Gut 1993; 34: 56-62

Anti-lactoferrin antibodies and other types ofANCAin ulcerative colitis, primary sclerosing cholangitis,and Crohn's disease

E Peen, S Almer, G Bodemar, B-O Ryden, C Sjolin, K Tejle, T Skogh

AbstractFifty two serum samples from patients withCrohn's disease, 24 from patients with ulcera-tive colitis, and 12 from patients with primarysclerosing cholangitis were analysed for thepresence of anti-neutrophil cytoplasm anti-bodies (ANCA) of IgG and IgA class by meansof enzyme linked immunosorbent assays usinglactoferrin, myeloperoxidase, and antigenextracted from azurophil granules, 'a antigen'(that is, an antigen preparation containingproteinase 3) as substrates. A high frequencyof positive tests for IgG anti-lactoferrin anti-bodies was found in sera from patients withulcerative colitis (50%) and primary sclerosingcholangitis (50%). In Crohn's disease only 4 of52 (8%) sera had anti-lactoferrin antibodies - inall four instances the sera belonged to patientswith disease involving the colon. AU patientswith sclerosing cholangitis and positive testsfor anti-lactoferrin had ulcerative colitis. Lowlevels of IgG antibodies against myeloperoxi-dase or a antigen were also found occasionallyin sera from patients with ulcerative colitis andprimary sclerosing cholangitis. IgA antibodiesdirected against lactoferrin and a antigen (butnot myeloperoxidase) were seen in all threeconditions.(Gut 1993; 34: 56-62)

Department of InternalMedicine,Gastroenterology andRheumatology Clinicsand Department ofMedical Microbiology,Transfusion Medicineand Clinical ImmunologyLaboratory, Faculty ofHealth Sciences,University Hospital,S-581 85 Linkoping,SwedenE PeenS AlmerG BodemarB-O RydenC SjolinK TeileT SkoghCorrespondence to:Dr Thomas Skogh,Department of InternalMedicine, RheumatologyClinic, Faculty of HealthSciences, UniversityHospital, Linkoping,S-581 85, Sweden.Accepted for publication12 June 1991

Since the description of anti-neutrophil cyto-plasm antibodies (ANCA) in active Wegener'sgranulomatosis,' the interest in anti-granulocyteantibodies has increased vastly. ANCA is nowrecognised as a family of autoantibodies directedagainst cytoplasmic antigens, mainly lysosomalenzymes, in polymorphonuclear neutrophilleukocytes (PMNL).2 In Wegener's granulo-matosis, ANCA are typically directed againstproteinase 3, a serine proteinase located inazurophilic granules of human PMNL.3 Anti-proteinase 3 antibodies may also occur inisolated, rapidly progressive glomerulone-phritis and occasionally in systemic vasculiticconditions other than Wegener's granulomato-sis.2 Antibodies directed against other lysosomalenzymes in PMNL azurophilic granules, forexample myeloperoxidase and elastase, alsooccur in primary systemic vasculitic diseases andrapidly progressive glomerulonephritis.34 Afterethanol fixation ofPMNL, proteinase 3 remainswithin the cytoplasmic granules, whereas myel-operoxidase and elastase become extracted andlocate close to or on the cell nucleus.24 At indirectimmunofluorescent microscopical determina-tion of ANCA, anti-proteinase 3 antibodiesproduce a typical pancytoplasmatic granular

staining pattern (C-ANCA), whereas anti-myeloperoxidase and anti-elastase antibodiesgive rise to a blurry perinuclear staining pattern(P-ANCA) or staining of the PMNL nuclei(granulocyte specific anti-nuclear antibodies,GS-ANA).24' A detergent extract of isolatedazurophil granules (a antigen)-can be used for thedetection of anti-proteinase 3 antibodies(C-ANCA) by means ofenzyme linked immuno-sorbent assay (ELISA) but does not allow detec-tion of anti-myeloperoxidase.5 6 Also, antibodiesagainst lactoferrin, an iron binding protein resid-ing in specific granules of PNML,7 produce aP-ANCA pattern owing to perinuclear/nuclearlocalisation of lactoferrin after ethanol fixation.8Apart from the artifactual GS-ANA stainingpattern caused by antibodies directed againstnucleophilic cytoplasmic antigens, it is possiblethat true GS-ANAs also exist.9

Apart from the occurrence in primarysystemic vasculitides and rapidly progressiveglomerulonephritis, P-ANCA/GS-ANA may beseen in other disease states, for example rheuma-toid arthritis without signs of vasculitis,'0 inflam-matory bowel disease, and primary sclerosingcholangitis."-'5 Recent evidence favours the ideathat inflammatory bowel disease may be causedby mesenteric vasculitis. I'l8

In a preliminary study of 16 frozen sera frompatients with Crohn's disease we found low levelsof anti-a antigen antibodies, anti-myeloperoxi-dase antibodies, and anti-lactoferrin antibodiesin some sera.'9 The present study was done toextend these observations and to include serafrom patients with ulcerative colitis and primarysclerosing cholangitis.

Patients and methods

CROHN S DISEASEFifty two patients, 27 men aged 21-71 years(mean 48 years) and 25 women aged 17-55 years(mean 38 years) were enrolled in the study. Themean duration of the disease was 17 years (range4-33 years). Eight patients had disease limited tothe colon and/or rectum, 19 patients had diseasein the small bowel alone, and 25 patients haddisease manifestations in both small bowel andcolon/rectum. Forty patients had been operatedon, 14 of whom had been subjected to onlyileocoecal resection. Three patients hadileorectal anastomosis, and six patients hadileostomy after proctocolectomy.

ULCERATIVE COLITISTwenty four patients, 11 men agedl9-65 years

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Figure 1: Diagram to illustrate the ELISA results ofIgG autoantibody directed againsta antigen, myeloperoxidase, and lactoferrin in serafrom patients with Crohn's disease (n=52),ulcerative colitis (n=24), and primary sclerosing cholangitis (n= 12). The lower limitforpositive ELISA (that is, +2 SD ofthe mean value of218 normal reference sera) is indicated.

(mean 46 years), and 13 women aged 20-68 years(mean 42 years) participated in the study. Nine-teen patients had left sided colitis and fivepatients had more extensive or total colitis.Fourteen of the patients had active disease - thatis at least five loose stools with visible blood (allhad visible inflammation at endoscopy) - and theremaining 10 patients were in clinical remission.

PRIMARY SCLEROSING CHOLANGITISTwelve patients, seven men aged 20-59 years(mean 38 years) and five women aged 35-63 years(mean 48 years), took part in the study. The

diagnosis had been made by endoscopic retro-grade cholangiography 3 years (range 1-6 years)before the study. Two patients had only intra-hepatic cholangitis and 10 had both extra- andintrahepatic cholangitis. In four patients cirrho-sis of the liver had been diagnosed at histopatho-logical examination of liver biopsies. One patienthad undergone liver transplantation 6 yearsearlier. Nine of the 12 patients had also sufferedfrom ulcerative colitis for 8 years (range 0-21years) and one had non-specific colitis. In the tworemaining patients no accompanying disease hadbeen diagnosed. None of the patients hadCrohn's disease.One of the patients originally participating in

the study had to be excluded as she proved tohave suffered from acute Campylobacter jejunicolitis.

ANTIGEN PREPARATIONSAzurophil granules were prepared from isolatednormal peripheral granulocytes after low pres-sure homogenisation of the cells and centrifuga-tion of the homogenate in a Percoll (PharmaciaFine Chemicals, Uppsala, Sweden) gradient asdescribed elsewhere.5 The granules were lysedwith 0-01% (final concentration) Triton X-100(Merck, Darmstadt, Germany).5 The extractthus achieved (a antigen) was diluted incarbonate-bicarbonate buffer pH 9 5 and used assubstrate for ELISA.5

Lyophilised preparations of myeloperoxidase(Calbiochem, La Jolla, CA, USA), human milklactoferrin (Sigma Chemical Co, St Louis, MO,USA), and bovine milk lactoferrin (Sigma) weredissolved in carbonate-bicarbonate buffer (10[tg antigen/ml) and used for ELISA.

ELISAHigh binding plastic microtitre plates (NuncImmunoplate, Roskilde, Denmark) were coatedwith a antigen, myeloperoxidase, or humanlactoferrin solutions by incubation at 40C for 12hours. After washing with buffer (phosphatebuffered saline, PBS, with 0 05% Tween 20),serum samples (diluted 1:10 in PBS-Tween)were applied for 30 minutes at room tempera-ture. Normal human serum diluted 1:10 servedas a blank. The microtitre plates were washedthoroughly with PBS-Tween and incubated foranother 30 minutes with alkaline-phospatase(ALP) conjungated rabbit anti-human y chainor rabbit anti-human a chain antisera (Dako,Glostrup, Denmark) diluted 1:400 in PBS-Tween. After washing, the substrate buffer wasapplied and the optical density (OD) read at405 nm when the positive reference samples hadreached OD 1.0. OD values exceeding 2 SDs ofthe reference material from healthy blood donors(n=218 for IgG tests; n=211 for IgA tests) wereconsidered positive. The IgG autoantibodyanalyses were performed on all sera from patientswith Crohn's disease (n=52), ulcerative colitis(n= 24), and primary sclerosing cholangitis(n=12). IgA autoantibody tests were done onsera from 51 patients with Crohn's disease, 21ulcerative colitis sera, and 11 sera from patientswith primary sclerosing cholangitis.

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Figure 2: Sodium dodecyl sulphate polyacrylamide gels (SDS-PAGE) electrophoresis ofthehuman milk lactoferrin preparation (lane 1) showed only material ofmolecular weight about 80kD when stained with Comassie blue. Lane 2 shows molecular weight markers for comparison.Immunoblotting, using polyclonal rabbit anti-human leukocyte lactoferrin, showed slightreaction with antigens oflower molecular weight, apartfrom the strong reaction with the 80 kDlactoferrin band (lane 3). Patient sera positivefor IgG anti-human milk lactoferrin, as tested byELISA, however, reacted only with lactoferin.

IgG anti-bovine lactoferrin antibodies weremeasured essentially as described above, exceptthat no negative or positive serum referenceswere used. OD values were read after 30 minutesincubation with substrate buffer. Eleven positiveand 10 negative sera in the test for IgG anti-human lactoferrin were selected for this analysis.

RABBIT ANTI-LACTOFERRIN ANTISERASerum containing polyclonal rabbit anti-humanleukocyte lactoferrin was the kind gift ofDr Johan Richter, Department of InternalMedicine, University Hospital, Lund, Sweden.To produce anti-human milk lactoferrin anti-bodies, rabbits were immunised by subcutane-ous injections of 2 mg human milk lactoferrin(that is, the same antigen as that used for theELISA and western blot assays) together withFreund's incomplete adjuvant (Sigma) on threeoccasions with 4 week intervals. The reason fornot using Freund's complete adjuvant was toavoid immunisation against hsp-65, because ofits immunological cross reaction with humanlactoferrin.20 Two weeks after the second boosterdose, the animals were anaesthetised withbarbiturate, and exsanguinised by heartpuncture. Serum was pooled. Anti-lactoferrinactivity of the serum was confimed by agarosedouble radial immunodiffusion against humanmilk lactoferrin.

WESTERN BLOTTINGLactoferrin (1 [ig per lane) was electrophoresedin 5-20% sodium dodecyl sulphate poly-acrylamide gels (SDS-PAGE) and transferred topolyvinylidene difluoride membranes (MilliporeContinental Water Systems, Bedford, MA,USA). Blocking was done with 5% bovine serumalbumin (Sigma) in PBS. Anti-lactoferrinpositive patient sera (as judged by ELISA) werediluted 1:20 or 1:200 in PBS-Tween with 1%bovine serum albumin, and applied for 1 hour.

The rabbit anti-human leukocyte lactoferrinserved as a reference. ALP conjugated anti-human IgG and anti-rabbit IgG (Dako) wereused as secondary antibodies.

INDIRECT IMMUNOFLUORESCENCE (IIF)MICROSCOPYTo study the distribution of lactoferrin inethanol fixed granulocytes cytocentrifuged ontomicroscope slides, the slides were incubated withrabbit anti-human milk lactoferrin in a moistchamber for 30 minutes. After washing with PBSand incubation for another 30 minutes withsheep fluorescein isothiocyanate (FITC) con-jugated anti-rabbit Ig (Wellcome Diagnostics,Temple Hill, Dartford, UK), the slides wereagain washed with PBS, mounted with PBS-glycerin, and inspected under a fluorescencemicroscope with a mercury lamp (HBO 50) epi-illumination and filters for FITC activation/emission.

STATISTICSDifferences in ELISA results between the con-trol sera and the patient sera were evaluated bythe X2 test, and divided into four groups: p.-005=not significant (NS); p<0O05 (*); p<0-01 (**);p<O.OOl (***)

ResultsThe frequencies of IgG antibodies directedagainst a antigen, myeloperoxidase, and lacto-ferrin are shown in Figure 1.

In Crohn's disease 4 of 52 sera had IgG anti-aantigen, which is not statistically different fromthe control group, although two of the sera hadremarkably high antibody levels. In ulcerativecolitis 4 of 24 of the sera contained IgG anti-aantigen antibodies (p<0Q05), and in primarysclerosing cholangitis 4 of 12 sera (p<0-001)were positive.IgG anti-myeloperoxidase antibodies occurred

in 3 of 52 of the sera taken from patients withCrohn's disease (not statistically different from

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Figure 3: Diagram to illustrate the lack ofcorrelation betweenELISA results for IgG anti-human lactoferrinautoantibodies and IgG antibodies directed against bovinemilk lactoferrin. Ten patient sera positive in the ELISA forIgG anti-human lactoferrin (-) and 12 randomly selectednormal reference sera (L) negative in the IgG anti-humanlactoferrin test were analysed.

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Figure 4: Results ofthe ELISA testsfor IgA autoantibodies directed against a antigen,myeloperoxidase, and lactoferrin. Fifty one serafrom patients with Crohn's disease, 21 serafrom patients with ulcerative colitis, and 1I serafrom patients with primary sclerosingcholangitis were analysed. The cut oflevels (that is, values exceeding 2 SD ofthe mean ofareference material of21 I normal sera) are indicated.

the control group), and in 7 of24 ofthe ulcerativecolitis sera (p<0001). Four of 12 sera frompatients with primary sclerosing cholangitis werepositive (p<0001).Four of 52 (8%) of the sera from patients with

Crohn's disease contained IgG anti-lactoferrinantibodies (not significantly different from thecontrol group). All of these sera proved to comefrom patients with Crohn colitis. However,colitis also occurred in 33 of 48 (69%) withoutIgG anti-lactoferrin antibodies. Both in ulcera-tive colitis and primary sclerosing cholangitis,

IgG anti-lactoferrin antibodies were found in50% of the sera (p<0-001 in both instances). Allof the anti-lactoferrin positive cholangitis serabelonged to patients suffering from concomitantulcerative colitis - that is, 6 of 9 (67%) sera frompatients with sclerosing cholangitis and con-comitant ulcerative colitis contained IgG anti-lactoferrin antibodies. Two of the sera frompatients with primary sclerosing cholangitis andone serum from a patient with ulcerative colitiswere positive in all three ELISA tests. However,in all three instances the ELISA tests gave quitedifferent OD values in the different assays,indicating true positive ELISA test results.

Positive anti-lactoferrin ELISA results wereblocked by preincubation of the lactoferrin-coated microtitre plates with rabbit IgG anti-lactoferrin (not illustrated). The occurrence ofIgG anti-lactoferrin was also confirmed bywestern blotting, revealing a single reaction bandwith an antigen ofmolecular weight about 80 000and corresponding to rabbit anti-human leuko-cyte lactoferrin (Fig 2).One patient originally included in the study

had acute colitis and a high level of IgG anti-lactoferrin at the first sampling occasion, and afurther raised anti-lactoferrin level in a laterserum sample (not illustrated). This patient was,however, excluded from the study since hersymptoms were explained by infection withCampylobacterjejuni.

Figure 3 illustrates the lack of correlationbetween levels ofIgG anti-human lactoferrin andIgG anti-bovine lactoferrin as tested by ELISAwith 10 sera positive in the anti-human lacto-ferrin and 12 sera negative in the same test.

Figure 4 shows the occurrence of IgA anti-bodies directed against a antigen, myeloperoxi-dase, and human lactoferrin.

Raised levels of IgA anti-a antigen were seenin 12 of 51 (24%) sera from Crohn's disease(p<0-001), in 5 of 11 (45%) sera from patientswith primary sclerosing cholangitis (p<0001),but in only 2 of 21 (10%) sera from patients withulcerative colitis (NS). The levels of IgA anti-myeloperoxidase did not differ from the controlgroup in any of the disease states, whereas IgAanti-lactoferrin was significantly (p<005) morecommon in Crohn's disease, although it was seenonly in a minority of the patient sera (5 of 51=10%). IgA anti-lactoferrin was found in signific-antly (p<0-001) increased frequency also inulcerative colitis (7 of 21=33%), but not insclerosing cholangitis (1 of 11=9%).The levels of IgA-ANCAs were not explained

by the occurrence of agglutinating rheumatoidfactors (not illustrated).

Figure 5 shows the frequency of positive IgGand/or IgA tests for either anti-a antigen, or anti-myeloperoxidase, or anti-lactoferrin in sera fromblood donors, Crohn's disease, ulcerative colitis,and primary sclerosing cholangitis. Takentogether, positive IgG tests for one or more of thethree granulocyte antigens were found in 11%(24 of 218) control sera, in 19% (10 of 52) ofCrohn's disease (NS), in 67% (16 of 24) ofulcerative colitis (p<0-001), and in 67% (8 of 12)of primary sclerosing cholangitis sera; IgA anti-granulocyte antibodies were found in 7% (15 of211) of the controls, in 29% (15 of 51) ofCrohn's

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lgG IgA IgG+lgA 19GFigure 5: Diagram summarising theELISA testsforIgG andlor IgA autoantibodiesgranulocyte antigens tested - that is, a antigen, myeloperoxidase, and human lactof

disease patients (p<O.OOl), in 48% (10 of 21) ofulcerative colitis patients (p<0001), and in 55%(6 of 11) of primary sclerosing cholangitis sub-jects (p<O.OOl). Antibodies of either IgG or IgAclass were found in 49% of the sera from patientswith Crohn's disease (p<0001), in 74% ofthe sera from patients with ulcerative colitis(p<0001), and in 82% of the cholangitis sera

(p<O-OOl).There was no obvious correlation between

disease activity or extraintestinal disease(arthritis, arthralgia, skin involvement) andthe presence of IgG or IgA anti-granulocyte

Figure 6: Granulocyte specific antinuclear antibodieslperinuclear staining anti-neutrophilcytoplasm antibodies (GS-ANA/P-ANCAI IIF staining pattern achieved after incubation ofanti-human milk lactoferrin antibodies with cytocentrifuged, ethanolfixed humanpolymorphonuclear granulocytes.

*

*

antibodies. The levels of IgG and IgA anti-granulocyte antibodies did not correlate tothe total serum levels of IgG and IgA (notillustrated).

Figure 6 shows the GS-ANA/P-ANCAimmunofluorescence pattern of cytocentrifugedethanol-fixed human polymorphonuclearneutrophil granulocytes after staining withrabbit anti-human milk lactoferrin. This, takentogether with the western blot analysis (Fig 2),shows that antibodies against human milk lacto-ferrin recognise human leukocyte lactoferrin andvice versa.

DiscussionIn this study we report high frequencies of IgGanti-lactoferrin antibodies (corresponding toP-ANCA) in sera from patients with ulcerativecolitis and primary sclerosing cholangitis. InCrohn's disease, however, serum anti-lactoferrinantibodies of IgG were rarely detected, and thenonly in patients with colonic disease manifesta-tions. IgA anti-lactoferrin antibodies were, how-ever, found in some cases of both ulcerativecolitis and Crohn's disease.

Several different types of autoantibodies havebeen described in inflammatory bowel diseaseand primary sclerosing cholangitis."- 1921-27

Although none of the autoantibodies have beenproved to be of pathogenetic significance, it hasbeen shown, both in Crohn's disease and inulcerative colitis, that IgG and complement can

be present on the apical surface of enterocytes invivo,2 29 possibly indicating a pathogenetic rolefor anti-epithelial/anti-brush border antibodiesin inflammatory bowel disease. In this connec-

tion, and considering the high frequency of anti-lactoferrin antibodies found in the present study,it is interesting to note that lactoferrin has beenreported to bind to intestinal brush border via a

specific receptor.30 Antibodies of IgA classdirected against Saccharomyces cerviciae are

common in Crohn's disease but not in ulcerativecolitis, and determination of such antibodiesmay be of diagnostic help.3" It is possible thatmany of the anti-microbial antibodies describedin inflammatory bowel disease and other inflam-matory disease states, may in fact be reflectionsof immunisation against microbial heat-shockproteins, which has been implied in the patho-genesis of several autoimmune disease states32and which may induce anti-lactoferrin anti-bodies22 (see below).

Wakefield et al presented evidence thatvasculitis and microthrombosis in mesentericvessels can be important pathogenetic factors ininflammatory bowel disease.' '8 Furthermore,P-ANCA/GS-ANA, which occurs in severalprimary vasculitis diseases, can frequently bedemonstrated in inflammatory bowel disease andprimary sclerosing cholangitis also."-'5 P-ANCA/GS-ANA is more common in ulcerative colitisthan in Crohn's disease," 13-'5 which is confirmed

in the present study by the high frequency ofIgGl anti-lactoferrin antibodies in ulcerative colitis

and primary sclerosing cholangitis but not inCrohn's disease. In Crohn's disease the fre-quency of IgG anti-a antigen and anti-myeloperoxidase antibodies did not differ

* Control

Cl Crohn's disease

O] Ulcerative colitis

B Primary sclerosing cholangitis

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statistically from the control group, although in afew instances the antibody levels were surpris-ingly high. In both ulcerative colitis and primarysclerosing cholangitis the frequency of positiveIgG anti-a/anti-myeloperoxidase tests differedstatistically from the control group, but ingeneral the levels were low and, considering thesmall number of patients with positive anti-cc/anti-myeloperoxidase tests, the relevance ofthese results is uncertain. However, it is interest-ing that these types ofANCAs were found at all,since they are considered to be reliable markersof primary systemic vasculitis.2 ANCAs of IgAclass have been reported in Hennoch-Schonleinspurpura and IgA nephropathy, although thesefindings are sometimes explained by thepresence of rheumatoid factor.33 The appearanceof antibodies directed against a antigen in somecases of ulcerative colitis (IgG) and in Crohn'sdisease (IgA) could possibly favour the hypo-thesis of primary vasculitis as a pathogeneticfactor in these diseases.""-8

Lactoferrin is an iron binding protein thatoccurs abundantly not only in the specificgranules ofgranulocytes,7 but also in tears, milk,and secretions at mucosal surfaces.3"36 Raisedlevels of circulating lactoferrin are seen duringactive inflammatory disease.37 It exerts anti-bacterial effects by depriving bacteria of ironrequired for growth, and it has anti-inflamma-tory properties - for example, by preventingcomplement activation through inhibition ofclassical C3 convertase.38 Lactoferrin can alsoprevent the formation of hydroxyl radicals byiron binding.3 139 Lactoferrin may thus be ofgreat importance as a non-specific anti-phlogisticdefence factor at the primary immunologicalbarriers. It has been shown experimentally thatbinding of lactoferrin by anti-lactoferrin anti-bodies increases the amount and duration ofhydroxyl radical formation by granulocytes.39Hypothetically, anti-lactoferrin autoantibodiescould, by counteracting the anti-inflammatoryeffects of lactoferrin, aggravate and prolongmucosal inflammation induced by several differ-ent mechanisms, and the antibodies may there-fore have pathogenetic significance even thoughtheir occurrence does not seem to correlate withdisease activity. Anti-lactoferrin antibodies mayalso have pathogenetic effects by activation ofprimed granulocytes infiltrating the gut mucosaor adhering to vessel walls, in analogy with theeffects of other types of ANCA.5I `' In addition,mucosal lactoferrin/anti-lactoferrin complexesmay stimulate intestinal goblet cells to excessivemucus secretion, as do other immune com-plexes.42 On the other hand, the occurrence ofanti-lactoferrin autoantibodies may, of coursemerely be an epiphenomenon without patho-genetic significance. The fact that the individualtypes of ANCA were found only in a minority(.50%) of the sera tested is an argument infavour of this. On the other hand, consumptionof circulating antibodies cannot be ruled out inthe seronegative instances.The origin of anti-lactoferrin autoantibodies is

unknown. Although bovine and human lacto-ferrin have molecular and antigenic similari-ties,35 immunisation of experimental animalswith bovine lactoferrin seldom seems to result in

cross immunisation against human lactoferrin,34and the lack of correlation between levels of anti-human lactoferrin and anti-bovine lactoferrinshown in this study contradicts the hypothesisthat cross immunisation against dietary bovinelactoferrin explains the appearance of anti-human lactoferrin autoantibodies. An excitingpossibility is the antigenic mimicry between the65 kD mycobacterial heat-shock protein (hsp-65) and human lactoferrin, and the fact thatimmunisation against hsp-65 results in the pro-duction of anti-lactoferrin antibodies.32 Myco-bacterial infection and immunisation againstmycobacterial antigens/hsp-65 have been impli-cated in several autoimmune disease states, bothexperimental and clinical.20"3 435 Consideringthe occurrence of IgA anti-lactoferrin antibodiesin some cases of Crohn's disease in the presentstudy, we find it interesting that IgA (but notIgG) anti-hsp-65 antibodies have been reportedin Crohn's disease.42 Furthermore, the registra-tion of anti-lactoferrin antibodies in a case ofCampylobacter colitis in this study is interestingwith regard to the possibility of cross immunisa-tion against microbial antigen(s). We thereforeintend to analyse additional sera from patientswith infectious gastroenteritis for the presence ofanti-lactoferrin antibodies.

In conclusion, we have shown high frequen-cies of IgG anti-lactoferrin antibodies in ulcera-tive colitis, and primary sclerosing cholangitis,but not in Crohn's disease, whereas IgA auto-antibodies directed against lactoferrin werefound in some cases of both ulcerative colitis andCrohn's disease. We suggest that anti-lactoferrinmay be of pathogenetic significance by counter-acting the antiphlogistic properties of lactoferrinat mucosal surfaces, thereby aggravating and/orsustaining mucosal inflammation initiated byother factors.

This study was financed by grants from Professor Nanna SvatrzFoundation, the Swedish Association against Rheumatism, KingGustaf Vth 80-Year Foundation, and the Swedish MedicalResearch Council.

1 van der Woude FJ, Rasmussen N, Lobatto S, et al. Auto-antibodies against neutrophils and monocytes: tool fordiagnosis and marker of disease activity in Wegener'sgranulomatosis. Lancet 1985; i: 425-9.

2 Kallenberg CGM, Cohen Tervaert JW, van der Woude FJ,et al. Autoimmunity to lysosomal enzymes: a new clue tovasculitis and glomerulonephritis? Immunol Today 1991; 12:61-4.

3 Jenne DE, Tschopp J, Ludemann J, Utecht B, Gross WL.Wegener's auto-antigen decoded. Nature 1990; 346: 520.

4 Falk RJ, Jenette JC. Anti-neutrophil-cytoplasmic auto-antibodies with specificity for myeloperoxidase in patientswith systemic vasculitis and idiopathic necrotizing andcrescentic glomerulonephritis. N Engl J Med 1988; 318:1651-7.

5 Skogh T, Dahlgren C, Holmgren K, et al. Anti-granulocyteantibodies (C-ANC, P-ANCA, GS-ANA) studied by con-focal scanning laser fluorescence microscopy, ELISA, andchemiluminescence techniques. Scand J Immunol 1991; 34:137-45.

6 Rasmussen N, Sjolin C, Isaksson B, et al. An ELISA for thedetection of anti-neutrophil cytoplasm antibodies (ANCA).J Immunol Meth 1990; 127: 139-45.

7 Furmanski P, Li Z-P. Multiple forms of Lactoferrin inNormal and Leukemic Human Granulocytes. Exp Hematol1990; 18: 932-5.

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