on new alleles and new antigens

In 1998, the ISBT Granulocyte Antigen Working Party established a new nomenclature for the human neutrophil antigens (HNA) which included five antigen systems and a total of seven antigens. In the meantime, new antigens and new alleles have been described and these now make it necessary for the ISBT HNA nomenclature subcommittee to present a proposal for an HNA nomenclature update. HNA‐1 includes four antigens, HNA‐1a, HNA‐1b, HNA‐1c and HNA‐1d, which can result from five different Fcγ receptor IIIb (FcγRIIIb)‐encoding alleles, FCGR3B*01, *02, *03, *04 and *05. Due to copy number variation, individuals may exhibit between zero and four alleles leading to either the HNA‐1 null type or combinations of the known HNA‐1 antigens. Current data indicate that HNA‐2 is an isoantigen without allelic variation, encoded by the CD177 gene. Individuals without any HNA‐2 expression on their neutrophils are addressed as HNA‐2 null. The two antithetical antigens of the choline‐transporter‐like protein 2 (CTL2), HNA‐3a and HNA‐3b, are encoded by the three different alleles SLC44A2*01, *02 and *03, which discriminate in one or two nucleotide substitutions. HNA‐4a, which is encoded by the ITGAM*01 allele, is an antigen of the αMβ2‐integrin. The αLβ2‐integrin contains HNA‐5a as the only antigen which is encoded by the ITGAL*01 allele. Antigens and alleles other than those listed have to be approved by the ISBT Granulocyte Immunobiology Working Party (GIWP) before they can be assigned to the official nomenclature.

Impact of priming on the response of neutrophils to human neutrophil alloantigen-3a antibodies.

Human neutrophil alloantigen-3a (HNA-3a) antibodies can induce transfusion-related acute lung injury (TRALI). The severity of TRALI varies largely among the affected patients. Severe comorbidity seems to increase the susceptibility for TRALI, potentially by priming of neutrophils. Thus, the impact of neutrophil priming on HNA-3a antibody-mediated neutrophil aggregation and CD11b surface expression was investigated. Neutrophils were primed using formyl-methionyl-leucyl-phenylalanine (fMLP) or bacterial lipopolysaccharide (LPS). Granulocyte aggregation and CD11b surface expression were evaluated by the granulocyte agglutination test and by flow cytometry (FC), respectively. Priming-induced changes in the surface expression of choline transporter-like protein 2 (CTL2) and the CTL2 mRNA expression were assessed by FC and quantitative real-time polymerase chain reaction, respectively. Priming of neutrophils lowered the amount of HNA-3a antibodies required for inducing granulocyte aggregation in a dose-dependent manner by 50% to 75%. The priming agent concentration necessary for this response differed between donors. Priming slightly enhanced binding of HNA-3a antibodies to neutrophils. However, CTL2 de novo synthesis was not induced after priming with LPS, indicating that increased HNA-3a antibody binding was likely caused by translocation of intracellular CTL2 to the surface or by increased affinity of HNA-3a antibodies to CTL2. HNA-3a antibodies influenced CD11b surface expression on neutrophils only marginally, which was also not potentiated by priming with fMLP or LPS. This study provides experimental evidence supporting the "threshold model" of TRALI. Priming of neutrophils with fMLP or LPS increases their aggregation response to HNA-3a antibodies by lowering the required antibody amount.

Membrane transport mechanisms of choline in human intestinal epithelial LS180 cells.

The aim of the present study was to investigate the membrane transport mechanisms of choline using human intestinal epithelial LS180 cells. The mRNA of choline transporter-like proteins (CTLs) was expressed significantly in LS180 cells, and the rank order was CTL1 > CTL4 > CTL3 > CTL2 > CTL5. In contrast, the mRNA expression of other choline transporters, organic cation transporter (OCT) 1, OCT2 and high-affinity choline transporter 1 (CHT1), was considerably lower in LS180 cells. Five mm unlabelled choline, hemicolinium-3 and guanidine, but not tetraethylammonium, inhibited the cellular uptake of 100 µm choline in LS180 cells. The uptake of choline into LS180 cells was virtually Na(+)-independent. The uptake of choline was significantly decreased by acidification of the extracellular pH; however, it was not increased by alkalization of the extracellular pH. In addition, both acidification and alkalization of intracellular pH decreased the uptake of choline, indicating that the choline uptake in LS180 cells is not stimulated by the outward H(+) gradient. On the other hand, the uptake of choline was decreased by membrane depolarization along with increasing extracellular K(+) concentration. In addition, the Na(+)-independent uptake of choline was saturable, and the Km value was estimated to be 108 µm. These findings suggest that the uptake of choline into LS180 cells is membrane potential-dependent, but not outward H(+) gradient-dependent.

CHOLINE TRANSPORTER-LIKE1 is required for sieve plate development to mediate long-distance cell-to-cell communication.

Phloem, a plant tissue responsible for long-distance molecular transport, harbours specific junctions, sieve areas, between the conducting cells. To date, little is known about the molecular framework related to the biogenesis of these sieve areas. Here we identify mutations at the CHER1/AtCTL1 locus of Arabidopsis thaliana. The mutations cause several phenotypic abnormalities, including reduced pore density and altered pore structure in the sieve areas associated with impaired phloem function. CHER1 encodes a member of a poorly characterized choline transporter-like protein family in plants and animals. We show that CHER1 facilitates choline transport, localizes to the trans-Golgi network, and during cytokinesis is associated with the phragmoplast. Consistent with its function in the elaboration of the sieve areas, CHER1 has a sustained, polar localization in the forming sieve plates. Our results indicate that the regulation of choline levels is crucial for phloem development and conductivity in plants.

Palmitic acid and oleic acid differentially regulate choline transporter-like 1 levels and glycerolipid metabolism in skeletal muscle cells.

Choline is an essential nutrient required for the biosynthesis of membrane lipid phosphatidylcholine (PtdCho). Here we elucidate the mechanism of how palmitic acid (PAM) and oleic acid (OLA) regulate choline transporter-like protein 1 (CTL1/SLC44A1) function. We evaluated the mechanism of extracellular and intracellular transport of choline, and their contribution to PtdCho and other glycerolipid-diacylglycerol (DAG) and triacylglycerol (TAG) homeostasis in differentiated skeletal muscle cells. PAM reduces total and plasma membrane CTL1/SLC44A1 protein by lysosomal degradation, and limits the choline uptake while increasing DAG and TAG synthesis. OLA maintains total and plasma membrane CTL1/SLC44A1, but increases PtdCho synthesis more than PAM. OLA does not increase the rate of DAG synthesis, but does increase TAG content. Thus, the CTL1/SLC44A1 presence at the plasma membrane regulates choline requirements in accordance with the type of fatty acid. The increased PtdCho and TAG turnover by OLA stimulates cell growth and offers a specific protection mechanism from the excess of intracellular DAG and autophagy. This protection was present after OLA treatments, but not after PAM treatments. The mitochondrial choline uptake was reduced by both FA; however, the regulation is complex and guided not only by the presence of the mitochondrial CTL1/SLC44A1 protein but also by the membrane potential and general mitochondrial function.

Choline transporter‐like proteins CTLs/SLC44 family as a novel molecular target for cancer therapy

Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine (PC), the methyl donor betaine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in various cancers. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. Previous studies have demonstrated abnormalities in choline uptake and choline phospholipid metabolism in cancer cells using the imaging of cancer with positron emission tomography (PET) and magnetic resonance spectroscopy (MRS). The aberrant choline metabolism in cancer cells is strongly correlated with their malignant progression. Using quantitative real-time PCR, the mRNA expression of choline transporters was measured, and it was found that choline transporter-like proteins CTLs/SLC44 family are highly expressed in various cancer cell lines. Choline uptake through CTLs is associated with cell viability, and the functional inhibition of CTLs could promote apoptotic cell death. Furthermore, non-neuronal cholinergic systems that include CTLs-mediated choline transport are associated with cell proliferation and their inhibition promotes apoptotic cell death in colon cancer, small cell lung cancer and human leukemic T-cells. The identification of this new CTLs-mediated choline transport system provides a potential new target for cancer therapy.

Functional analysis of [methyl-3H]choline uptake in glioblastoma cells: Influence of anti-cancer and central nervous system drugs

Positron emission tomography (PET) and PET/computed tomography (PET-CT) studies with 11C- or 18F-labeled choline derivatives are used for PET imaging in glioblastoma patients. However, the nature of the choline transport system in glioblastoma is poorly understood. In this study, we performed a functional characterization of [methyl-3H]choline uptake and sought to identify the transporters that mediate choline uptake in the human glioblastoma cell lines A-172 and U-251MG. In addition, we examined the influence of anti-cancer drugs and central nervous system drugs on the transport of [methyl-3H]choline. High- and low-affinity choline transport systems were present in A-172 cells, U-251MG cells and astrocytes, and these were Na+-independent and pH-dependent. Cell viability in A-172 cells was not affected by choline deficiency. However, cell viability in U-251MG cells was significantly inhibited by choline deficiency. Both A-172 and U-251MG cells have two different choline transporters, choline transporter-like protein 1 (CTL1) and CTL2. In A-172 cells, CTL1 is predominantly expressed, whereas in U-251MG cells, CTL2 is predominantly expressed. Treatment with anti-cancer drugs such as cisplatin, etoposide and vincristine influenced [methyl-3H]choline uptake in U-251MG cells, but not A-172 cells. Central nervous system drugs such as imipramine, fluvoxamine, paroxetine, reboxetine, citalopram and donepezil did not affect cell viability or [methyl-3H]choline uptake. The data presented here suggest that CTL1 and CTL2 are functionally expressed in A-172 and U-251MG cells and are responsible for [methyl-3H]choline uptake that relies on a directed H+ gradient as a driving force. Furthermore, while anti-cancer drugs altered [methyl-3H]choline uptake, central nervous system drugs did not affect [methyl-3H]choline uptake.

Antibodies Specific For Human Neutrophil Antigen 3a (HNA-3a) Recognize Complex Epitopes On Choline Transporter Protein 2 (CTL2): Implications For HNA-3a Antibody Detection

Transfusion-related acute lung injury (TRALI), the leading cause of mortality associated with blood transfusion, usually results from passive transfer of antibodies present in donated blood to a patient. TRALI can be triggered by antibodies specific for Class I or Class II HLA antigens, human neutrophil antigens (HNA) and possibly other targets. For reasons not well understood, antibodies specific for the leukocyte antigen HNA-3a cause particularly severe, often fatal TRALI. It would be highly desirable therefore to be able to screen blood donors routinely for HNA-3a antibodies.HNA-3a/b antigens are carried on choline transporter-like protein 2 (CTL2), an apparent 10 membrane-spanning protein with 5 extracellular loops and N and C intracellular termini. The HNA-3a/b polymorphism is created by an R/Q substitution at position 154 in the first of these extracellular loops (Loop 1). In solid phase assays, about one-half of HNA-3a antibodies implicated in TRALI recognize Loop 1 peptides containing R154 (Type 1 antibodies). The remaining antibodies (Type 2) are non-reactive with peptides despite reacting well against full length CTL2. We studied reactions of Type 1 and Type 2 HNA-3a antibodies against soluble recombinant CTL2 fragments, human CTL2, mouse CTL2, and human/mouse CTL2 chimeras expressed in HEK293 cells to characterize the basis for HNA-3a antibody heterogeneity. The following observations were made:1) Only Type 1 antibodies react with detergent-solublized CTL2 in solid phase assays.2) A soluble recombinant fragment derived from the first extracellular (EC) loop (R154) of human CTL2 reacts only with Type 1 antibodies.3) Mouse CTL2 is 91% identical to human CTL2 and contains the R154 residue critical for HNA-3a expression. Type 1 antibodies recognize mouse CTL2, but Type 2 antibodies do not.4) Chimeric CTL2 containing human sequence in EC Loop 1 and mouse sequence in Loops 2-5 (H1M) reacts only with Type 1 antibodies. The reciprocal construct with mouse sequence in EC loop 1 and human sequence in Loops 2-5 (M1H) reacts with both Type 1 and Type 2 antibodies.5) Chimeric CTL2 containing human sequence in EC Loops 1-2 and mouse sequence in Loops 3-5 (H2M) reacts only with Type 1 antibodies.6) Chimeric CTL2 containing human sequence in EC Loops 1-3 and mouse sequence in Loops 4-5 (H3M) reacts with both Type 1 and Type 2 antibodies.These findings show that Loop 1 peptides containing R154 are sufficient for Type 1 antibodies to recognize CTL2 and the Type 1 epitope survives detergent solubilization of the protein. However, Type 2 antibodies require human sequence in EC Loops 1-3 for binding and the epitope they recognize does not survive detergent treatment. Moreover both Type 1 and Type 2 recognize the M1H chimera with the entire EC loop 1 sequence derived from mouse. The simplest explanation for these observations is that Type 2 HNA-3a antibodies need to contact human amino acid residues in EC Loop 3 in addition to Loop 1 for tight binding to CTL2. An alternative possibility is both Type 1 and 2 antibodies recognize only Loop1, but EC Loops 2 and 3 are required to hold Loop 1 in a configuration suitable for Type 2 antibody binding. In either case, it appears that at least the first 3 EC loops of CTL2 (R154) need to be in a configuration that closely mimics their natural state in the cell membrane in order to be recognized by Type 2 HNA-3a antibodies. Considerable ingenuity will be required to engineer a target capable of detecting both Type 1 and Type 2 HNA-3a antibodies in a format suitable for large-scale blood donor screening. Disclosures:No relevant conflicts of interest to declare.

Anti–Human Neutrophil Antigen-3a Induced Transfusion-Related Acute Lung Injury in Mice by Direct Disturbance of Lung Endothelial Cells

Antibodies against human neutrophil antigen-3a (HNA-3a) located on choline transporter-like protein 2 induce severe transfusion-related acute lung injury (TRALI). This study aims to identify the mechanism implicated in anti-HNA-3a-mediated TRALI. Our analysis shows that anti-HNA-3a recognizes 2 choline transporter-like protein 2 isoforms (P1 and P2) on human microvascular endothelial cells from lung blood vessels but reacts only with the P1 isoform on neutrophils. Direct treatment of HNA-3a-positive endothelial cells with anti-HNA-3a, but not with anti-HNA-3b, leads to reactive oxygen species production, increased albumin influx, and decreased endothelial resistance associated with the formation of actin stress filaments and loosening of junctional vascular endothelium-cadherin. In a novel in vivo mouse model, TRALI was documented by significant increase in lung water content, albumin concentration, and neutrophil numbers in the bronchoalveolar lavage on injection of human anti-HNA-3a in lipopolysaccharides-treated, as well as nontreated mice. Interestingly, although neutrophil depletion alleviated severity of lung injury, it failed to prevent TRALI in this model. Infusion of anti-HNA-3a F(ab')2 fragments caused moderate TRALI. Finally, mice lacking nicotinamide adenine dinucleotide phosphate oxidase (NOX2(y/-)) were protected from anti-HNA-3a-mediated TRALI. These data demonstrate the initiation of endothelial barrier dysfunction in vitro and in vivo by direct binding of anti-HNA-3a on endothelial cells. It seems, however, that the presence of neutrophils aggravates barrier dysfunction. This novel mechanism of TRALI primarily mediated by endothelial cell dysfunction via choline transporter-like protein 2 may help to define new treatment strategies to decrease TRALI-related mortality.