Clinical, radiological, and pathological features of anti-asparaginyl tRNA synthetase antibody-related interstitial lung disease.

BACKGROUND: Myositis and interstitial lung disease (ILD) frequently occur in patients with anti-aminoacyl-tRNA synthetase (ARS) antibodies. Nearly half of ARS-ILD patients have the acute or subacute form of the disease, and one-third of these patients show a deterioration in pulmonary function over the long-term course because of frequent recurrences and refractoriness to therapy. Several reports recently described different characteristics depending on the individual anti-ARS antibodies, and the anti-asparaginyl tRNA synthetase (KS) antibody was strongly linked to ILD rather than to myositis. We therefore hypothesized that KS-ILD may have clinical characteristics that differ from those of other ARS-ILDs. The aim of this study was to clarify the clinical, radiological, and pathological features of KS antibody-positive ILD. METHODS: We retrospectively analyzed 19 consecutive patients with KS-ILD who underwent initial clinical measurements and high-resolution computed tomography and pathological assessments. We also analyzed disease behavior based on pulmonary function test results during the follow-up period. RESULTS: Our KS-ILD cohort included patients with dermatomyositis (10.5%), primary Sjögren syndrome (5.3%), and idiopathic ILD (84.2%). Most patients presented with chronic onset (89.5%) and a nonspecific pattern of interstitial pneumonia at each radiological and pathological assessment (89.4% and 85.7%, respectively). The pulmonary function test results showed that the mean changes from the initial %forced vital capacity and %diffusing capacity of the lung for carbon monoxide at 3 years were 3.7% ± 2.9% and 9.35% ± 3.0%, respectively. CONCLUSIONS: Most KS-ILD patients showed a tendency for chronic disease onset and long-term stabilization of pulmonary function.

Immune Response to Brugia malayi Asparaginyl-tRNA Synthetase in Balb/c Mice and Human Clinical Samples of Lymphatic Filariasis.

Background: Lymphatic filariasis (LF) is a global health problem, with a peculiar nature of parasite-specific immunosuppression that promotes long-term pathology and disability. Immune modulation in the host by parasitic antigens is an integral part of this disease. The current study attempts to dissect the immune responses of aminoacyl-tRNA synthetases (AARS) with emphasis on Brugia malayi asparaginyl-tRNA synthetase (BmAsnRS), since it is one among the highly expressed excretory/secretory proteins expressed in all stages of the parasite life cycle, whereas its role in filarial pathology has not been elaborately studied. Methods and Results: In this study, recombinant BmAsnRS (rBmAsnRS) immunological effects were studied in semipermissive filarial animal model Balb/c mice and on clinically defined human samples for LF. In mice study, humoral responses showed considerable titer levels with IgG2a isotype followed by IgG2b and IgG1. Immunoreactivity studies with clinical samples showed significant humoral responses especially in endemic normal with marked levels of IgG1 and IgG2 followed by IgG3. The cell-mediated immune response, evaluated by splenocytes and peripheral blood mononuclear cells proliferation, did not yield significant difference when compared with control groups. Cytokine profiling and qRT-PCR analysis of mice samples immunized with rBmAsnRS showed elevated levels of IFN-γ, IL-10, inhibitory factor-cytotoxic T lymphocyte-associated protein-A (CTLA-4) and Treg cell marker-Forkhead Box P3 (FoxP3). Conclusions: These observations suggest that rBmAsnRS has immunomodulatory effects with modified Th2 response along with suppressed cellular proliferation indicating the essence of this molecule for immune evasion by the parasite.

Unique N-terminal extension domain of human asparaginyl-tRNA synthetase elicits CCR3-mediated chemokine activity.

Asparaginyl-tRNA synthetase (NRS) is not only essential in protein translation but also associated with autoimmune diseases. Particularly, patients with antibodies that recognize NRS often develop interstitial lung disease (ILD). However, the underlying mechanism of how NRS is recognized by immune cells and provokes inflammatory responses is not well-understood. Here, we found that the crystal structure of the unique N-terminal extension domain of human NRS (named as UNE-N, where -N denotes NRS) resembles that of the chemotactic N-terminal domain of NRS from a filarial nematode, Brugia malayi, which recruits and activates specific immune cells by interacting with CXC chemokine receptor 1 and 2. UNE-N induced migration of CC chemokine receptor 3 (CCR3)-expressing cells. The chemokine activity of UNE-N was significantly reduced by suppressing CCR3 expression with CCR3-targeting siRNA, and the loop3 region of UNE-N was shown to interact mainly with the extracellular domains of CCR3 in nuclear magnetic resonance perturbation experiments. Based on these results, evolutionarily acquired UNE-N elicits chemokine activities that would promote NRS-CCR3-mediated proinflammatory signaling in ILD.

The pulmonary histopathology of anti-KS transfer RNA synthetase syndrome.

CONTEXT: The clinical spectrum of the antisynthetase syndromes (AS) has been poorly defined, although some frequently present with pulmonary manifestations. The anti-KS anti-asparaginyl-transfer RNA synthetase syndrome is one in which pulmonary interstitial lung disease is almost always present and yet the histopathologic spectrum is not well described. OBJECTIVE: To define the morphologic manifestations of pulmonary disease in those patients with anti-KS antiasparaginyl syndrome. DESIGN: We reviewed the connective tissue disorder registry of the University of Pittsburgh and identified those patients with anti-KS autoantibodies who presented with interstitial lung disease and had surgical lung biopsies. RESULTS: The 5 patients with anti-KS antisynthetase syndrome were usually women presenting with dyspnea and without myositis, but with mechanic's hands (60%) and Raynaud phenomenon (40%). They most often presented with a usual interstitial pneumonia pattern of fibrosis (80%), with the final patient displaying organizing pneumonia. CONCLUSIONS: Pulmonary interstitial lung disease is a common presentation in patients with the anti-KS-antisynthetase syndrome, who are often women with rather subtle or subclinical connective tissue disease, whereas the literature emphasizes the nonspecific interstitial pneumonia pattern often diagnosed clinically. Usual interstitial pneumonia and organizing pneumonia patterns of interstitial injury need to be added to this clinical differential diagnosis.

Crucial role of macrophage selenoproteins in experimental colitis.

Inflammation is a hallmark of inflammatory bowel disease (IBD) that involves macrophages. Given the inverse link between selenium (Se) status and IBD-induced inflammation, our objective was to demonstrate that selenoproteins in macrophages were essential to suppress proinflammatory mediators, in part, by the modulation of arachidonic acid metabolism. Acute colitis was induced using 4% dextran sodium sulfate in wild-type mice maintained on Se-deficient (<0.01 ppm Se), Se-adequate (0.08 ppm; sodium selenite), and two supraphysiological levels in the form of Se-supplemented (0.4 ppm; sodium selenite) and high Se (1.0 ppm; sodium selenite) diets. Selenocysteinyl transfer RNA knockout mice (Trsp(fl/fl)LysM(Cre)) were used to examine the role of selenoproteins in macrophages on disease progression and severity using histopathological evaluation, expression of proinflammatory and anti-inflammatory genes, and modulation of PG metabolites in urine and plasma. Whereas Se-deficient and Se-adequate mice showed increased colitis and exhibited poor survival, Se supplementation at 0.4 and 1.0 ppm increased survival of mice and decreased colitis-associated inflammation with an upregulation of expression of proinflammatory and anti-inflammatory genes. Metabolomic profiling of urine suggested increased oxidation of PGE2 at supraphysiological levels of Se that also correlated well with Se-dependent upregulation of 15-hydroxy-PG dehydrogenase (15-PGDH) in macrophages. Pharmacological inhibition of 15-PGDH, lack of selenoprotein expression in macrophages, and depletion of infiltrating macrophages indicated that macrophage-specific selenoproteins and upregulation of 15-PGDH expression were key for Se-dependent anti-inflammatory and proresolving effects. Selenoproteins in macrophages protect mice from dextran sodium sulfate-colitis by enhancing 15-PGDH-dependent oxidation of PGE2 to alleviate inflammation, suggesting a therapeutic role for Se in IBD.

Nematode asparaginyl-tRNA synthetase resolves intestinal inflammation in mice with T-cell transfer colitis.

The therapeutic effects of a controlled parasitic nematode infection on the course of inflammatory bowel disease (IBD) have been demonstrated in both animal and human models. However, the inability of individual well-characterized nematode proteins to recreate these beneficial effects has limited the application of component immunotherapy to human disease. The nematodes that cause chronic human lymphatic filariasis, Brugia malayi and Wuchereria bancrofti, are among the parasites that induce immune suppression. Filarial lymphatic pathology has been shown to involve NF-κB pathway-dependent production of vascular endothelial growth factor (VEGF), and stimulation of VEGF expression has also been reported by interleukin 8 (IL-8) via NF-κB pathways. Previously, we have shown that the filarial asparaginyl-tRNA synthetase (rBmAsnRS) interacts with IL-8 receptors using a combination of extracellular loops that differ from those bound by IL-8. To test the hypothesis that rBmAsnRS might induce an anti-inflammatory effect in vivo, we studied the effects of rBmAsnRS in an established murine colitis model using T-cell transfer mice. T-cell transfer colitis mice treated intraperitoneally with 100 µg of rBmAsnRS four times over 2 weeks showed resolution of cellular infiltration in the colonic mucosa, along with induction of a CD8(+) cellular response. In addition, rBmAsnRS induced a rise in IL-10 production from CD3(+) and lipopolysaccharide (LPS)- and cytosine phosphate guanosine (CPG)-stimulated splenic cells. In summary, this work demonstrates a novel anti-inflammatory nematode protein, supports the hygiene hypothesis, and supports continued refinement of alternative immunotherapies for treatment of IBD.

Interleukin-8-like activity in a filarial asparaginyl-tRNA synthetase.

A wide range of secondary biological functions have been documented for eukaryotic aminoacyl-tRNA synthetases including roles in transcriptional regulation, mitochondrial RNA splicing, cell growth, and chemokine-like activities. The asparaginyl-tRNA synthetase (AsnRS) of the filarial nematode, Brugia malayi, is a highly expressed excretory-secretory molecule which activates interleukin 8 (IL-8) receptors via extracellular domains that are different from those used by IL-8. Recent success in determining the complete atomic structure of the B. malayi AsnRS provided the opportunity to map its chemokine-like activity. Chemotaxis assays demonstrated that IL-8-like activity is localized in a novel 80 amino acid amino terminal substructure. Structural homology searches revealed similarities between that domain in B. malayi AsnRS and substructures involved in receptor binding by human IL-8. These observations provide important new insights into how parasite-derived molecules may play a role in the modulation of immune cell function.

Identification of modifications in microbial, native tRNA that suppress immunostimulatory activity.

Naturally occurring nucleotide modifications within RNA have been proposed to be structural determinants for innate immune recognition. We tested this hypothesis in the context of native nonself-RNAs. Isolated, fully modified native bacterial transfer RNAs (tRNAs) induced significant secretion of IFN-α from human peripheral blood mononuclear cells in a manner dependent on TLR7 and plasmacytoid dendritic cells. As a notable exception, tRNA(Tyr) from Escherichia coli was not immunostimulatory, as were all tested eukaryotic tRNAs. However, the unmodified, 5'-unphosphorylated in vitro transcript of tRNA(Tyr) induced IFN-α, thus revealing posttranscriptional modifications as a factor suppressing immunostimulation. Using a molecular surgery approach based on catalytic DNA, a panel of tRNA(Tyr) variants featuring differential modification patterns was examined. Out of seven modifications present in this tRNA, 2'-O-methylated G(m)18 was identified as necessary and sufficient to suppress immunostimulation. Transplantation of this modification into the scaffold of yeast tRNA(Phe) also resulted in blocked immunostimulation. Moreover, an RNA preparation of an E. coli trmH mutant that lacks G(m)18 2'-O-methyltransferase activity was significantly more stimulatory than the wild-type sample. The experiments identify the single methyl group on the 2'-oxygen of G(m)18 as a natural modification in native tRNA that, beyond its primary structural role, has acquired a secondary function as an antagonist of TLR7.

SLA/LP/tRNP((Ser)Sec) antigen in autoimmune hepatitis: identification of the native protein in human hepatic cell extract.

A diagnostic subgroup of AIH type 1 is characterized by specific serum antibodies against soluble liver protein. The respective autoantigen was named SLA/LP/tRNP((Ser)Sec), after three homologous recombinant polypeptides were isolated from expression gene libraries. We analyzed human cultured liver cells for the human homologue of recombinant SLA/LP/tRNP((Ser)Sec) by antigen purification. In addition, a monoclonal antibody was generated against recombinant SLA-p35, a truncated recombinant SLA-reactive polypeptide. With a positive patient serum, immune affinity chromatography was performed on the 52 kD-SLA main antigenic determinant pre-enriched by ion exchange chromatography. By mass spectrometry, the 52 kD-SLA/LP/tRNP ((Ser)Sec) autoantigen was unambiguously identified in the purification product. The identity of the recombinant SLA-p35 and its human homologue was further confirmed by a specific signal of the anti SLA-p35 monoclonal antibody with purified human SLA/LP/tRNP((Ser)Sec). The 48 kD-SLA species frequently comigrating in SLA-immunoblotting however was not identified by either approach. We conclude that the native counterpart of recombinant tRNP((Ser)(Sec)) indeed is detectable with a molecular weight of 52 kD in soluble liver extract of human cells as the major antigenic component of SLA/LP/tRNP((Ser)Sec).

Nonspecific interstitial pneumonia (NSIP) associated with anti-KS antibody: differentiation from idiopathic NSIP.

We report two cases of biopsy-proven nonspecific interstitial pneumonia (NSIP) with anti-KS (asparaginyl-tRNA) antibody. Anti-KS antibody is the sixth anti-aminoacyl-tRNA synthetase (ARS) antibody. They showed interstitial pneumonia without clinical symptoms, and high resolution computed tomography (HRCT) of the chest demonstrated consolidation along bronchovascular bundles and volume loss in the bilateral lower lobes, which were suggestive of connective tissue diseases (CTD). One case of cellular NSIP responded to corticosteroid, but the other case of fibrotic NSIP required corticosteroid and cyclosporin. In patients with these HRCT findings, the measurement of anti-ARS antibodies could be helpful even in the absence of clinical symptoms suggestive of CTD.

Clinical and immunogenetic features of patients with autoantibodies to asparaginyl-transfer RNA synthetase.

OBJECTIVE: We have previously described anti-KS autoantibodies and provided evidence that they are directed against asparaginyl-transfer RNA (tRNA) synthetase (AsnRS). The aim of the present study was to identify patients with anti-AsnRS autoantibodies and elucidate the clinical significance of this sixth antisynthetase antibody. In particular, we studied whether it was associated with the syndrome of myositis (polymyositis or dermatomyositis [DM]), interstitial lung disease (ILD), arthritis, and other features that had been previously associated with the 5 other anti-aminoacyl-tRNA synthetase autoantibodies. METHODS: More than 2,500 sera from patients with connective tissue disease (including myositis and ILD) and controls were examined for anti-AsnRS autoantibodies by immunoprecipitation (IP). Positive and control sera were tested for the ability to inhibit AsnRS by preincubation of the enzyme source with the serum. The HLA class II (DRB1, DQA1, DQB1, DPB1) alleles were identified from restriction fragment length polymorphism of polymerase chain reaction-amplified genomic DNA. RESULTS: Anti-AsnRS antibodies were identified in the sera of 8 patients (5 Japanese, 1 American, 1 German, and 1 Korean) by IP of the same distinctive set of tRNA and protein that differed from those precipitated by the other 5 antisynthetases, and these antibodies showed specific inhibition of AsnRS activity. Two of these patients had DM, but 7 of 8 (88%) had ILD. Four patients (50%) had arthritis, and 1 had Raynaud's phenomenon. This antisynthetase was very rare among myositis patients (present in 0% of Japanese myositis patients), but it was found in 3% of Japanese ILD patients. Thus, most patients with anti-AsnRS had chronic ILD with or without features of connective tissue disease. Interestingly, all 4 Japanese patients tested had DR2 (DRB1*1501/1502), compared with 33% of healthy controls. CONCLUSION: These results indicate that anti-AsnRS autoantibodies, like anti-alanyl-tRNA synthetase autoantibodies, have a stronger association with ILD than with myositis and may be associated with the DR2 phenotype.

Inhibition of in vitro amino acylation and translation by adenosine antibodies.

Adenosine antibodies markedly inhibited in vitro amino acylation of tRNA in a dose-dependent manner. The inhibition was specific as it was reversed by the homologous hapten. Addition of excess tRNA reversed the inhibition indicating that binding of antibodies to tRNA is responsible for inhibition. Adenosine antibodies also inhibited in vitro translation of endogenous mRNAs in rabbit reticulocyte lysate in a dose-dependent manner. The homologous hapten reversed the inhibition showing thereby the immunospecificity of inhibition.

A human auto-immune antibody specifically recognizing initiator methionine tRNA from yeast and higher eucaryotes.

Analysis of sera from 168 patients with autoimmune disorders revealed that one patient with Sjôgren's syndrome produced antibodies against deproteinized initiator methionine tRNA in addition to those against La protein. This anti-tRNAimet recognizes also tRNAimet from yeast but not from Phaseolus vulgaris chloroplasts (bean) or E. coli. It appears therefore that the epitope could be located in the TF loop in which an A residue in position 60 and the AUCG sequence are the only common features in yeast and human tRNAimet.

Autoantibodies specific for U1 RNA and initiator methionine tRNA.

Autoantibodies reactive with specific nuclear and cytoplasmic small RNAs were identified by immunoprecipitation of HeLa cell RNA. Approximately 30% of antisera examined from patients with autoimmune disorders contained anti-RNA antibodies. Two previously undescribed specificities--anti U1 RNA and anti-initiator methionine tRNA--were identified. Anti-RNA antibodies were reactive with gel-purified species as well as with RNA synthesized in vitro using the SP-6 transcription system. Antigenic mapping using two sera specific for the human initiator methionine tRNA revealed separate epitopes, one of which is conserved in formyl-methionine initiator tRNA from Escherichia coli. RNA fragmentation studies further suggested that secondary or tertiary tRNA structure is required for antibody recognition. The mammalian U1 RNA specific antibodies did not precipitate small RNAs of yeast but were highly reactive with yeast ribosomal RNA, thus indicating a possible relationship between these RNA species. Results obtained with these antisera are discussed in terms of higher order structure of small RNA molecules as well as the role of nucleic acid antibodies in the autoimmune phenomenon.

Isolation of a novel antibody, which precipitates ribonucleoprotein complex containing threonine tRNA from a patient with polymyositis.

We have isolated and characterized a novel antibody from a patient with polymyositis, which precipitates threonine tRNA and an unknown small RNA molecule from 32P-labeled HeLa extract. Fingerprint analysis showed that the human threonine tRNA precipitated was nearly identical to the murine tRNA described by Harada [(1978) Seikagaku 50, 397-411]. Nucleotide analysis suggested that the other small RNA molecule might be transfer RNA. Since phenol extraction resulted in the loss of immunoprecipitability, the protein portion was presumed to be involved in the recognition of the antigen. Immunofluorescence staining of HeLa cells with the antibody clearly demonstrated that the antigen was located predominantly in the cytoplasm.

Immunoelectron microscopic localization of the S19 site on the 30 S ribosomal subunit which is crosslinked to A site bound transfer RNA.

Phe-tRNA of Escherichia coli, specifically derivatized at the S4U8 position with the 9 A long p-azidophenacyl photoaffinity probe, was crosslinked exclusively to protein S19 of the 30 S ribosomal subunit when the transfer RNA occupied the ribosomal A site (Lin et al., 1983). Two antigenic sites for S19 are known, on opposite sides of the head of the subunit. In this work, discrimination between these two sites was accomplished by affinity immunoelectron microscopy. A dinitrophenyl group was placed on the acp3U47 residue of the same tRNA molecules bearing the photoprobe on S4U8. Addition of this group affected neither aminoacylation, A site binding, nor crosslinking. It also made possible specific affinity purification of crosslinked tRNA-30 S complexes from unreactive 30 S. Reaction of the 2,4-dinitrophenyl-labeled tRNA-30 S complex with antibody was followed by immunoelectron microscopy to reveal the sites of attachment. All of the bound antibody was associated with the ribosome region corresponding to only one of the two known antigenic sites for S19, namely the one closer to the large side projection of the 30 S subunit. A site within this region must be within 10 A of the S4U8 residue of tRNA when it is bound in the ribosomal A site.

A mammalian tRNAHis-containing antigen is recognized by the polymyositis-specific antibody anti-Jo-1.

The mammalian cell antigen reactive with the autoantibody anti-Jo-1 has been shown to contain tRNAHis. The RNA sequence of this human and mouse cell tRNA was determined in a search for unusual features that might be related to antigenicity. The 5' terminal nucleotide is unique among other sequenced tRNAs in that it is a methylated guanine. The presence of the hypermodified base queuine, which occurs in the wobble position of the anticodon of tRNAHis from several species, was not detected in the tRNAHis immunoprecipitated by anti-Jo-1 from either human HeLa or mouse Friend erytholeukemia cell extracts. The binding of protein(s) appears to confer antigenicity on tRNAHis since either proteinase K treatment or phenol extraction resulted in the loss of immunoprecipitability. However, we have not succeeded in identifying an antigenic protein, and we find that the antigenic complex is not resolved from purified tRNAHis by Sephacryl S-200 column chromatography. Immunofluorescence studies indicate that the antigenic form of tRNAHis is located preferentially in the mammalian cell cytoplasm. The results presented here are discussed in light of an earlier report (1) on the nature of the Jo-1 antigen.