Clin. exp. Immunol. (1991) 85, 396-401

ADONIS 0009910491 00249J

The effect of anti-neutrophil cytoplasm autoantibodies signal transduction in human neutrophils

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K. N. LAI & C. M. LOCKWOOD Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge, England

(Acceptedfor publication 18 March 1991)

SUMMARY The effect of anti-neutrophil cytoplasm autoantibodies (ANCA) on neutrophil activation was studied by pre-incubating neutrophils with IgG and F(ab')2 prepared from ANCA patients with Wegener's granulomatosis or microscopic polyarteritis. We measured the generation of inositol triphosphate (IP3) (a product of hydrolysis of membrane phospholipid which acts as a second intracellular messenger), and the translocation of protein kinase C (PKC) upon stimulation by a chemotactic peptide, fMet-Leu-Phe (fMLP). ANCA+ F(ab')2 did not induce a significant increase in IP3 generation. Nonetheless, ANCA+ F(ab')2 and ANCA+ IgG pretreatment of human neutrophils reduced the production of inositol phosphates upon subsequent fMLP stimulation compared with experiments performed when cells were pretreated with F(ab')2 and IgG prepared from ANCAhealthy subjects. A significantly reduced generation of IP3 and inositol biphosphate (IP2) was observed. ANCA+ F(ab')2 pretreatment of neutrophils inhibited fMLP-stimulated IP3 generation in a dose-dependent manner. The membrane-bound PKC activity upon stimulation by FMLP and PMA was reduced in neutrophils pretreated with ANCA+ F(ab')2 and IgG. These results indicate that ANCA affect in vitro signal transduction (IP3 generation, and translocation of PKC) in human neutrophils. Apparently, further activation of signal transduction by chemotactic peptide is significantly blunted in cells pre-incubated with ANCA+ F(ab')2 but not with F(ab')2 from healthy controls.

Keywords systemic vasculitis Wegener's granulomatosis anti-neutrophil cytoplasm autoantibodies neutrophils signal transduction.

INTRODUCTION Wegener's granulomatosis and microscopic polyarteritis (MPA) are systemic vasculitides of uncertain aetiology characterized by chronic polymorphonuclear leucocyte infiltration within and around vessel walls at various sites throughout the body. Recently, the finding of circulating autoantibodies to neutrophil cytoplasm antigens (ANCA) has implicated autoimmune mechanisms in the pathogenesis of vasculitis (van der Woude et al., 1985) although it is uncertain whether these play a primary role, or rise secondary to polymorph injury. We tested the effect of incubation of ANCA with neutrophils to see whether this altered their subsequent ability to be activated by chemotactic peptides, since activation is an essential feature of leucocyte participation in inflammatory events. We found that prior exposure to ANCA (either whole immunoglobulins or F(ab')2 fragments) would inhibit fMet-Leu-Phe (fMLP) or

phorbol myristate acetate (PMA) induced inositol phosphate metabolism and translocation of protein kinase C, both important steps in signal transduction pathways leading to neutrophil activation. This pathophysiological effect may contribute to the vascular injury found in ANCA-associated diseases. SUBJECTS AND METHODS Isolation of IgG, preparation of F(ab')2 fragments Purified IgG preparations were made by ammonium sulphate precipitation from nine randomly selected ANCA + patients with systemic vasculitis (five with Wegener's granulomatosis and four with MPA), and nine ANCA- healthy subjects. The IgG preparation from the nine vasculitic patients had high titres measured by immunofluorescence (van der Woude et al., 1985) and radioimmunoassay (Lockwood et al., 1987). Immunofluorescence studies revealed cytoplasmic staining in six and perinuclear staining in three. F(ab')2 fragments of IgG were produced from six randomly selected ANCA+ (four with Wegener's granulomatosis and two with MPA) and six control preparations by pepsin digestion and column chromatography with

Correspondence: K. N. Lai, Reader in Medicine, Department of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong.

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ANCA and signal transduction in neutrophil Sephadex G-1 50. The preparations were further purified by passing through a Protein G column (Pharmacia, Uppsala, Sweden). The purity of F(ab')2 fragments was ascertained by polyacrylamide gel electrophoresis. Preparation of human neutrophils Neutrophils were prepared from fresh blood samples obtained from healthy volunteers by centrifugation through a solution of 2% methylcellulose and 34% hypaque as described previously (Lockwood et al., 1987). The neutrophils were then preincubated with F(ab')2 at a concentration of 0-5 mg/ml or with IgG preparation at a concentration of 10 mg/ml at 370C for 20 min prior to measurement of inositol triphosphate (IP3) and protein kinase C (PKC). The concentration of IgG (10 mg/ml) used for incubation was similar to the serum IgG levels measured in these vasculitic patients. All experiments were performed in pairs with cells incubated with IgG or F(ab')2 from vasculitic patients and healthy controls under identical experimental conditions. The results, expressed as percentage of the values measured in cells incubated with only culture medium (i.e. without antibody or fMLP), were then compared. The viability of neutrophils incubated with IgG/F(ab')2 was examined by Trypan blue staining. The possible cytotoxicity caused by these antibodies was studied by chromium release from 5'Crlabelled neutrophils incubated with either ANCA or control F(ab')2 preparations.

Measurement of inositol phosphates Inositol phosphates were labelled by incubating 8 x 106 neutrophils in RPMI 1640 medium (GIBCO) containing myo-(2-3H) inositol (10 pCi/ml) (New England Nuclear, Boston, MA) and 10% heat-inactivated fetal calf serum (FCS) for 18 h in 5% C02 and at 37°C. The prelabelled cells were washed by suspending twice at 37°C for 10 min in RPMI 1640 without inositol (GIBco). The cells were resuspended in calcium medium (138 mm NaCI, 6 mM KCI, I mM MgSO4, 1 1 mm CaCI2, 100 M EGTA, I mm I Na2HPO4, 5 mm NaHCO3, 5 5 mm glucose, 20 mm HEPES, pH 7-4), warmed for 5 min at 37°C, followed by the incubation with F(ab')2, IgG or calcium medium for 20 min. The neutrophils were thoroughly washed twice with calcium medium before I /M fMLP (Sigma, St Louis, MO) was added. The reaction was stopped after 20 s and inositol phosphates were extracted by adding equal volume of ice-cold 10% perchloric acid to the incubation buffer. The cell debris was pelleted by centrifugation, and the supernatant was desalted by addition of 1-2 ml of a freon (1,1 ,2-trichlorotrifluoroethane)/tri-N-octylamine mixture (1:1 v/v, Sigma), for each 1-0 ml of 10% perchloric acid added. The soluble inositol phosphates were contained in the upper of the three phases after thorough mixing and centrifugation. The inositol phosphates were separated by stepwise elution from Dowex formate column (BioRad, Richmond, CA) as described (Berridge et al., 1983). Radioactivity in the fractions was determined by liquid scintillation counting with 80% (v/v) aquasol. Measurement of PKC Human neutrophils (10 x 106) were resuspended in 10 ml of RPMI 1640 containing 10% FCS and pre-incubated for 10 min in a water bath at 37°C. fMLP (1 gM) was added for 20 s followed by phorbol myristate acetate (PMA) (100 ng/ml) and incubation was continued for 10 min. The cells were sedimented

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by centrifugation, resuspended in 2 ml ice-cold buffer A (20 mm Tris-HCI, pH 7-5, 0-5 mm EGTA, 0.5 mm EDTA, 01 mM phenylmethylsulphonyl fluoride, I mg/ml soybean trypsin inhibitor, I mm benzamidine, and I mm dithiothreitol; Sigma) and ultrasonically disrupted (amplitude 20 pm for 20 s). The crude homogenate was centrifuged at 50 OOOg for 60 min and the pellet (particulate fraction) was resuspended in 1 5 ml buffer B (20 mM Tris-HCI, pH 7*5, 5 mm EGTA, 2 mm EDTA, 0-1 mM phenylmethylsulphonyl fluoride, 20 pg/ml soybean trypsin inhibitor, I mm benzamidine, and I mm dithiothreitol) containing 1% Triton X-100 (BDH, Poole, UK), stored on ice for 60 min, and centrifuged at 50 000 g for 30 min. The solubilized particulate fraction was chromatographed on DEAE-cellulose (Sigma) columns (0-5 x I cm) equilibrated with buffer B. The column was washed with 5 ml buffer B followed by 5 ml buffer C (20 mM Tris-HCl, pH 7 5,0*5 mM EGTA, 0 5 mM EDTA, 20 pg/ ml soybean trypsin inhibitor). PKC was eluted with 0.5 ml buffer C containing 100 mm NaCl and the column was further washed with 5 ml of buffer C containing 100 mm NaCl. Phospholipid-independent PKC activity was eluted with 0 5 ml buffer C containing 400 mm NaCl. PKC enzymatic activity was assayed following a modification of previously described method (Kikkawa et al., 1983): 120 p1 of PKC eluate were added to 130 p1 of a reaction mixture containing 5 ,umol Tris-HCl, pH 7.5, 1-25 pM MgCI2, 125 nmol CaC12, 50 ug histone type III (Sigma), 10 pg phosphatidyl-L-serine (Sigma), 0-2 pg Diolein (Sigma), and 10 nmol (gamma-32P) ATP, 1 Ci/mmol (Amersham International, Amersham, UK). Background activity was measured in the presence of 250 nmol of EGTA instead of CaC12, diolein, and phosphatidyl-L-serine. After a 3-min incubation at 30°C, the assay was terminated by adding 3 ml of 25% trichloroacetic acid. Acid-precipitable material was collected on a nitrocellulose membrane filter in a suction apparatus and extensively washed with 25% trichloroacetic acid. Radioactivity was assayed by an automatic liquid scintillation counter. Results (mean + s.e.m.) are expressed as percentage of values of cells incubated without IgG/F(ab')2/fMLP. Non-parametric statistical analyses were used as appropriate.

RESULTS IgG and F(ab'h preparation F(ab')2 preparations from the six ANCA + patients gave a strong cytoplasmic immunofluorescence staining at a concentration of 10 pg/ml but similar staining was not observed with F(ab')2 from ANCA- controls even up to a concentration of 500 pg/ml. Endotoxin was not detected in either the IgG or F(ab')2 preparation by the Limulus ameobocyte lysate test. Neutrophils incubated with these preparation at 37° for 2 h demonstrated 95% viability by 04% Trypan blue staining. The chromium release from neutrophils incubated with ANCA and control F(ab')2 preparations for 20 min were 13-1 +0 6% and 142+0-8% of labelled chromium prior to incubation with antibody, respectively, and these values were lower than those measured in neutrophils incubated with culture medium (273 + 0-5%), suggesting these antibodies did not cause cytotoxicity. Kinetic studies of IP3 generation Extensive experimental evidence indicates IP3 is a second messenger, which exerts its action by releasing calcium from

K. N. Lai & C. M. Lockwood

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The effect of anti-neutrophil cytoplasm autoantibodies on the signal transduction in human neutrophils.

The effect of anti-neutrophil cytoplasm autoantibodies (ANCA) on neutrophil activation was studied by pre-incubating neutrophils with IgG and F(ab')2 ...
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