Proc. Nati. Acad. Sci. USAVol. 85, pp. 4015-4019, June 1988Medical Sciences

Anti-endothelial cell antibodies: Detection and characterization insera from patients with autoimmune hypoparathyroidism

(parathyroid gland/cell culture)

ANDREA FATTOROSSI*, GERALD D. AURBACH*, KAZUSHIGE SAKAGUCHI*, ALESSANDRO CAMAt,STEPHEN J. MARX*, ELIZABETH A. STREETEN*, LORRAINE A. FITZPATRICK*, AND MARIA LUISA BRANDI*t*Metabolic Diseases Branch, and tDiabetes Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda,MD 20892

Contributed by Gerald D. Aurbach, December 31, 1987

ABSTRACT In a previous report, we described antibodiesin autoimmune hypoparathyroidism (AHP) that are cytotoxicfor cultured bovine parathyroid cells. In the present study, weshow that sera from six AHP patients, but not from 26 patientswith other autoimmune diseases or from 7 healthy subjects,react with bovine endothelial cells in culture (by flow cytometryand fluoresence microscopy) and in tissue sections (by immu-nohistology). We found uniformly that the immunoglobulinclass reacting is IgM. Adsorption experiments showed that theantigenic determinants reacting with AHP sera were similar onbovine cultured endothelial cell membranes and in tissuesections of bovine parathyroid glands. The AHP sera alsoreacted with endothelial cells cultured from bovine adrenalmedulla and pulmonary artery. Immunoblot analysis showedantibody binding. to two major bands of 200 and 130 kDasolubilized from the membrane fraction of bovine parathyroidendothelial cells. Only one AHP serum consistently recognizedendothelium-related structures on frozen sections of threedifferent human parathyroid adenomas; two other sera reactedwith one adenoma each; and three did not react with humanadenomas. This indicates that human material is less suitablethan bovine in detecting endothelium-related immune phenom-ena in AHP sera. The anti-endothelium IgM antibodies appearto be disease-specific but are not organ- or species-specific. Theidentification of endothelial cells as the target for antibodies inAHP raises the possibility that the endothelium subserves animportant local function for endocrine epithelium.

In an earlier study, we found that antibodies in the sera ofpatients with autoimmune hypoparathyroidism (AHP) (1-5)reacted with and were cytotoxic for bovine parathyroid cellsin culture (6). We have now utilized fluorescence flowcytometry and immunohistology with class-specific antiserato study the nature of antibodies reacting with antigen(s) onhuman as well as bovine cells. We found that it is primarilyendothelial cells of parathyroid tissue that react with anti-bodies from patients with AHP.

(7, 8). In this study, we used PT-b cells from the same primaryculture between the 3rd and the 15th passage. Endothelialcells from bovine parathyroid tissue (BPE) were cloned bylimiting dilutions (M.L.B., K.S., and G.D.A., unpublishedresults) and cultured in Coon's modified Ham's F12 mediumthat contained antibiotics, 10%o Nu-Serum (CollaborativeResearch, Waltham, MA), 2% Ultroser-G (IBF, Savage,MD), 200 ,ug/ml of D-galactose (Sigma), 1.0 mM Ca2-, and0.5 mM Mg2 . Bovine adrenal medullary endothelial(BAME) cells (obtained from P. Lelkes, National Institutesof Health) were grown in minimal essential medium supple-mented with antibiotics and 10% (vol/vol) heat-inactivatedfetal calf serum (FCS) (Biofluids, Rockville, MD) as de-scribed (9). Bovine pulmonary artery endothelial (BPAE)cells (ATCC no. CCL 209) were maintained in minimalessential medium containing antibiotics and 20% FCS.Cloned human MCF-7 mammary cancer cells (obtained fromM. Lippman, National Institutes of Health) and normalhuman skin fibroblasts (HSF) (ATCC no. CRL 1229) weremaintained in Dulbecco's modified Eagle's medium supple-mented with antibiotics and 20% FCS.Immunofluorescence in Cultured Cells. Sera were tested in

an indirect immunofluorescence assay against a series ofcultured cells using flow cytometry and/or fluorescencemicroscopy. For flow cytometry experiments, cells werereleased from culture plates using 0.05% trypsin in phos-phate-buffered saline (PBS)/2mM EDTA and resuspended incold PBS (without Ca2" or Mg2+)/2% FCS. Cells (5 x 105in 1 ml) were incubated for 45 min in an ice bath with a 1:20dilution ofserum from AHP patients or normal patients. Afterrepeated washing with PBS, cells were incubated for anadditional 45 min in an ice bath with a 1:40 dilution of amonospecific, affinity-purified, fluorescein-conjugated goatanti-human IgG, IgM, or IgA (Kirkegaard and Perry Labo-ratories, Gaithersburg, MD). The cells were washed againwith PBS and used for flow cytometry (FACS 440, BectonDickinson; operating with an argon laser at 488 nm and 200mV) (10). Ethidium bromide (50 Ag/ml) was used to gate out

MATERIALS AND METHODSPatient Groups. Subjects were studied under approved

protocols at the National Institutes of Health. Serum sampleswere obtained from 42 patients with the following diagnoses:6, AHP§; 7, autoimmune thyroid disease; 4, Addison disease;3, idiopathic pulmonary fibrosis; 4, primary biliary cirrhosis;3, rheumatoid arthritis; 8, systemic lupus erythematosus; and7, normal subjects.

Cell Culture. Bovine (PT-b) and cloned rat (PT-r) parathy-roid cells were prepared and cultured as previously described

Abbreviations: AHP, autoimmune hypoparathyroidism; PT-b cells,bovine parathyroid cells; BPE cells, bovine parathyroid endothelialcells; BAME cells, bovine adrenal medullary endothelial cells; PT-rcells, rat parathyroid cells; HSF cells, human skin fibroblasts; BPAEcells, bovine pulmonary artery endothelial cells; FCS, fetal calfserum.tTo whom reprint requests should be addressed at: MetabolicDiseases Branch, National Institute of Diabetes, Digestive andKidney Diseases, Building 10, Room 9C101, Bethesda, MD 20892.§Patients 1-6 represent patients 2-7, respectively, from a previousreport (6). As previously described (6), sera from all AHP subjectsmediated specific lysis of PT-b cells as determined by a 51Cr releaseassay. Patients 1-6 were clinically hypoparathyroid, patients 3-6expressed mucocutaneous candidiasis, patients 5 and 6 were alo-pecic, and patients 4 and 6 displayed adrenal insufficiency. Patients1 and 2 are twin sisters; patients 3-5 are siblings in another family.There were no other affected members in the family of patient 6.

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dead cells from final counts (11). Quantitative data from flowcytometry are reported as the mean peak of fluorescenceexpressed in units of channel number. Comparable prepara-tions were also taken for fluorescence microscopy (6).

Immunohistologic Staining. Immunofluorescence was per-formed as described (12) on cold methanol/acetone-fixedfrozen sections obtained from several bovine and rat tissues,including parathyroid, adrenal, pituitary, kidney, and liver.Three human parathyroid adenomas were also used. Sec-tions, 6-8 ,um thick, were incubated with fluorescein-conjugated goat anti-human IgG, IgM, or IgA (1:20 in PBS).Double fluorescence staining was performed in human para-thyroid adenomas to compare the distribution of vascularendothelium and antigen(s) recognized by AHP serum. Fac-tor VIII-related antigen was chosen as a marker for endo-thelium (13). Sections were sequentially incubated with AHPserum and with a 1:30 dilution of rabbit antiserum for factorVIII-related antigen (Calbiochem-Behring). After washingwith PBS, sections were incubated with a mixture of fluo-rescein-conjugated goat anti-human IgM and fluorescein- orrhodamine-conjugated goat anti-rabbit IgG (1:30) (AccurateChemicals, Westbury, NY), washed, and scanned by fluo-rescence microscopy.

Adsorption Studies. Cells (10 x 106/ml) were resuspendedin PBS supplemented with 2% FCS and were incubated withAHP serum (1:10 final dilution) for 4 hr at 4°C. Suspensionswere then centrifuged, and the supernatant was used forimmunofluorescence studies. Adsorption was also carriedout using 10-fold concentrations of conditioned medium fromPT-b, BPE, BAME, PT-r, and HSF cell cultures. Theconditioned medium was concentrated by centrifugation onCentricon filters (Amicon). Concentrated medium was incu-bated with AHP serum (1:10 final dilution) for 4 hr at 4°C.This mixture was then immediately used for immunofluo-rescence analysis.

Statistical Evaluation. The significance of intergroup dif-ferences was evaluated by the nonparametric Wilcoxon ranksum test for independent samples (14).Immunoblot Studies. Serum IgM from AHP patient 6 and

from one healthy donor was purified by boric acid precipi-tation and Sephadex G-200 column chromatography (15).Membranes were prepared according to Thom et al. (16).Cells were lysed in hypoosmotic borate buffer, filteredthrough nylon gauze, and centrifuged at 450 x g for 10 minto separate large particles. The supernatant was centrifugedat 12,000 x g for 30 min, and the pellet was resuspended inPBS, layered above a 35% sucrose cushion in PBS, andcentrifuged at 24,000 x g for 1 hr. Purified membranes werecollected at the sucrose/PBS interface. Membrane proteinswere prepared for electrophoresis and fractionated on a 6%NaDodSO4/polyacrylamide gel according to Laemmli (17).Electrophoretic transfer was carried out as described (18).The nitrocellulose membrane was blocked with a buffercontaining 10% nonfat milk (Carnation, Los Angeles), 20 mMTris (pH 7.5), and 500 mM NaCI. Blots were incubatedovernight at 4°C with purified IgM from AHP patient 6. Afterextensive washing, blots were sequentially incubated at 4°C

with affinity-purified goat anti-human IgM (Kirkegaard andPerry Laboratories), affinity-purified rabbit anti-goat IgG(Kirkegaard and Perry Laboratories), and 125I-labeled affin-ity-purified goat anti-rabbit IgG (2 x i0s cpm/ml) (NewEngland Nuclear). After extensive washing, blots were driedand submitted to radioautography.

RESULTSIndirect Immunofluorescence in Cultured Cells. Immuno-

fluorescence analysis on trypsinized resuspended PT-b,BPE, or BAME cells revealed that sera from AHP patientsreacted with the cell membrane (Fig. 1A). No detectablestaining was observed with normal sera (Fig. 1B) or with serafrom 28 patients displaying autoimmune diseases other thanAHP. We found only IgM antibodies reacting with the bovinecells. PT-r, MCF7, and HSF cells were completely nonreac-tive (data not shown). Flow cytometry analysis confirmed thefluorescence microscopy observations and showed a singlepeak of fluorescence with PT-b, BPE, BAME, and BPAEcells (Fig. 2A). The intensity of fluorescence varied some-what among the bovine cell lines (data not shown) and amongpatients, probably reflecting different degrees of antigenicexpression and different antibody titer, respectively (Table1). Statistical analysis indicated that the fluorescence inten-sity in the bovine cell lines was significantly higher (P < 0.01)than in the other cell types tested (Table 1 and Fig. 2B).Among the non-AHP patients, serum from only one, anaddisonian, was reactive (Table 1). The reactivity in all caseswas ascribable solely to IgM antibodies. The intensity offluorescence obtained in bovine cells with the sera from otherautoimmune disease patients and healthy donors was signif-icantly (P < 0.01) lower than that obtained with sera fromAHP patients. As illustrated in Fig. 3, adsorption of AHPserum with BAME cells completely abolished reactivity onBPE cells. Comparable results were obtained by preincubat-ing AHP sera with PT-b and BPAE cells; adsorption with celltypes other than bovine was without effect (data not shown).A detectable reduction in fluorescence intensity was ob-served after incubating AHP sera with conditioned mediumfrom PT-b, BPE, and BAME cells. In this instance, thereduction in specific fluorescence intensity ranged between50% and 75%, as calculated by the shift in the mean peak offluorescence toward the control value. Conditioned mediumfrom PT-r or HSF cells were inert (data not shown).

Immunohistology. Results of immunohistological studieson frozen sections of tissue were reasonably consistent withthe immunofluorescent findings on cultured cells. All serafrom AHP patients contained IgM antibodies that reactedwith determinants in bovine parathyroid tissue closely re-lated to the vascular endothelium and in close apposition tothe epithelial cell membrane in the region of vascular cells(Table 2 and Fig. 4A and B). The non-AHP addisonian patientdisplaying anti-endothelial antibodies also showed positivereactivity in the immunohistochemical analysis. A similarendothelium-related pattern was also observed in bovineadrenal, pituitary, and kidney, but there was much lower

FIG. 1. Staining patterns of BPEcells in suspension. Reaction of serumfrom AHP patient 6 (A) and a normalsubject (B). Indirect immunofluores-cence staining was obtained with fluo-rescein-conjugated anti-human IgM asthe second antibody. The exposuretimes for the micrographs in A and B.(x 300.)

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a)m

E

CD

0

A

llllI,III

,I

11I

4w

B

E

0

Fluorescence intensity

FIG. 2. Flow cytometry profile of PBE cells (A) and Pl-r cells(B) incubated with serum from AHP patient 2 ( ) or a normalsubject (--- ). Indirect immunofluorescence staining was obtainedwith fluorescein-conjugated anti-human IgM as the second antibody.Results are displayed as cell number versus relative fluorescenceintensity. The mean peaks of fluorescence (in units of channelnumbers) were 35 and 81 in A and 29 and 32 in B for normal and AHPserum, respectively.

intensity of immunohistochemical staining than was found inthe parathyroid tissue (data not shown). In these othertissues, antigenic material was not as evenly distributed as inthe parathyroid sections; it was detectable in certain areasbut not in others, and it had no obvious relation to theabundance of vascular endothelium. Sera from other autoim-mune disease patients and healthy subjects did not react withsuch endothelium-related structures in any tissue examined,although sera from lupus erythematosus and Graves diseasepatients did react with the expected nuclear or cytoplasmicdeterminants. Bovine liver and all rat tissues includingparathyroid, adrenal, pituitary, kidney, and liver were uni-formly unreactive (data not shown). Incubation of AHPserum with PT-b, BPE, BAME, or BPAE cells dramaticallyreduced or completely abolished the fluorescent staining onbovine parathyroid sections. Adsorption with PT-r or HSFcells was without effect (data not shown).Less consistent results were obtained from testing sera

from AHP patients on three human parathyroid adenomas.Only serum from patient 2 gave positive results with all threespecimens. Two other sera were positive with one specimeneach (Table 2 and Fig. 4C), and three sera were reactive withnone of the adenomas. The immunofluorescence patternclosely resembled the one observed in bovine parathyroidtissue. In some areas, however, the immunohistochemicalstaining was also detectable on the surface or in the cyto-plasm of parathyroid epithelial cells. Adsorption of positive

Table 1. Reactivity of serum from subjects with AHP or otherautoimmune diseases and from healthy subjects on cultured cellsby flow cytometry

IgM fluorescence

Diagnosis Bovine cells* Other cellstAHP 1 49-67 33-38AHP 2 65-81 32-38AHP 3 57-64 31-35AHP 4 51-58 31-36AHP 5 49-55 28-33AHP 6 58-70 28-32Other autoimmune

diseases (n = 28)t 31-38 NDAddison disease (n = 1) 41-54 31-36Normal (n = 7) 29-36 29-35

Reactivity was detected with fluorescein-conjugated anti-IgM.Values represent the range of intensity expressed as the mean peakof fluorescence in units of channel numbers. ND, not determined.*PT-b, BPE, BAME, and BPAE cells.tPT-r, MCF-7, and HSF cells.tThe positive patient affected by Addison disease is not included.

Fluorescence intensity

FIG. 3. Flow cytometry profile ofBPE cells incubated with AHPserum (from patient 6) preadsorbed on BAME cells (- ) or withnormal serum similarly preabsorbed (- -). Indirect immunofluores-cence staining was obtained with fluorescein-conjugated anti-humanIgM as the second antibody. Results are displayed as cell numberversus relative fluorescence intensity. The mean peak of fluores-cence (in units of channel numbers) was 31 for preadsorbed AHPpatient serum and 32 for control serum.

sera with PT-b, BPE, BAME, or BPAE cells markedlyreduced the intensity of fluorescence closely related to thevascular endothelium on human parathyroid adenomas, leav-ing unchanged the epithelial staining (data not shown).Adsorption with PT-r or HSF cells had no effect.Immunoblot Studies. Immunoblot analysis of purified

membrane preparations showed that serum from AHP pa-tient 6 identified major bands of 200 and 130 kDa in BPE cellmembranes (Fig. 5). The high background reactivity ob-served in this analysis is characteristic of immunoblotsexposed to IgM antibodies. Membranes from HSF cellsshowed complete absence of these bands (Fig. 5).

DISCUSSIONThe current studies elucidate further our earlier findings thatantibodies circulating in AHP react with cultured PT-b cells(6). We now show that antigenic determinants reacting withsuch antibodies are found predominantly on endothelial cellsand exist in human as well as in bovine tissue. The closesimilarity of antigen(s) in cell cultures and whole tissues isillustrated by the relationship between immunofluorescenceintensity between cells in suspension and in tissue slices andby results of adsorption studies. This rules out the possibilitythat the expression of the determinants recognized by AHP

Table 2. Reactivity of serum from subjects with AHP or otherautoimmune diseases and from healthy subjects on parathyroidtissue of bovine and human origin

Reactivity on parathyroid tissue

Human adenoma

Diagnosis Bovine A B CAHP 1 + + ND ND + + +AHP 2 +++ +++ +++ +++AHP 3 +++ - - -AHP 4 + - - NDAHP 5 ++ ND - -AHP 6 +++ - +-Other autoimmune

disease (n = 20)Normal (n = 6) - - ND

Indirect immunofluorescence was developed with fluorescein-conjugated anti-human IgM as the second antibody. Each experi-ment was carried out at least twice with the same specimen.Reactivity was characterized as being negative/weak (-), moderate(+), strong (+ +), or very strong (+ + +). The tissues were normalbovine parathyroid and three human parathyroid adenomas. ND, notdetermined.

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20--4 - 200

- 130

97.4 -

68 -

43 -

A B

FIG. 5. Immunoblots with extracts ofBPE cell membranes and human fibro-blast membranes reacted with IgM prep-aration from AHP patient 6. Lanes: A,human fibroblasts; B, BPE cells. Themajor bands of 200 and 130 kDa areindicated. A third band (in lane B), justbelow the 200-kDa marker, also wasobserved, but its significance is unclearbecause the background is also increasedin that area. The positions of the molec-ular size standards (in kDa) are alsoindicated.

FIG. 4. Section of bovine parathyroid tissue stained with AHPserum (patient 6) using fluorescein-conjugated anti-human IgM as thesecond antibody (A) or with rabbit anti-human factor VIII-relatedantigen using fluorescein-conjugated anti-rabbit IgG as the secondantibody (B). The highest concentration of the antigenic materialrecognized by serum from AHP patients was found distributed inregions corresponding to interstitium or vascular endothelium (arrowsin A). However, the reaction product was not restricted to theendothelium but was diffusely distributed throughout the whole glandgiving the appearance of an external matrix in close apposition to theepithelial cell membrane (arrowheads and Inset in A). The distributionof endothelium is outlined by the reactivity with factor VIII-relatedantigens in B (arrows). Illustrations have been taken from comparablesections of the same parathyroid tissue. The intense yellowish auto-fluorescence of intracytoplasmatic granules is an intrinsic property ofthe unstained tissue. (A) (x 420.) (Inset) (x 630.) (B) (x 315.) (C)Section of human parathyroid adenoma doubly stained using AHPserum (from patient 2) plus fluorescein-conjugated anti-human IgM

antibodies on cultured cells represents an artifact due tononphysiological in vitro conditions rather than an intrinsicendothelial cell characteristic. In flow cytometry experi-ments, PT-b cells gave a single peak of fluorescence withAHP patients' sera, indicating a uniform distribution ofreacting antibodies on the cell population. PT-b cells arecomprised ofboth epithelial and endothelial parathyroid cells(M.L.B., K.S., and G.D.A., unpublished data). The singlepeak offluorescence in this mixed cell population may reflecttransfer of this antigen from endothelial to epithelial cells.This possibility is supported by the observation that concen-trated conditioned medium from such cultures can adsorb theanti-bovine endothelium antibodies and that endothelium-related antigenic material in frozen parathyroid sections wasfound in close apposition to epithelial cells. In a previouspaper we showed that sera from the same AHP patientscaused complement-mediated lysis ofPT-b cells (6). Analysisof earlier and current findings indicates that the magnitude ofcytotoxic effect is proportional to the intensity of the immu-nofluorescent staining. In our previous report, we alsoobserved that adsorbing AHP patients' sera with bovineorgan extracts other than parathyroid decreased the cyto-toxic activity on PT-b cells to a variable extent (6). Immu-nohistological data obtained here reveal that the distributionofantigenic material in certain bovine organs such as adrenal,kidney, and pituitary is not uniform. This suggests thattissues vary in their content of antigen(s) that react with AHPantibodies. The antibodies identified here reacting withendothelium are in the IgM class. We note that Haspel et al.(19), who studied monoclonal antibodies obtained from miceaffected by autoimmune polyendocrine disease, also foundsuch antibodies to be of the IgM class.We found variable reactivity of AHP sera with human

parathyroid adenoma tissue. Inconsistencies in detecting anti-parathyroid antibodies by using human substrata in idiopathichypoparathyroidism have already been noted (1). Recently, ithas been reported that human parathyroid adenomas may notbe representative of normal expression of tissue antigens (5);variation in tumor tissue expression of antigens may account,at least in part, for the inconsistent reactivities that weobserved in our series of patients and for the variable and lowyield for detection of anti-parathyroid antibodies in AHP (1-5,20-22). The age distribution of our patients was 12-24; in thestudy of Posillico et al. (20), it was 56-71.

anti-serum and also factor VIII-related antigen and rhodamine-conjugated anti-rabbit IgG antiserum. Two consecutive exposuresfrom the same microscopic field were made on the same frame usingfilter sets for fluorescein or rhodamine. Fluorescence (green and red)codistributes in most areas (arrows) and sometimes appears asyellow staining due to superimposition of green and red fluoro-chromes. Deposits of IgM only are also visible. ( x 630.)

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Immunofluorescent deposits, similar, but not identical, inpattern, were found in bovine and human parathyroids. Thissuggests that the antigenic determinants in the two kinds oftissues do not completely overlap. Adsorption experimentsconfirmed this view by showing that AHP serum preincu-bated with bovine endothelial cells still gave detectablereactions with human parathyroid adenoma epithelium.The significance of the anti-endothelium antibodies in the

pathogenesis of AHP cannot be conclusively established.The reactivity with the outer cell membrane in culture andwith accessible determinants in whole tissues fulfills one ofthe essential prerequisites for a pathogenic antibody (5). Theanti-endothelial antibodies do not appear to be organ-specificsince reactivity is found in nonparathyroid tissues. The lackof reactivity with bovine liver and all rat tissues might reflectlow concentrations of antigen that are undetectable with ourmethods, lack of the antigen in those tissues, or existencetherein of related antigens distinct in immunoreactivity.Although not organ-specific, the anti-endothelium antibodiesappear to be disease-specific; in only one subject withoutclinical evidence of parathyroid dysfunction were character-istic IgM antibodies detectable. This patient displayed overtAddison disease. Adrenocortical insufficiency is an ex-tremely frequent finding in AHP patients with type I poly-glandular autoimmune disease (21, 22). A 20% incidence ofparathyroid antibodies in patients with isolated idiopathicadrenocortical insufficiency has been reported (22). In addi-tion, experimental immune hypoparathyroidism in dogs isassociated with immunopathological features of the adrenalcortex (23). As envisioned by others (24), the possibilityexists that in some cases damage to the parathyroid glandsmay be subclinical or that circulating antibodies appearbefore glandular dysfunction becomes fully developed.We have used immunoblotting techniques to gain further

insight into the nature of the antigen(s) recognized by AHPpatients' serum. These studies identified major bands of 200and 130 kDa in BPE cell membranes that reacted with AHPserum. Although more AHP patients clearly need to beinvestigated to allow final conclusions, the possibility arisesthat these structures represent the major target for AHPantibodies.

Recently, Posillico et al. (20) described three cases ofidiopathic hypoparathyroidism displaying antibodies thatinhibit secretion of parathyroid hormone from dispersedcells. They also reported that these sera reacted with com-ponents on tissue sections of human parathyroid adenomas.However, one could not determine whether endothelial orepithelial cells contained the major reactive component.

Complement-fixing, non-species-specific, anti-endothelialcell antibodies have been detected in a number of autoim-mune diseases and are often reported to be reactive withdiverse organs (25-29). Major differences exist, however,between those findings and our results. With few exceptions,previously described anti-endothelium antibodies were pri-marily IgG or, more rarely, IgA. Most important, all reportsknown to us deal with non-organ-specific autoimmune dis-eases, such as systemic lupus erythematosus, scleroderma,and rheumatoid arthritis, and/or with long-term chronicinfectious disorders.By using immunofluorescence techniques, we have de-

tected, in the sera of AHP patients, antibodies of the IgMclass directed against antigen(s) found on bovine endothelialcells. The exact significance of these antibodies in regard tothe pathophysiology ofthe disease remains to be clarified andcorrelated with clinical findings in AHP and other patients.Forsberg et al. (30) have suggested that endocrine cells, byreleasing ATP to interact with purinergic receptors, canregulate endothelial cells. Control of blood flow might be one

function influenced thereby (30). Specific antibodies againstendothelial cells might explain the involvement of severalorgans in AHP. Do endothelial cells, the entity bearing thetarget antigen for antibodies of the type studied here, providea key function required for normal endocrine epithelial cellphysiology?

We thank Drs. Penelope Feuillan, Carolyn Bondy, Merily Poth,and Allen Spiegel for referral of their patients. The skillful technicalassistance of Ms. Susan A. Barbieri for fluorescence-activated cellsorter analysis and the excellent secretarial work of Mrs. Joan M.Glass are also gratefully acknowledged. We are grateful to Dr. PeterLelkes for a critical reading ofthe manuscript. This work was carriedout, in part, under a grant to A.F. from the Burroughs-WellcomeFund.

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