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Anti-Lactoferrin Antibodies and Other Types Ofanca 56 Gut 1993; 34: 56-62 Anti-lactoferrin antibodies and other types ofANCA in ulcerative colitis, primary sclerosing cholangitis, Gut: first published as 10.1136/gut.34.1.56 on 1 January 1993. Downloaded from and Crohn's disease E Peen, S Almer, G Bodemar, B-O Ryden, C Sjolin, K Tejle, T Skogh Abstract staining pattern (C-ANCA), whereas anti- Fifty two serum samples from patients with myeloperoxidase and anti-elastase antibodies Crohn's disease, 24 from patients with ulcera- give rise to a blurry perinuclear staining pattern tive colitis, and 12 from patients with primary (P-ANCA) or staining of the PMNL nuclei sclerosing cholangitis were analysed for the (granulocyte specific anti-nuclear antibodies, presence of anti-neutrophil cytoplasm anti- GS-ANA).24' A detergent extract of isolated bodies (ANCA) of IgG and IgA class by means azurophil granules (a antigen)-can be used for the of enzyme linked immunosorbent assays using detection of anti-proteinase 3 antibodies lactoferrin, myeloperoxidase, and antigen (C-ANCA) by means ofenzyme linked immuno- extracted from azurophil granules, 'a antigen' sorbent assay (ELISA) but does not allow detec- (that is, an antigen preparation containing tion of anti-myeloperoxidase.56 Also, antibodies proteinase 3) as substrates. A high frequency against lactoferrin, an iron binding protein resid- of positive tests for IgG anti-lactoferrin anti- ing in specific granules of PNML,7 produce a bodies was found in sera from patients with P-ANCA pattern owing to perinuclear/nuclear ulcerative colitis (50%) and primary sclerosing localisation of lactoferrin after ethanol fixation.8 cholangitis (50%). In Crohn's disease only 4 of Apart from the artifactual GS-ANA staining 52 (8%) sera had anti-lactoferrin antibodies - in pattern caused by antibodies directed against all four instances the sera belonged to patients nucleophilic cytoplasmic antigens, it is possible with disease involving the colon. AU patients that true GS-ANAs also exist.9 with sclerosing cholangitis and positive tests Apart from the occurrence in primary for anti-lactoferrin had ulcerative colitis. Low systemic vasculitides and rapidly progressive levels of IgG antibodies against myeloperoxi- glomerulonephritis, P-ANCA/GS-ANA may be dase or a antigen were also found occasionally seen in other disease states, for example rheuma- http://gut.bmj.com/ in sera from patients with ulcerative colitis and toid arthritis without signs ofvasculitis,'0 inflam- primary sclerosing cholangitis. IgA antibodies matory bowel disease, and primary sclerosing directed against lactoferrin and a antigen (but cholangitis."-'5 Recent evidence favours the idea not myeloperoxidase) were seen in all three that inflammatory bowel disease may be caused conditions. by mesenteric vasculitis.I'l8 (Gut 1993; 34: 56-62) In a preliminary study of 16 frozen sera from patients with Crohn's disease we found low levels on September 29, 2021 by guest. Protected copyright. of anti-a antigen antibodies, anti-myeloperoxi- Since the description of anti-neutrophil cyto- dase antibodies, and anti-lactoferrin antibodies plasm antibodies (ANCA) in active Wegener's in some sera.'9 The present study was done to granulomatosis,' the interest in anti-granulocyte extend these observations and to include sera antibodies has increased vastly. ANCA is now from patients with ulcerative colitis and primary Department of Internal recognised as a family ofautoantibodies directed sclerosing cholangitis. Medicine, against cytoplasmic antigens, mainly lysosomal Gastroenterology and enzymes, in Rheumatology Clinics polymorphonuclear neutrophil and Department of leukocytes (PMNL).2 In Wegener's granulo- Patients and methods Medical Microbiology, matosis, ANCA are typically directed against Transfusion Medicine proteinase 3, a serine proteinase located in and Clinical Immunology CROHN S DISEASE Laboratory, Faculty of azurophilic granules of human PMNL.3 Anti- Fifty two patients, 27 men aged 21-71 years Health Sciences, proteinase 3 antibodies may also occur in (mean 48 years) and 25 women aged 17-55 years University Hospital, isolated, rapidly progressive glomerulone- (mean 38 years) were enrolled in the S-581 85 Linkoping, study. The Sweden phritis and occasionally in systemic vasculitic mean duration of the disease was 17 years (range E Peen conditions other than Wegener's granulomato- 4-33 years). Eight patients had disease limited to S Almer sis.2 Antibodies directed against other lysosomal the colon and/or rectum, 19 patients had G Bodemar disease B-O Ryden enzymes in PMNL azurophilic granules, for in the small bowel alone, and 25 patients had C Sjolin example myeloperoxidase and elastase, also disease manifestations in both small bowel and K Teile occur in primary systemic vasculitic diseases and colon/rectum. Forty patients had been operated T Skogh rapidly progressive glomerulonephritis.34 After on, 14 of whom had been subjected to only Correspondence to: Dr Thomas Skogh, ethanol fixation of PMNL, proteinase 3 remains ileocoecal resection. Three patients had Department of Internal within the cytoplasmic granules, whereas myel- ileorectal anastomosis, and six patients had Medicine, Rheumatology Clinic, Faculty of Health operoxidase and elastase become extracted and ileostomy after proctocolectomy. Sciences, University locate close to or on the cell nucleus.24 At indirect Hospital, Linkoping, S-581 85, Sweden. immunofluorescent microscopical determina- Accepted for publication tion of ANCA, anti-proteinase 3 antibodies ULCERATIVE COLITIS 12 June 1991 produce a typical pancytoplasmatic granular Twenty four patients, 11 men agedl9-65 years Anti-lactoferrin antibodies andother types ofANCA in ulcerative colitis, primary sclerosing cholangitis, and Crohn's disease 57 IgG anti-a antigen diagnosis had been made by endoscopic retro- grade cholangiography 3 years (range 1-6 years) Crohn's Ulcerative Primary before the study. Two patients had only intra- disease colitis sclerosing hepatic cholangitis and 10 had both extra- and Gut: first published as 10.1136/gut.34.1.56 on 1 January 1993. Downloaded from 100 cholangitis intrahepatic cholangitis. In four patients cirrho- sis of the liver had been diagnosed at histopatho- logical examination ofliver biopsies. One patient had undergone liver transplantation 6 years earlier. Nine of the 12 patients had also suffered 50 from ulcerative colitis for 8 years (range 0-21 years) and one had non-specific colitis. In the two remaining patients no accompanying disease had been diagnosed. None of the patients had Crohn's disease. Z_~~~oooooooo pp...... One of the patients originally participating in the study had to be excluded as she proved to have suffered from acute Campylobacter jejuni IgG anti-myeloperoxidase colitis. Crohn's Ulcerative Primary ,o disease colitis sclerosing ANTIGEN PREPARATIONS 4)0 co cholangitis Azurophil granules were prepared from isolated e. 100 normal peripheral granulocytes after low pres- 0 sure homogenisation of the cells and centrifuga- C.) .0> tion of the homogenate in a Percoll (Pharmacia 01)Co Fine Chemicals, Uppsala, Sweden) gradient as described elsewhere.5 The granules were lysed .-0 50 with 0-01% (final concentration) Triton X-100 Q (Merck, Darmstadt, Germany).5 The extract co thus achieved (a antigen) was diluted in n0L carbonate-bicarbonate buffer pH 9 5 and used as substrate for ELISA.5 0@@-@@@@::@t- 0 * 0 0 Lyophilised preparations of myeloperoxidase (Calbiochem, La Jolla, CA, USA), human milk lactoferrin (Sigma Chemical Co, St Louis, MO, http://gut.bmj.com/ IgG anti-lactoferrin USA), and bovine milk lactoferrin (Sigma) were dissolved in carbonate-bicarbonate buffer (10 Crohn's Ulcerative Primary [tg antigen/ml) and used for ELISA. disease colitis sclerosing .P ..cholangitis 100 ELISA High binding plastic microtitre plates (Nunc on September 29, 2021 by guest. Protected copyright. .00 gO~~~~~ Immunoplate, Roskilde, Denmark) were coated with a antigen, myeloperoxidase, or human lactoferrin solutions by incubation at 40C for 12 50 hours. After washing with buffer (phosphate buffered saline, PBS, with 0 05% Tween 20), *g @0 0 0@ serum samples (diluted 1:10 in PBS-Tween) *v@@@@@@@@@@@** ~ ~ 0 g were applied for 30 minutes at room tempera- 00000000000000e ture. Normal human serum diluted 1:10 served as a blank. The microtitre plates were washed Figure 1: Diagram to illustrate the ELISA results ofIgG autoantibody directed against thoroughly with PBS-Tween and incubated for a antigen, myeloperoxidase, and lactoferrin in sera from patients with Crohn's disease (n=52), another 30 minutes with alkaline-phospatase ulcerative colitis (n=24), andprimary sclerosing cholangitis (n= 12). The lower limitfor positive ELISA (that is, +2 SD ofthe mean value of218 normal reference sera) is indicated. (ALP) conjungated rabbit anti-human y chain or rabbit anti-human a chain antisera (Dako, Glostrup, Denmark) diluted 1:400 in PBS- (mean 46 years), and 13 women aged 20-68 years Tween. After washing, the substrate buffer was (mean 42 years) participated in the study. Nine- applied and the optical density (OD) read at teen patients had left sided colitis and five 405 nm when the positive reference samples had patients had more extensive or total colitis. reached OD 1.0. OD values exceeding 2 SDs of Fourteen of the patients had active disease - that the reference material from healthy blood donors is at least five loose stools with visible blood (all (n=218 for IgG tests; n=211 for IgA tests) were had visible inflammation at endoscopy) - and the considered positive. The IgG autoantibody remaining 10 patients were in clinical remission. analyses were performed on all sera from patients with Crohn's disease (n=52), ulcerative colitis (n=24), and primary sclerosing cholangitis PRIMARY SCLEROSING CHOLANGITIS (n=12). IgA autoantibody tests were done on Twelve patients, seven men aged 20-59 years sera from 51 patients with Crohn's disease, 21 (mean 38 years) and five women aged 35-63 years ulcerative colitis sera, and 11 sera from patients (mean 48 years), took part in the study. The with primary sclerosing cholangitis. 58 Peen, Almer, Bodemar, Ryden, Sjdlin, Tejile, Skogh The rabbit anti-human leukocyte lactoferrin kD 1 2 3 4 5 served as a reference.
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