Antigen-specific T(h)1 cells as direct effectors of Propionibacterium acnes-primed lipopolysaccharide-induced hepatic injury.

T(h)1 cells are cytotoxic effector cells that utilize Fas ligand (FasL) and tumor necrosis factor. The physiological roles of cytotoxic T(h)1 cells are considered to be immunoregulation by eliminating autoreactive lymphocytes or hyper-activated foreign antigen-specific lymphocytes. Their pathological roles, however, remain to be clarified. To investigate whether T(h)1 cells can destroy organs, we generated a Propionibacterium acnes-specific T(h)1 clone from C57BL/6 mice and tested whether the clone could serve as an effector in a P. acnes-primed lipopolysaccharide (LPS)-induced hepatic injury system, one of the septic shock models. B6SMN:C3H-FasL(gld) (B6-gld) mice, which were deficient in functional FasL, were resistant to P. acnes/LPS-induced hepatic shock. The T(h)1 clone rendered B6-gld mice sensitive to the hepatic shock after the i.v. transfer. The hepatic injury in the clone-transferred B6-gld mice, which was evaluated by both biochemical and histological examination, was inhibited by an anti-FasL mAb that we developed. These results suggested that bacterial antigen-specific T(h)1 cells like this clone can participate in organ destruction in vivo as one of the cytotoxic effectors and play a critical role in endotoxin-induced hepatic injury.

Cloning of novel soluble gp130 and detection of its neutralizing autoantibodies in rheumatoid arthritis.

In an attempt to isolate disease-associated autoantigens in rheumatoid arthritis (RA), we cloned a new autoantigen named gp130-RAPS, which is a novel soluble form of the IL-6 signal-transducing molecule gp130. gp130-RAPS is a 50-kDa protein translated from alternatively spliced mRNA and has a truncated form of gp130 with a unique sequence, Asn-Ile-Ala-Ser-Phe (NIASF), in its COOH-terminus. We observed serum antibodies to this NIASF sequence frequently in patients with RA, but not in those with other systemic rheumatic diseases or in healthy subjects. In RA, detection of those antibodies was significantly associated with disease activity indices such as serum C-reactive protein (CRP) levels, erythrocyte sedimentation rate, blood platelet counts, and serum IL-6 concentration. In vitro experiments revealed that gp130-RAPS inhibited IL-6 activity, and this inhibition was neutralized by antibodies to the COOH-terminus of gp130-RAPS derived from patients with RA. Thus, autoantibody to gp130-RAPS may play an important role in the progression of RA by promoting IL-6 activity. Inspection of autoantibodies to gp130-RAPS may become a practical clinical test for RA. gp130-RAPS and its autoantibody provide a new clue to the complicated pathogenesis of RA.

Detection of anti-neutrophil cytoplasmic antibodies in MRL/Mp-lpr/lpr mice and analysis of their target antigens.

Anti-neutrophil cytoplasmic antibodies (ANCA) have been widely studied and recognized to be clinically very important for some human diseases including systemic rheumatic diseases. We analyzed ANCA response and their target antigens in MRL/Mp-lpr/lpr (MRL-lpr) mice, an animal model of systemic rheumatic disease. P-ANCA was detected in 57% of the mice. Antibodies to the known P-ANCA target antigens at the same age were examined. Among these, antibodies to high mobility group (HMG) proteins HMG1 and HMG2 were detected in 57% of the mice, 75% of which were also positive for P-ANCA. These anti-HMG1/HMG2 activities were absorbed by preincubation with a mixture of HMG1 and HMG2. In contrast, antibodies to myeloperoxidase and cathepsin G were detected in 14% and 7%, respectively, but these activities were not inhibited by preincubation with corresponding antigens. In addition, the titers of P-ANCA and anti-HMG1/HMG2 antibodies in MRL-lpr mice were significantly correlated with each other. Thus, HMG1 and HMG2 were considered to be significant target antigens of P-ANCA in MRL-lpr mice.

High mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 are significant target antigens of perinuclear anti-neutrophil cytoplasmic antibodies in autoimmune hepatitis.

BACKGROUND: High mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 have been identified as novel antigens of perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCAs), and the existence of anti-HMG1 and anti-HMG2 antibodies in a population of patients with ulcerative colitis has been reported. AIMS: To investigate whether HMG1 and HMG2 are target antigens for p-ANCAs in autoimmune hepatitis (AIH). PATIENTS: Serum samples from 28 patients with AIH, 44 patients with primary biliary cirrhosis (PBC), 27 patients with chronic hepatitis C, and 23 patients with chronic hepatitis B were tested. METHODS: ANCAs were detected by routine indirect immunofluorescence (IIF). Anti-HMG1 and anti-HMG2 antibodies were assayed by enzyme linked immunosorbent assay. RESULTS: p-ANCAs were detected in 89% (25/28) of patients with AIH, 36% (16/44) of patients with PBC, 11% (3/27) of patients with chronic hepatitis C, and 13% (3/23) of patients with chronic hepatitis B. Anti-HMG1 and/or anti-HMG2 antibodies were detected in 89% (25/28) of patients with AIH, 70% (31/44) with PBC, 26% (7/27) with chronic hepatitis C, and 9% (2/23) with chronic hepatitis B. In AIH, anti-HMG1 and/or anti-HMG2 antibodies were detected in 96% (24/25) of p-ANCA positive patients. The p-ANCA staining pattern detected by IIF using sera from patients with AIH disappeared or decreased in titre after preincubation with a mixture of HMG1/HMG2. The presence and titres of those antibodies in AIH correlated significantly with those of p-ANCA, but not with those of anti-nuclear antibody or anti-smooth muscle antibody. CONCLUSIONS: HMG1 and HMG2 are significant target antigens of p-ANCA in AIH.

Cloning of follistatin-related protein as a novel autoantigen in systemic rheumatic diseases.

In an attempt to identify autoantigens of synovium in rheumatoid arthritis (RA), we constructed lambda phage expression cDNA libraries from synovium and screened them by IgG purified from synovial fluids, both of which were derived from RA patients. As a result of this unique combination of the libraries and probes, we cloned follistatin-related protein (FRP) as a novel autoantigen in systemic rheumatic diseases. FRP is a secreted protein containing a similar amino acid sequence to follistatin, an inhibitor of activin. FRP was first cloned as a transforming growth factor-beta1-inducible protein (called TSC-36) from a mouse osteoblastic cell line and was suggested to have some roles in the negative regulation of cellular growth. Immunoblotting analyses detected synovial fluid and serum anti-FRP antibodies of IgG class more frequently in RA than any other systemic rheumatic diseases and controls. Synovial fluid anti-FRP antibodies appeared in 44% of RA (n = 18) and none of osteoarthritis (OA) (n = 15) patients. Serum antibodies were detected in 30% of RA (n = 67), 17% of systemic sclerosis (n = 18), 10% of systemic lupus erythematosus (n = 51) and Sjögren's syndrome (n = 10), and none of polymyositis/dermatomyositis (n = 13) patients and healthy subjects (n = 30). These antibodies recognized an EC domain, an extracellular Ca2+ binding module. In anti-FRP antibody-positive RA patients, serum C-reactive protein level and erythrocyte sedimentation rate were more elevated than negative patients (P < 0.05 and P < 0.01, respectively). FRP gene expression was higher in RA than OA synovium (P < 0.05). However, there was no difference between these groups in the amount of synovial FRP, suggesting its elevated turnover in RA. As follistatin inhibits activin, FRP might inhibit some growth factor-like molecule. Detection of anti-FRP antibodies, possibly having disease-promoting effects as the blocking antibodies, could be one of the markers for clinical evaluation of systemic rheumatic diseases.

Prevalence and characterization of novel pANCA, antibodies to the high mobility group non-histone chromosomal proteins HMG1 and HMG2, in systemic rheumatic diseases.

OBJECTIVE: To determine the immunodiagnostic value of antibodies to the high mobility group non-histone chromosomal proteins HMG1 and HMG2, which have been identified as novel target antigens of perinuclear antineutrophil cytoplasmic antibodies (pANCA), in sera from patients with systemic rheumatic diseases. METHODS: Anti-HMG1 or HMG2 antibody was assayed by ELISA and Western blotting in sera from patients with systemic rheumatic diseases. These antibodies were analyzed for the relationship with pANCA detected by indirect immunofluorescence in these diseases, and with clinical features in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). RESULTS: Anti-HMG1 or HMG2 antibody was frequently detected in sera from patients with RA (48%), SLE (45%), Sjögren's syndrome (SS) (44%), and systemic sclerosis (SSc) (41%). In these diseases, anti-HMG1 antibody was detected more frequently than anti-HMG2 antibody. In sera from patients with RA, the positivity for anti-HMG1 and HMG2 antibodies was significantly correlated with the positivity for pANCA (p < 0.0001). Anti-HMG1/HMG2 antibodies were associated with some disease activity variables, e.g., erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, joint score and hand grip strength in RA, and CH50, C3, C4, and IgG in SLE. CONCLUSION: Anti-HMG1/HMG2 antibodies are detected commonly in systemic rheumatic diseases, particularly in RA, SLE, SS, and SSc. HMGI and HMG2 seem to be the significant target antigens of pANCA in RA. These antibodies are significantly associated with disease activity indices in RA and SLE.

Prevalence and characterization of perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCA) directed against HMG1 and HMG2 in ulcerative colitis (UC).

In a previous study, we reported that the high mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 were novel target antigens of P-ANCA. In this study, we determined the immunodiagnostic value of anti-HMG1/HMG2 antibodies in patients with UC. Sixty sera from patients with UC were tested for reactivity with HMG1 and HMG2 by means of ELISA. Anti-HMG1 antibody was detected in 32% of patients (40% of P-ANCA+ patients). Anti-HMG2 antibody was detected in 33% (40% of P-ANCA+ patients). Thirty-five percent of sera were positive for antibody to either HMG1 or HMG2 (43% of P-ANCA+ patients). P-ANCA+ patients expressed anti-HMG1/HMG2 antibodies with significantly greater frequency compared with P-ANCA- patients. Furthermore, the anti-HMG1/HMG2 antibodies were significantly related to disease activity in UC. Sixteen of the 18 UC patients, who had high titres of anti-HMG1 or -HMG2 antibody during the active phase, showed lower titres in the inactive phase. Anti-HMG1/HMG2 antibodies appear to be useful as a marker for disease activity in UC.

Novel autoantigens of perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCA) in ulcerative colitis: non-histone chromosomal proteins, HMG1 and HMG2.

Anti-neutrophil cytoplasmic antibodies (ANCA) in sera from ulcerative colitis (UC) patients have been described as reacting with proteins in the granules of human neutrophils such as cathepsin G and lactoferrin and with yet unidentified antigens. Here we report the existence of a new member of perinuclear ANCA (P-ANCA) in UC patients. In the previous study, we found that UC patients had a novel P-ANCA against neutrophil 28-kD protein. In this study, we purified the same antigens from HL-60 lysates by using reversed phase high-performance liquid chromatography, and revealed that the 28-kD antigen consisted of two different proteins. The N-terminus amino acids of these proteins are identical with those of high mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2. Immunoblotting analysis of human neutrophil lysates using rabbit anti-HMG1/2 antisera revealed a single band of 28 kD, and the 28-kD band detected by immunoblotting analysis using patient's serum IgG completely disappeared after preincubation with a mixture of HMG1 and HMG2. Furthermore, rabbit anti-HMG1/2 antisera showed a perinuclear staining pattern in indirect immunofluorescence studies using ethanol-fixed neutrophils. These data demonstrate that HMG1 and HMG2 are novel target antigens of P-ANCA. HMGI and HMG2 are distributed in the nuclei and cytoplasm of eukaryotic cells and act as transcription factors. Their intracellular localization and functions are distinct from those of the previously reported granular antigens of P-ANCA.

Anti-neutrophil cytoplasmic antibodies (ANCA) in ulcerative colitis: anti-cathepsin G and a novel antibody correlate with a refractory type.

We analysed the clinical significance of ANCA in patients with ulcerative colitis. On either an indirect immunofluorescence assay or an ELISA with fixed neutrophils, 71% (25/35) of the patients were positive for ANCA. However, only half of them reacted with either cathepsin G or lactoferrin. Western blot assays revealed positive bands in 40% (10/25) of the antibody-positive patients. The sizes of the bands detected were approximately 58, 47, 44, 40, and 28 kD. No significant correlation was found between the ANCA positivity and various variables, i.e. disease activity, extent of lesion, or treatment of the disease. The anti-cathepsin G and 28-kD positivity, however, significantly correlated with a refractory type of ulcerative colitis.

Behavioural effects of intrathecally injected tachykinins in rats with peripheral nerve transection.

The effect of unilateral sciatic nerve transection on behavioural responses produced by intrathecal administration of substance P (SP), neurokinin A, eledoisin and physalaemin was investigated in the rat. The injection of SP (3 nmol/rat) into the subarachnoid space was followed by reciprocal scratching, biting and licking of the fore- and hind-limbs. There was no observable difference in the behavioural response to SP between rats with nerve transection and sham operated rats at 5 days after operation. Whereas at 10, 20, and 30 days after nerve transection the response to SP was significantly increased as compared with sham operated rats. This phenomenon was also observed with neurokinin A (1.5, 3.0 and 6.0 nmol/rat), eledoisin (0.05 and 0.10 nmol/rat) and physalaemin (0.05 and 0.10 nmol/rat) at 10 days after operation. Ipsilateral depletion of SP from the lumbar (L4-L6) spinal cord was observed at 5, 10, 20, and 30 days after the unilateral transection of the sciatic nerve. These results suggest that sciatic nerve transection may produce an increased response to tachykinins through an enhanced sensitivity of tachykinin receptors in the lumbar cord.

Structure-activity studies of heptapeptide derivatives related to substance P, neurokinin A, B and other tachykinins on smooth muscles.

Diverse C-terminal heptapeptide derivatives related to substance P, kassinin, physalaemin, neurokinin A and B were synthesized and the contracting activities on the guinea pig ileum as well as rat duodenum were compared. In the partial sequence of C-terminal of tachykinin peptides, -I-II-Phe-III-Gly-Leu-Met-NH2, the combination of amino acid residues at positions I and III have significant roles in contraction of smooth muscle. For the activation of rat duodenal muscle (SP-E), Asp(I) and aliphatic amino acid(III), and for guinea pig ileal muscle(SP-P), Gln(I) and aromatic amino acid(III) are essential. Moreover, guinea pig ileum is sensitive to a full sequence of neurokinin peptides.

The contractile activities of neurokinin A, B and related peptides on smooth muscles.

The contractile activities of neurokinin A (NKA), neurokinin B (NKB) and related peptides on the guinea-pig ileum, rat vas deferens and rat duodenum were compared to the activity of substance P (SP). The potencies of NKA and NKB on the guinea-pig ileum (SP-P tissue) were nearly the same as that of SP. NKA was approximately 250-400 times more potent than SP on the rat vas deferens (EC50 = 59.5 nM; SP, EC50 = 1500 nM) and rat duodenum (EC50 = 1.8 nM; SP, EC50 = 674 nM) (SP-E tissues). NKB also showed high contracting activity on the rat duodenum (EC50 = 3.1 nM) but was 10 fold less active than NKA on the rat vas deferens. These results suggest that neurokinin peptides are possible endogenous agonists for the SP-E tissues. The contractile potency of NKA and NKB remained nearly complete after removal of N-terminal tripeptide portions, i.e., His-Lys-Thr and Asp-Met-His from the native peptides, respectively. However, the removal of the Asp residue from both NKA7 and NKB7 decreased activity until it was similar to that of SP.