Development of Antihuman Killer Cell Lectin-Like Receptor Subfamily G Member 1 Monoclonal Antibodies for Flow Cytometry.

Killer cell lectin-like receptor subfamily G member 1 (KLRG1), a type II transmembrane protein, was identified as an inhibitory receptor expressed on natural killer (NK) cells and certain T cells. The protein regulates effector functions and developmental processes in these cells. In this study, we established a specific and sensitive monoclonal antibody (mAb) for human KLRG1 (hKLRG1), which is useful for flow cytometry, using a Cell-Based Immunization and Screening (CBIS) method. The established anti-hKLRG1 mAb, KLMab-1 (mouse IgG1, kappa), reacted with overexpressed hKLRG1 in Chinese hamster ovary-K1 (CHO/hKLRG1) and human NK cells, which also expressed endogenous hKLRG1 as confirmed by flow cytometry. KLMab-1, which was established by the CBIS method, could be useful for elucidating the hKLRG1-related biological response by flow cytometry.

Generation of novel trimeric fragments of human SP-A and SP-D after recombinant soluble expression in E. coli.

Surfactant treatment for neonatal respiratory distress syndrome has dramatically improved survival of preterm infants. However, this has resulted in a markedly increased incidence of sequelae such as neonatal chronic inflammatory lung disease. The current surfactant preparations in clinical use lack the natural lung defence proteins surfactant proteins (SP)-A and D. These are known to have anti-inflammatory and anti-infective properties essential for maintaining healthy non-inflamed lungs. Supplementation of currently available animal derived surfactant therapeutics with these anti-inflammatory proteins in the first few days of life could prevent the development of inflammatory lung disease in premature babies. However, current systems for production of recombinant versions of SP-A and SP-D require a complex solubilisation and refolding protocol limiting expression at scale for drug development. Using a novel solubility tag, we describe the expression and purification of recombinant fragments of human (rfh) SP-A and SP-D using Escherichia coli without the need for refolding. We obtained a mean (± SD) of 23.3 (± 5.4) mg and 86 mg (± 3.5) per litre yield of rfhSP-A and rfhSP-D, respectively. rfhSP-D was trimeric and 68% bound to a ManNAc-affinity column, giving a final yield of 57.5 mg/litre of highly pure protein, substantially higher than the 3.3 mg/litre obtained through the standard refolding protocol. Further optimisation of this novel lab based method could potentially make rfhSP-A and rfhSP-D production more commercially feasible to enable development of novel therapeutics for the treatment of lung infection and inflammation.

The Orphan Immune Receptor LILRB3 Modulates Fc Receptor-Mediated Functions of Neutrophils.

Neutrophils are critical to the generation of effective immune responses and for killing invading microbes. Paired immune receptors provide important mechanisms to modulate neutrophil activation thresholds and effector functions. Expression of the leukocyte Ig-like receptor (LILR)A6 (ILT8/CD85b) and LILRB3 (ILT5/CD85a) paired-receptor system on human neutrophils has remained unclear because of the lack of specific molecular tools. Additionally, there is little known of their possible functions in neutrophil biology. The objective of this study was to characterize expression of LILRA6/LILRB3 receptors during human neutrophil differentiation and activation, and to assess their roles in modulating Fc receptor-mediated effector functions. LILRB3, but not LILRA6, was detected in human neutrophil lysates following immunoprecipitation by mass spectrometry. We demonstrate high LILRB3 expression on the surface of resting neutrophils and release from the surface following neutrophil activation. Surface expression was recapitulated in a human PLB-985 cell model of neutrophil-like differentiation. Continuous ligation of LILRB3 inhibited key IgA-mediated effector functions, including production of reactive oxygen species, phagocytic uptake, and microbial killing. This suggests that LILRB3 provides an important checkpoint to control human neutrophil activation and their antimicrobial effector functions during resting and early-activation stages of the neutrophil life cycle.

Comparative analysis of viral RNA signatures on different RIG-I-like receptors.

The RIG-I-like receptors (RLRs) play a major role in sensing RNA virus infection to initiate and modulate antiviral immunity. They interact with particular viral RNAs, most of them being still unknown. To decipher the viral RNA signature on RLRs during viral infection, we tagged RLRs (RIG-I, MDA5, LGP2) and applied tagged protein affinity purification followed by next-generation sequencing (NGS) of associated RNA molecules. Two viruses with negative- and positive-sense RNA genome were used: measles (MV) and chikungunya (CHIKV). NGS analysis revealed that distinct regions of MV genome were specifically recognized by distinct RLRs: RIG-I recognized defective interfering genomes, whereas MDA5 and LGP2 specifically bound MV nucleoprotein-coding region. During CHIKV infection, RIG-I associated specifically to the 3' untranslated region of viral genome. This study provides the first comparative view of the viral RNA ligands for RIG-I, MDA5 and LGP2 in the presence of infection.

Identification of a Novel Splice Variant Isoform of TREM-1 in Human Neutrophil Granules.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is critical for inflammatory signal amplification. Humans have two forms of TREM-1: a membrane receptor, associated with the adaptor DAP12, and a soluble receptor detected at times of infection. The membrane receptor isoform acts synergistically with the TLR pathway to promote cytokine secretion and neutrophil migration, whereas the soluble receptor functions as a counterregulatory molecule. In multiple models of sepsis, exogenous administration of soluble forms of TREM-1 attenuates inflammation and markedly improves survival. Despite intense interest in soluble TREM-1, both as a clinical predictor of survival and as a therapeutic tool, the origin of native soluble TREM-1 remains controversial. Using human neutrophils, we identified a 15-kDa TREM-1 isoform in primary (azurophilic) and secondary (specific) granules. Mass spectrometric analysis, ELISA, and immunoblot confirm that the 15-kDa protein is a novel splice variant form of TREM-1 (TREM-1sv). Neutrophil stimulation with Pseudomonas aeruginosa, LPS, or PAM(3)Cys4 resulted in degranulation and release of TREM-1sv. The addition of exogenous TREM-1sv inhibited TREM-1 receptor-mediated proinflammatory cytokine production. Thus, these data reveal that TREM-1 isoforms simultaneously activate and inhibit inflammation via the canonical membrane TREM-1 molecule and this newly discovered granular isoform, TREM-1sv.

Identification of 21 novel immune-type receptors in miiuy croaker and expression pattern of three typical inhibitory members.

Novel immune-type receptor (NITR) genes belong to the immunoglobulin superfamily and are encoded by clusters of multigene families. NITRs encode type I transmembrane proteins and are only found in teleosts. In the current study, total 21 NITR genes are identified from miiuy croaker (Miichthys miiuy) and named as MmNITR1 to MmNITR21. Miiuy croaker NITR genes that encoded one or two extracellular immunoglobulin (Ig) domains, a transmembrane (TM) region, an immunoreceptor tyrosine-based inhibitor motif (ITIM) in the cytoplasmic (Cyt) region. The majority of MmNITRs possess cytoplasmic ITIM that can be classified as inhibitory receptors. However, a smaller number of NITRs (MmNITR8, MmNITR15 and MmNITR16) can be classified as activating receptors by the lack of cytoplasmic ITIMs and presence of a positively charged residue within their transmembrane domain. As typical inhibitory receptors, MmNITR1, MmNITR2 and MmNITR3 have different characteristics of the structure. In MmNITR1 gene, variable (V) and intermediate (I) domains are encoded by two separate exons. In contrast to MmNITR1, MmNITR3 gene encode V and I domains in a single exon. And MmNITR2 gene is characterized by the presence of only one Ig-like (V-type) extracellular domain and lack of J or J-like motifs. Also MmNITR2 gene displays an additional exon which is 48bp long between the V domain and the TM region. Two and four potential N-link giycosylation sites (N-X-S/T) are present in the extracellular Ig domains. Real-time RT-PCR results showed that upon induction with Vibrio anguillarum, NITR gene expressions were induced by bacteria in kidney, liver and spleen. Meanwhile, NITRs are also primarily detected in different tissues. Phylogenetic analyses of NITR V domains indicate that MmNITR1 and MmNITR2 are more similar than MmNITR3.

Clearance kinetics and matrix binding partners of the receptor for advanced glycation end products.

Elucidating the sites and mechanisms of sRAGE action in the healthy state is vital to better understand the biological importance of the receptor for advanced glycation end products (RAGE). Previous studies in animal models of disease have demonstrated that exogenous sRAGE has an anti-inflammatory effect, which has been reasoned to arise from sequestration of pro-inflammatory ligands away from membrane-bound RAGE isoforms. We show here that sRAGE exhibits in vitro binding with high affinity and reversibly to extracellular matrix components collagen I, collagen IV, and laminin. Soluble RAGE administered intratracheally, intravenously, or intraperitoneally, does not distribute in a specific fashion to any healthy mouse tissue, suggesting against the existence of accessible sRAGE sinks and receptors in the healthy mouse. Intratracheal administration is the only effective means of delivering exogenous sRAGE to the lung, the organ in which RAGE is most highly expressed; clearance of sRAGE from lung does not differ appreciably from that of albumin.

Preparation and characterization of monoclonal antibodies against VSTM1.

VSTM1 (V-set and transmembrane domain containing 1) is a novel membrane molecule identified from immunogenomics, which has two main isoforms, VSTM1-v1 and VSTM1-v2. VSTM1-v1 is a type I transmembrane protein, and VSTM1-v2 is a classical secretory protein, lacking only the transmembrane domain compared with VSTM1-v1. This study was designed to generate VSTM1-specific monoclonal antibodies (MAbs) for further exploration of its expression and function. Mice were immunized with two recombinant prokaryotic proteins of GST-VSTM1-v2 and VSTM1-v2 without any tag. Hybridomas were generated by the fusion of the splenocytes to Sp2/0 myeloma cells. Three hybridoma cell lines (2C11, 6E11, and 7A8) stable in secreting anti-VSTM1 MAb were obtained and further characterized. All three MAbs were IgG2b isotype and effective in detecting the overexpressed VSTM1 in both Western blot and flow cytometry assays, while recognizing the endogenous VSTM1 in Western blot analysis only. These MAbs could be helpful in the basic study of VSTM1 and in revealing the interesting conformation difference between the overexpressed and endogenous proteins.

Cloning, expression and purification of the SRCR domains of glycoprotein 340.

Glycoprotein 340 (gp340), an innate immunity molecule is secreted luminally by monolayered epithelia and associated glands within the human oral cavity. Gp340 contains 14 scavenger receptor cysteine rich (SRCR) domains, two CUB (C1r/C1s Uegf Bmp1) domains and one zona pellucida (ZP) domain. Oral streptococci are known to adhere to the tooth immobilized gp340 via its surface protein Antigen I/II (AgI/II), which is considered to be the critical first step in pathogenesis that eventually results in colonization and infection. In order to decipher the interactions between gp340's domains and oral streptococcal AgI/II domains, we undertook to express human gp340's first SRCR domain (SRCR1) and the first three tandem SRCR domains (SRCR123) in Drosophila S2 cells. While our initial attempts with human codons did not produce optimal results, codon-optimization for expression in Drosophila S2 cells and usage of inducible/secretory Drosophila expression system (DES) pMT/BiP/V5-HisA vector greatly enhanced the expression of the SRCR domains. Here we report the successful cloning, expression, and purification of the SRCR domains of gp340. Recognition of expressed SRCRs by the conformational dependent gp340 antibody indicate that these domains are appropriately folded and furthermore, surface plasmon resonance studies confirmed functional adherence of the SRCR domains to AgI/II.

Anti-inflammatory role of PGD2 in acute lung inflammation and therapeutic application of its signal enhancement.

We investigated the role of prostaglandin D2 (PGD2) signaling in acute lung injury (ALI), focusing on its producer-effector interaction in vivo. Administration of endotoxin increased edema and neutrophil infiltration in the WT mouse lung. Gene disruption of hematopoietic PGD synthase (H-PGDS) aggravated all of the symptoms. Experiments involving bone marrow transplantation between WT and H-PGDS-deficient mice showed that PGD2 derived from alveolar nonhematopoietic lineage cells (i.e., endothelial cells and epithelial cells) promotes vascular barrier function during the early phase (day 1), whereas neutrophil-derived PGD2 attenuates its own infiltration and cytokine expression during the later phase (day 3) of ALI. Treatment with either an agonist to the PGD2 receptor, DP, or a degradation product of PGD2, 15-deoxy-Δ(12,14)-PGJ2, exerted a therapeutic action against ALI. Data obtained from bone marrow transplantation between WT and DP-deficient mice suggest that the DP signal in alveolar endothelial cells is crucial for the anti-inflammatory reactions of PGD2. In vitro, DP agonism directly enhanced endothelial barrier formation, and 15-deoxy-Δ(12,14)-PGJ2 attenuated both neutrophil migration and cytokine expression. These observations indicate that the PGD2 signaling between alveolar endothelial/epithelial cells and infiltrating neutrophils provides anti-inflammatory effects in ALI, and suggest the therapeutic potential of these signaling enhancements.

VSTM1-v2, a novel soluble glycoprotein, promotes the differentiation and activation of Th17 cells.

Cytokines are soluble proteins that mediate immune reactions and are responsible for communication among immune cells. CD4(+) T cells are the principle sources of cytokines of adaptive immunity. Cytokines play critical roles in the differentiation and effector function of CD4(+) T cells. They also play key roles in diseases, and some of them have been developed into drugs in the forms of recombinant cytokines, soluble receptors and neutralizing antibodies. Therefore, identifying novel potential cytokines is necessary and beneficial for better understanding immunology and enhancing human health. To find novel potential cytokines, we carried out an integrated bioinformatics analysis on the whole human genome. Cytokine candidates were selected for cDNA cloning, sub-cloning, secretion verification, expression profile analysis and functional study. Here, we report a novel soluble protein, VSTM1-v2, which is a classical secretory glycoprotein mainly expressed in immune tissues, and can promote the differentiation and activation of Th17 cells.

A one-step affinity-purification protocol to purify NB-LRR immune receptors from plants that mediate resistance to fungal pathogens.

Nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors from plants confer resistance to fungal pathogens and many other pathogenic organisms. Their low expression makes it challenging to purify these receptors from plants in sufficient quantities to be able to identify interacting proteins by mass spectrometry. Here we describe a protocol to affinity-purify recombinant NB-LRR immune receptors, fused to the streptavidin-binding peptide tag.

Proteomic studies on receptor for advanced glycation end product variants in idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease.

PURPOSE: Proteomic screening revealed declined levels of the receptor for advanced glycation end products (RAGE) in human idiopathic pulmonary fibrosis (IPF). This study was undertaken to investigate the different RAGE isoforms in two lung diseases with destruction of the lung parenchyma, i.e. IPF and chronic obstructive pulmonary disease (COPD). EXPERIMENTAL DESIGN: RAGE was analyzed by 2-DE, MS and Western blotting using lung tissues from non-smokers, smokers, patients with IPF, COPD and α-1-antitrypsin deficiency (AAT) and by ELISA from the bronchoalveolar lavage fluid samples. RESULTS: RAGE, detected by 2-DE in the control lung, was confirmed to be glycosylated, soluble, C-truncated RAGE with characteristics indicative of the presence of endogenous secretory RAGE (esRAGE). Further studies revealed a decrease of the full length-RAGE (FL-RAGE) and its C-terminal processed variant (cRAGE) in the lung tissues of IPF and COPD patients but not in AAT. The esRAGE level was reduced in IPF but was unchanged in COPD. CONCLUSIONS AND CLINICAL RELEVANCE: This study shows an involvement of the three RAGE variants (FL-RAGE, cRAGE, esRAGE) in IPF. The decline of FL-RAGE and cRAGE, but not esRAGE, in COPD lungs is evidence of involvement of specific RAGE variants also in this disease.

A protocol for the production of KLRG1 tetramer.

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells. Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca(2+)-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition.

The cytolytic activity of natural killer (NK) cells is regulated by inhibitory receptors that detect the absence of self molecules on target cells. Structural studies of missing self recognition have focused on NK receptors that bind MHC. However, NK cells also possess inhibitory receptors specific for non-MHC ligands, notably cadherins, which are downregulated in metastatic tumors. We determined the structure of killer cell lectin-like receptor G1 (KLRG1) in complex with E-cadherin. KLRG1 mediates missing self recognition by binding to a highly conserved site on classical cadherins, enabling it to monitor expression of several cadherins (E-, N-, and R-) on target cells. This site overlaps the site responsible for cell-cell adhesion but is distinct from the integrin alpha(E)beta(7) binding site. We propose that E-cadherin may coengage KLRG1 and alpha(E)beta(7) and that KLRG1 overcomes its exceptionally weak affinity for cadherins through multipoint attachment to target cells, resulting in inhibitory signaling.

Measurement of the bacteriophage inactivation kinetics with purified receptors.

Practical methods are described for studying the interaction between bacterial viruses and their surface receptors.

Identification and characterization of a transmembrane isoform of CD160 (CD160-TM), a unique activating receptor selectively expressed upon human NK cell activation.

CD160 has been initially identified as a GPI-anchored MHC-class I activating receptor mainly expressed on peripheral blood NK cells. Herein, we report the identification of three additional CD160-related mRNAs generated through alternative splicings of the CD160 gene, among which one encoded a putative CD160 transmembrane isoform (CD160-TM). We first establish that CD160-TM surface expression is highly restricted to NK cells and is activation-dependent. Additionally, we provide evidence that CD160-TM represents a novel activating receptor, as assessed by the increased CD107a NK cell surface mobilization observed upon its engagement. Finally, we demonstrate that the CD160-TM cytoplasmic tail is by itself sufficient to mediate the recruitment of Erk1/2 signaling pathway, and that the initiation of this activation process is dependent on the Src-family kinase p56(lck). The identification of CD160-TM therefore provides new possibilities regarding the role of CD160 isoforms in the regulation of NK cell functions.

Expression, crystallization and preliminary X-ray diffraction analysis of human paired Ig-like type 2 receptor alpha (PILRalpha).

Human paired immunoglobulin-like (Ig-like) type 2 receptor alpha (PILRalpha) is a type I membrane protein that is mainly expressed in immune-related cells such as monocytes, granulocytes and dendritic cells. PILRalpha can suppress the functions of such immune cells because it has the immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracellular region, which recruits the phosphatase Src homology-2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP-2) to inhibit phosphorylations induced by activation signals. The extracellular region of human PILRalpha comprises one immunoglobulin superfamily V-set domain and a stalk region. The V-set domain (residues 13-131) of human PILRalpha was overexpressed in Escherichia coli as inclusion bodies, refolded by rapid dilution and purified. The PILRalpha protein was successfully crystallized at 293 K using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.3 A resolution at SPring-8 BL41XU; they belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 40.4, b = 45.0, c = 56.9 A, and contain one molecule per asymmetric unit.

[Comparison of the binding affinity of LAIR-1(CD305) and LAIR-2(CD306) with their ligands].

AIM: To compare the binding affinity of LAIR-1 and LAIR-2 with their ligands. METHODS: The ligand-expressing cells were incubated with the LAIR-1 and/or LAIR-2 fusion proteins at different concentration at the same time or in succession. Then the interaction changes and competition were measured by influorescent staining and flow cytometric analysis with the specific mAbs against LAIR-1 or LAIR-2. RESULTS: The membrane ligand for LAIR-1 and LAIR-2 was expressed extensively and ligand recognition was of cross-species. LAIR-2 blocked the interaction of LAIR-1 with its ligand but LAIR-1 didn't block the interaction of LAIR-2 with its ligand. CONCLUSION: LAIR-1 and LAIR-2 probably bind to the same ligand with different affinity, which provides some significant evidence for investigating the molecular mechanisms of LAIR-1 family in modulating immune response.

[Construction and expression of the fusion vector for HA-tagged human RAGE gene].

OBJECTIVE: To construct a mammalian expression vector for HA-tagged receptor of advanced glycation end products (RAGE). METHODS: Human RAGE cDNA codon region was amplified by PCR from human cDNA library and cloned into the pcDNA3 vector following routine procedures. After identification by enzyme digestion, PCR and sequencing, the correct recombinant vector RAGE/pcDNA3 was inserted with HA sequence behind the signal peptide sequence of RAGE. After identification by sequencing, HA-RAGE/pcDNA3 was transfected into HEK293 cells, and its expression was detected by Western blotting. RESULTS: Identification by enzyme digestion, PCR and sequencing, confirmed the validity of the recombinant vector RAGE/pcDNA3, and HA-RAGE/pcDNA3 was highly expressed in HEK 293 cells. CONCLUSION: HA-tagged RAGE is successfully constructed and expressed in mammalian cells, which may facilitate functional study of RAGE in cell signal transduction.