Neutrophil autophagy and extracellular DNA traps contribute to airway inflammation in severe asthma.

BACKGROUND: Autophagy and neutrophil extracellular DNA traps (NETs) are implicated in asthma; however, their roles in asthma pathogenesis have not been elucidated. OBJECTIVES: We compared autophagy and NET production levels from peripheral blood neutrophils (PBNs) of patients with severe asthma (SA) and non-severe asthma (NSA). Additionally, we investigated the inflammatory effects of NETs on human airway epithelial cells (AECs) and peripheral blood eosinophils (PBEs). METHODS: Peripheral blood neutrophils from patients with SA (n = 30) and NSA (n = 38) were treated with interleukin (IL)-8 (100 ng/mL). Autophagy (light chain 3-II expression) and NET production levels were evaluated by Western blot, immunofluorescence microscopy, and PicoGreen assay. The effects of NETs on AECs were assessed by investigating cell death, cell detachment, expression of occludin and claudin-1, and IL-8 production; the effects of NETs on PBEs were examined by investigating the activation and release of eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). RESULTS: Untreated and IL-8-treated PBNs from the SA group produced higher autophagy and NET levels compared with those from the NSA group (P < 0.01). IL-8 increased autophagy and NET levels in PBNs from the SA group, but not from the NSA group. NET levels were correlated with autophagy levels in PBNs (P < 0.001). IL-8-induced NET production levels negatively were correlated with FEV1/FVC (r = -0.700, P = 0.016). NETs induced cell death, detachment, degradation of occludin and claudin-1, and IL-8 production from AECs. Higher levels of NET-induced ECP and EDN were released from PBEs in SA compared with NSA groups. CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophil autophagy and NETs could enhance asthma severity by damaging airway epithelium and triggering inflammatory responses of AECs and PBEs. Modulating neutrophil autophagy and NET production may be a new target therapy for SA.

Autophagy mechanisms in sputum and peripheral blood cells of patients with severe asthma: a new therapeutic target.

BACKGROUND: Autophagy and genetic predisposition have been suggested to potentially play roles in the development of asthma. However, little is known about the role of autophagy in the pathogenesis of severe asthma. OBJECTIVE: We compared autophagy in the sputum granulocytes, peripheral blood cells (PBCs) and peripheral blood eosinophils (PBEs) between patients with severe asthma and those with non-severe asthma and investigated the functional effects of autophagy. METHODS: We enrolled 36 patients with severe asthma, 14 with non-severe asthma and 23 normal healthy controls in this study. Sputum granulocytes, PBCs and PBEs were isolated from each subject. Autophagy was evaluated based on the expression of microtubule-associated protein light chain 3 (LC3) by Western blot, confocal microscopy, transmission electron microscopy and flow cytometry. IL-8 levels were measured by ELISA. To induce autophagy, HL-60 cells, human primary small airway epithelial cells (SAECs) and A549 cells were treated with IL-5, IL-1ß and TNF-α. To inhibit autophagy, PI3K inhibitors (LY29400 and 3-methyladenine [3-MA]) and hydroxychloroquine (HCQ) were used. Knockdown of ATG5 and Beclin-1 was performed in A549 cells, and the therapeutic effects of dexamethasone were evaluated. RESULTS: Higher autophagy levels were noted in sputum granulocytes, PBCs and PBEs from patients with severe asthma than from patients with non-severe asthma and healthy controls (P < 0.05 for all). IL-5 increased autophagy levels in both PBCs and PBEs (P < 0.05). 3-MA attenuated the increased expression of LC3-II and eosinophil cationic protein in HL-60 cells induced by IL-5 (P = 0.034 for both). Dexamethasone did not affect autophagy levels in PBEs. IL-1ß increased LC3-II expression and IL-8 production (P < 0.01) in SAECs, and this was attenuated by LY294002, 3-MA, HCQ and knockdown of ATG5 and Beclin-1 (in A549 cells) (P < 0.01). CONCLUSIONS AND CLINICAL RELEVANCE: Autophagy could play a role in the pathogenesis of severe asthma. Autophagy modulation may be a novel therapeutic target for conventional therapy-resistant severe asthma.

Effects of immunization with the rNfa1 protein on experimental Naegleria fowleri-PAM mice.

Free-living Naegleria fowleri causes primary amoebic meningoencephalitis (PAM) in humans and animals. To examine the effect of immunization with Nfa1 protein on experimental murine PAM because of N. fowleri, BALB/c mice were intra-peritoneally or intra-nasally immunized with a recombinant Nfa1 protein. We analysed Nfa1-specific antibody and cytokine induction, and the mean survival time of infected mice. Mice immunized intra-peritoneally or intra-nasally with rNfa1 protein developed specific IgG, IgA and IgE antibodies; the IgG response was dominated by IgG1, followed by IgG2b, IgG2a and IgG3. High levels of the Th1 cytokine, IFN-γ, and the regulatory cytokine, IL-10, were also induced. The mean survival time of mice immunized intra-peritoneally with rNfa1 protein was prolonged compared with controls, (25.0 and 15.5 days, respectively). Similarly, the mean survival time of mice immunized intra-nasally with rNfa1 protein was 24.7 days, compared with 15.0 days for controls.

Identification of the amino acid sequence motif for conventional PKC-mediated regulation of NKp46 surface expression.

A number of surface receptors expressed on NK cells are related to the regulation of NK cell activity and characterized by either inhibitory or activating properties. Natural cytotoxicity receptors (NCR) are one major family of activating receptors involved in NK cytotoxicity. The three family members of NCR are NKp46, NKp44, and NKp30. The surface density of these receptors might vary with the activation state of NK cells, and their density may directly correlate with their natural cytotoxicity. In this study, we investigated the regulation of NKp46 expression and determined the amino acid sequence motif for protein kinase C (PKC)-mediated regulation of its surface expression. We produced stable cell lines expressing full-length NKp46 and investigated the change in expression after PMA or IL-2 treatment using flow cytometry, RT-PCR, and immunoblotting methods. Expression of NKp46 on Jurkat T-cell transfectants appeared to be induced by PMA treatment until 8-h post-treatment and then gradually decreased afterwards to levels that were less than those measured at pretreatment. Parallel to surface expression of NKp46, total NKp46 protein expression also appeared to fluctuate after PMA treatment, but the expression of mRNA transcripts was not significantly affected. Experiments with mutant NKp46-expressing stable cell lines demonstrated that Ser288 might be critical for the surface expression of NKp46 and the PKC-mediated regulation of NKp46 expression. However, NKp46 surface expression was not influenced by IL-2 in stable cell lines expressing wild-type and mutant NKp46.

Diversity of the repertoire of p58 killer cell inhibitory receptors in a single individual.

p58 Killer cell inhibitory receptors (KIRs) recognize HLA-C molecules on target cell surface and transmit an inhibitory signal to prevent cell-mediated cytotoxicity. p58 KIR family is composed of multiple receptors whose amino acid sequences are similar but their ligand specificity is different. However, it is not clear how diverse the repertoire of p58 KIR is, particularly in a single individual. To address this question, cDNAs were cloned encoding the extracellular domain of p58 KIR from a single individual. Twelve different p58 KIRs were identified suggesting that the repertoire in a single individual is highly diverse. Interestingly, seven of them have hybrid sequences of three previously reported p58 KIRs. Using an RNase protection assay, it was demonstrated that the mRNA transcripts of the hybrid KIRs are present in peripheral blood mononuclear cells (PBMCs). Four differently spliced forms of p58 KIR were also identified indicating that the repertoire is diverse in size as well as in sequence. Putative splicing sites found in p58 KIR cDNAs suggest that the differently spliced forms are produced by alternative splicing mechanism, and that the hybrid KIRs may also be generated by alternative splicing of two consecutive genes and/or by trans-splicing mechanism found in Ig genes in B cells.

Clonal expansion of T-cells in measles.

Some autoimmune complications such as postinfectious encephalomyelitis are associated with immunologic abnormalities induced by measles virus infection. To address the superantigenic stimulation in measles which might be related with autoimmune complications, T-cells bearing the TCRBV5S2 or TCRBV8 chains and the expression of activation markers were analyzed by monoclonal antibodies. To estimate clonal expansions, the CDR3 length profile in T-cells bearing the TCRBV5S2 or TCRBV8 chains was analyzed by two-stage PCR. Results showed that the expression of DR molecules in CD3+ cells was increased significantly in measles patients (19.6 +/- 20.7%) compared to healthy children (2.9 +/- 1.4%). The mean percentage (7.1 +/- 4.4%) of T-cells bearing the TCRBV8 chain was increased in measles patients compared to healthy children (5.6 +/- 3.1%). The percentage of T-cells bearing the TCRBV5S2 chain in measles patients (3.0 +/- 1.2%) was similar to that in healthy children (2.7 +/- 0.6%). By analysis of the CDR3 length we found that there was no evidence of clonal expansions in T-cells bearing the TCRBV8 chain and that there were clonal expansions in T-cells bearing the TCRBV5S2 chain. These data suggest a conventional antigenic stimulation with T-cells bearing the TCRBV5S2 chain and a superantigenic stimulation with T-cells bearing the TCRBV8 chain may occur in the acute stage of measles infection.

Serologic responses of Korean soldiers serving in malaria-endemic areas during a recent outbreak of Plasmodium vivax.

Anti-Pv200 antibody levels were assessed in samples from endemic areas of Plasmodium vivax malaria in the Republic of Korea (ROK), using an indirect enzyme-linked immunosorbent assay (ELISA) method. Asymptomatic carriers of P. vivax were detected using nested polymerase chain reaction (PCR) of blood samples. Anti-Pv200 antibody levels in 20 vivax malaria patients (optical density +/- standard deviation [OD +/- SD] values 1.85 +/- 0.29 of IgG isotype and 1.33 +/- 1.33 of IgM isotype) were markedly higher than those of uninfected, malaria-naive controls (0.08 +/- 0.16 of IgG isotype and 0.04 +/- 0.04 of IgM isotype). Antibody levels for 7 out of 8 soldiers with a recent malaria infection were sustained above the cut-off values for 4 months after successful treatment. Analysis of serum collected from 40 healthy, asymptomatic soldiers who had a P. vivax malaria attack within 3 months after our sampling, revealed 11 antibody-positive samples (27.5%), compared to 5 positive samples (12.5%) collected from a random selection of 40 soldiers. Among a larger pool of 1,713 soldiers who had served in high-risk areas for P. vivax transmission, 15% were antibody positive. Among 1,000 blood samples from asymptomatic soldiers who had served in the high-risk areas, 4 samples (0.4%) were parasite positive, as determined by nested PCR. Our results show that anti-Pv200 antibody levels can provide useful information in the late diagnosis of P. vivax malaria infection in a previously naive population and also in large seroepidemiologic studies. Furthermore, our results suggest that asymptomatic P. vivax carriers could be important in the current outbreak of malaria in Korea.

Diversity of the p70 killer cell inhibitory receptor (KIR3DL) family members in a single individual.

NK cells and some T cells express members of a multigenic family of killer cell inhibitory receptors (KIRs) including p70 KIR (KIR3DL) and p58 KIR (KIR2DL) family that recognize polymorphic class I MHC molecules on target cells and transmit an inhibitory signal to prevent killer cell-mediated cytoxicity. The cDNA sequences of p70 KIR family members reported so far suggest that the p70 KIR gene consists of a multigene complex and that each gene may exhibit certain degrees of polymorphism. However, it is not clear how diverse the repertoire of the p70 KIR family is, particularly in a single individual. To address this question in more detail and to determine some indication as to the origin of the diversity, we cloned p70 KIR cDNAs from a single individual. We identified nine new KIRs that are different from the previously reported ones. A comparison of the amino acid sequences with published sequences of p70 KIRs showed that two clones belonged to the KIR3DL1 family, five clones belonged to the KIR3DL2 family, one clone belonged to the KIR2DL4 family, and one clone appeared to be an alternatively spliced form of p70 KIR. These results suggested that the repertoire of p70 KIR family members in a single individual is highly diverse. It is not clear how the diverse receptors are generated in a single individual, but a comparison of amino acid sequences of p70 KIR family members suggested that some of them may be encoded by distinct genes or their alleles, while others may be generated by a recombination mechanism and/or an alternative splicing mechanism at the maturation of the mRNA transcripts.

Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns.

Apoptosis, which is anti-inflammatory, and necrosis, which is pro-inflammatory, represent the extremes of the cell death spectrum. Cell death is complex and both apoptosis and necrosis can be observed in the same cells or tissues. Here, we introduce a novel combined mode of cellular demise--caspase-dependent regulated necrosis. Most importantly, it is mainly characterized with release of marked amount of oligo- or poly-nucleosomes and their attached damage-associated molecular patterns (DAMPs) and initiated by caspase activation. Caspase-activated DNase has dual roles in nucleosomal release as it can degrade extracellularly released chromatin into poly- or oligo-nucleosomes although it prohibits release of nucleosomes. In addition, osmotically triggered water movement following Cl(-) influx and subsequent Na(+) influx appears to be the major driving force for nucleosomal and DAMPs release. Finally, Ca(2+)-activated cysteine protease, calpain, is an another essential factor in nucleosomal and DAMPs release because of complete reversion to apoptotic morphology from necrotic one and blockade of nucleosomal and DAMPs release by its inhibition.

NF-κB and STAT3 cooperatively induce IL6 in starved cancer cells.

A number of genes involved in tumorigenesis have been known to be controlled by signal transducer and activator of transcription 3 (STAT3) and NF-κB, either synergistically or individually. In starved cancer cells, we found that NF-κB was activated through endoplasmic reticulum stress signals, which depend on reactive oxygen species, cytosolic calcium and preserved translation of NF-κB p65 subunit, but independent of IκBα serine phosphorylation, thereby resulting in IL6 induction. STAT3 was required for proper induction of IL6 by NF-κB. They existed as identical nuclear complexes in proximal IL6 promoters, and STAT3 had critical roles in binding to IL6 promoters as well as nuclear retention of NF-κB. The conditioned media from starved cancer cells contained various secretory factors, such as IL6, IL9, VWF (von Willebrand factor), FREM1 (FRAS1 related extracellular matrix 1), SAA1 (serum amyloid A1), SDK1 (sidekick homolog 1) and ADAM12 (ADAM metallopeptidase domain 12), induced by NF-κB and STAT3 and promoted clonogenic capacities of cancer cells, and proliferation and migration of human umbilical vein endothelial cells. These results suggest novel survival strategies of cancer cells by which two oncogenic transcriptional factors, NF-κB and STAT3, are activated simultaneously by an intrinsic mechanism during stressful conditions of cancer cells, and they cooperatively induce various survival factors.

Reconstitution of class I MHC molecules expressed in E. coli and complexed with single antigenic peptides.

The HLA-Cw3 heavy chain has been expressed at high level as insoluble protein aggregates in E. coli. The protein aggregates dissolved in strong denaturant solution were efficiently reconstituted by removal of denaturant in the presence of an HLA-Cw3 binding peptide (FAM) and beta 2m. The reconstituted HLA-Cw3/FAM protein binds specifically to a p58 natural killer cell inhibitory receptor, a natural ligand. The HLA-A2 molecule has also been reconstituted in complex with either of a peptide from myelin associated glycoprotein (MAG) or a peptide from the GAG protein of human immunodeficiency virus. The HLA-A2/MAG protein crystallized under the identical conditions as HLA-A2 purified from human lymphoblastoid cells. The reconstitution method has yielded an abundant supply of HLA molecules complexed with single antigenic peptides, and may be of general utility in reconstituting any class I MHC molecules. However, the HLA molecules could not be reconstituted either without a peptide or with an irrelevant peptide. Using this property, the reconstitution method could be used to determine whether a peptide is restricted/bound to certain class I MHC molecule.

Molecular basis of HLA-C recognition by p58 natural killer cell inhibitory receptors.

NK cells express several inhibitory receptors that recognize class I MHC molecules expressed on target cells. The NK cell inhibitory receptors (KIRs) provide a key regulatory function for NK cells via specific interaction with MHC/peptide complexes, but the molecular details for recognition of class I MHC molecules by KIRs still remain unclear. Here we report cDNA cloning and expression of p58 KIRs and a p50 killer cell activatory receptor (KAR) from a Korean blood donor and demonstrate direct binding between recombinant soluble p58 KIRs and recombinant soluble HLA-C molecules. We identified three p58/p50 killer cell receptors (KAR-K1, KIR-K6, and KIR-K7), which are homologous to p50 cl-39, p58 47.11, and p58 cl-6, respectively. Native gel shift assay revealed that p58 KIR-K6 and KIR-K7 bind both HLA-Cw3 and HLA-Cw6 molecules, but p50 KAR-K1 binds neither of the HLA-C molecules. However, binding of HLA-C molecule by p58 KIR is affected by the antigenic peptide bound on the MHC molecule, suggesting that the p58 KIR binding to the HLA-C molecule may be dependent on the peptide. In addition, the binding interaction requires the presence of both p58 Ig domains, suggesting that the binding mode of HLA-C and p58 KIR may have some similarity to that of the neonatal Fc receptor and the Fc fragment of Ab and may be distinct from that of TCR and MHC.

Evidence for selection of 11 amino acid CDR3 domains in V kappa III-derived immunoglobulin light chains in Kawasaki disease.

Kawasaki disease (KD) is a rheumatic disease that occurs during childhood. Although T cells have been implicated as having an important role in the pathogenesis of KD, the role of B cells is unclear. To detect preferential expression of 11 amino acid complementarity determining region (CDR)3 domains, we used two-stage PCR (polymerase chain reaction) to analyze the CDR3 lengths of VkIII-derived immunoglobulin kappa light chains expressed in peripheral blood B cells during the acute, subacute, and convalescent phase of this disease. As controls, the peripheral blood B cells of age-matched normal and children with acute febrile diseases other than KD were tested. In 5 of 7 KD patients, expression of kappa light chains containing 11 amino acid codon CDR3 intervals was increased during the acute and subacute phase of KD but decreased during the convalescent phase. Two of the 7 KD patients showed the same pattern during the subacute and convalescent phase, but not during the acute phase. Two of the 5 patients with acute febrile diseases other than KD showed increased expression of kappa chains with 11 amino acid codon CDR3 intervals, but it was not a major fraction. Three of the 5 patients with acute febrile diseases other than KD and all normal control subjects showed only 9 and 10 amino acid CDR3 domains. These results strongly suggest that B cells expressing kappa light chains with the 11 amino acid CDR3 domains might be involved in the pathogenesis of KD.

Clonal expansion of CD8+ T cells in Kawasaki disease.

Kawasaki disease (KD) is the major cause of acquired heart disease in children. KD is suspected of being an infectious disease, but the etiology has not yet been clarified. Immunologically, the disease is associated with the activation of T cells, monocytes, and macrophages resulting in highly elevated levels of several cytokines. Recently, expansions of T cells expressing TCRBV2 and TCRBV8 chains have been reported, and this suggests the involvement of a superantigen in the pathogenesis of KD. To address the role of a superantigen in KD, we investigated clonal expansion of T cells by estimating the complementarity-determining region 3 size profile among T cells expressing TCRBV1, TCRBV2, TCRBV4, TCRBV5, TCRBV8, TCRBV14, TCRBV16, TCRBV17, TCRBV18, and TCRBV20 chains during acute KD, during subacute KD, and during the long term follow-up period. During the acute phase of KD, several clonal expansions were found mainly in the CD8+ T cells that disappeared during the long term follow-up period. Our data suggest that the conventional Ags rather than a superantigen were involved in the pathogenesis of acute KD.

Enhanced expression of immunoglobulin kappa light chains with unusually long CDR3 regions in patients with rheumatoid arthritis.

OBJECTIVE: Our previous sequence analysis of immunoglobulin kappa light chains revealed that some patients with rheumatoid arthritis (RA) expressed repertoires enriched for transcripts containing unusually long CDR3 lengths of 11 amino acid codons. This was due, in part, to N region addition at the Vkappa-Jkappa joins. In this study, we analyzed a larger number of individuals to determine how often enrichment of kappa light chain repertoires for 11 amino acid CDR3 occurs in synovial lymphocytes and peripheral blood lymphocytes (PBL) of individuals with RA. METHODS: To measure length variability of kappa chain CDR3 regions, we performed a 2 stage polymerase chain reaction amplification and polyacrylamide gel electrophoresis. We sampled PBL and synovial lymphocytes of 9 patients with longstanding RA, and used PBL of 9 age and sex matched healthy individuals as controls. RESULTS: In PBL of healthy individuals, there was low level but consistent expression of kappa chains containing CDR3 with 11 amino acids. In patients, there was enhanced expression of kappa chains containing CDR3 with 11 amino acids compared to healthy individuals. This enhanced expression of kappa chains containing CDR3 of 11 amino acids was more pronounced in synovial lymphocytes compared to PBL of the same patients. CONCLUSION: These findings suggest that there is antigenic selection of B cells bearing antibodies with unusually long kappa light chain CDR3 in RA.