Endothelial Injury in Internal Organs of University of California at Davis Line 200 (UCD 200) Chickens, an Animal Model for Systemic Sclerosis (Scleroderma)

Journal of Autoimmunity (2000) 14, 143–149doi: 10.1006/jaut.1999.0355, available online at http://www.idealibrary.com on

Endothelial Injury in Internal Organs of University ofCalifornia at Davis Line 200 (UCD 200) Chickens, anAnimal Model for Systemic Sclerosis (Scleroderma)

Van Anh Nguyen, Roswitha Sgonc, Hermann Dietrich and Georg Wick

Institute for General and ExperimentalPathology, University of Innsbruck,Medical School, Innsbruck, Austria

Received 9 August 1999Accepted 22 October 1999

Systemic sclerosis (SSc) is a multisystem disorder characterized by mono-nuclear cell infiltration and fibrosis. Using skin samples from human SSc andUCD 200 chickens, which spontaneously develop a hereditary disease closelyresembling human SSc, we have shown previously that endothelial cellapoptosis is a primary event in the pathogenesis of SSc. The aim of the presentstudy was to investigate the initial disease stage in visceral organs of UCD 200chickens with special emphasis on endothelial apoptosis, mononuclear cellinfiltration and collagen deposition using tissue samples from oesophagus,lung, heart, kidney and liver. Apoptotic endothelial cells were detected byterminal deoxynucleotidyl transferase-mediated FITC-dUTP nick end labeling(TUNEL), mononuclear cell infiltrates were stained with hematoxylin andeosin, and increased collagen deposition was demonstrated by Goldnerstaining. Apoptotic endothelial cells were detected in oesophagus, lung andkidney of UCD 200 chickens at the initial stage of the disease. No apoptoticendothelial cells were found in heart or liver of UCD 200 or in visceral organsof healthy normal UCD 058 control chickens. Oesophagus of UCD 200chickens, which was the most affected internal organ, showed mononuclearcell infiltrations and increased deposition of collagen. Perivascular inflamma-tory infiltrates and collagen deposition appeared later than endothelial cellapoptosis. These data support the hypothesis that endothelial cell apoptosisinitiates the disease process, followed by mononuclear cell infiltration andfibrosis. © 2000 Academic Press

Key words: apoptosis,systemic sclerosis, vasculardamage, endothelial cells,fibrosis, visceral organs

Correspondence to: Roswitha Sgonc, PhD, Institute for General andExperimental Pathology, University of Innsbruck, Medical School,Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria. Fax: 0043/512–507–2867. E-mail: [email protected]

Introduction

Systemic sclerosis (SSc), or scleroderma, is a general-ized autoimmune disorder of the connective tissueassociated with increased deposition of collagen andother matrix components that cause extensive fibroticdestruction of the skin as well as internal organs,including gastrointestinal tract, lung, heart andkidney [1–3]. Other hallmarks of the disease in-clude pathologic changes in the small arteries andthe microvasculature, prominent abnormalities inhumoral and cellular immunity, and perivascularmononuclear cell infiltration [4–6]. Vascular pathologyconsists essentially of intimal proliferation and dupli-cation of basal lamina leading to luminal narrowingand complete capillary occlusion. Finally, these alter-ations induce devascularization with subsequentunderperfusion and chronic ischaemia of involved

1430896–8411/00/020143+07 $35.00/0

organ systems. The humoral immunological abnor-malities are characterized by increased B lymphocyteactivation and expression of autoantibodies [7, 8]. Themost prominent cellular immunological abnormalityis the imbalance of Th1/Th2 cell function, probablydue to an excess of Th2 cell activity [9, 10].

SSc is a disorder that progresses through manystages. Vascular and immunological abnormalitiespredominate in the early stages, whereas fibrosis ofaffected organs is most prominent in the later stages ofthe disease. Investigations of the early inflammatorystage are likely to be of great importance for theelucidation of the initiating factors and pathogeneticprocess of this disease. Since initial developments arenot accessible for analysis in humans and are, there-fore, often missed, appropriate animal models arerequired. University of California at Davis line 200(UCD 200) chickens, a well-established animal modelfor human SSc, spontaneously develop an inheritedfibrotic syndrome with features similar to thoseobserved in human scleroderma, which permits studyof the initial stage of the disease [11–14]. Thesechickens exhibit the entire clinical, histopathological

© 2000 Academic Press

144 V. A. Nguyen et al.

Materials and Methods

Animals

UCD 200 and healthy UCD 058 normal WhiteLeghorn chickens were bred and maintained in theCentral Laboratory Animal Facilities of the MedicalSchool of the University of Innsbruck, Austria. Thenatural history, care, and maintenance of these UCDlines have been extensively described [11].

Biopsies

Animals were killed by cardiac exsanguination underNembutal anaesthesia (20 mg/kg). Biopsy specimenswere taken from oesophagus, lung, heart, kidney andliver of 30 UCD 200 and 25 control UCD 058 chickensat different disease stages at 1, 2 and 5 weeks, and at2 and 11 months of age. Biopsy material was immedi-ately frozen in liquid nitrogen and stored at −196°Cuntil sectioning.

Antibodies and conjugates

Rabbit anti-von Willebrand factor (vWf), which isconsidered an endothelial cell marker, was diluted1:50 in PBS/1% BSA and tetramethylrhodamineisothiocyanate (TRITC)-swine Ig to rabbit Ig 1:30(Dakopatts, Glostrup, Denmark).

Figure 1. Histology of 2-month-old UCD 200 chicken combshowing narrowing of the lumen of a small artery withintimal proliferation and hyperplasia of the media (originalmagnification 400×).

Simultaneous detection and characterization ofapoptotic cells

Characterization of endothelial cells was performedby standard indirect immunofluorescence tests (IIF),

and serological spectrum of SSc studied thus far. Theinherited scleroderma-like disease starts at 1–2 weeksafter hatching with inflammation of the skin, mostprominently in the comb and to a lesser degree inthe neck. Initially, the comb becomes swollen anderythematous, and subsequently necrosis and loss ofthe comb (so-called ‘self-dubbing’) occurs in about90% of birds by 4 weeks of age. Histological examin-ation at this stage shows occlusion of small arteriesand arterioles with thickening of the muscle wall,narrowing of the lumen and distinctive intimal pro-liferation (Figure 1). These vascular lesions areaccompanied with a severe perivascular mononuclearcell infiltration and excessive accumulation of colla-gen types I, II and III in skin and viscera. Subsequenttissue fibrosis occurs later in the course of the disease,usually as the inflammatory infiltrates begin todecrease. At 6 months of age, the majority of line 200chickens have extensive pathology of visceral organs,including oesophagus, lung, heart, kidney and testes.In addition, the affected birds develop multiple sero-logical abnormalities with production of a variety ofautoantibodies, i.e., rheumatoid factors, anticardio-lipin, antiendothelial cell antibodies (AECA), anti-cytoplasmic and antinuclear antibodies [11, 15, 16].

The underlying pathogenetic events of SSc are stillnot fully understood, and several mechanisms havebeen proposed. It is clear that three cell types areinvolved in the disease process and responsible formost of the clinical and pathologic manifestations ofSSc. These are: (1) endothelial cells, (2) mononuclearcells, and (3) fibroblasts. Vascular and immune dys-function and the interstitial fibrosis are thought toresult from an interaction of these cells with a varietyof cytokines, which may trigger a cascade of eventsleading to uncontrolled fibroblast activation.

Primary alterations of endothelial cells of smallvessels have been thought to play a principal rolein initiating or perpetuating the immunologic andconnective tissue alterations characteristic of SSc.Although the mechanism of vascular damage in SSc

has long been investigated, it remains unclear. Severalfactors have been implicated to cause endothelialinjury, including granzyme A and B [17–20], AECA[21, 22] complement factors [23] and oxygen freeradicals [24]. Recent findings in a study of skin biop-sies of UCD 200 chickens and human SSc patientsrevealed that endothelial cell apoptosis is a primaryevent in the pathogenesis of SSc, possibly induced byAECA-dependent cellular cytotoxity (ADCC) [25, 26].The aim of the present study was to investigatewhether endothelial cell apoptosis is a localized phe-nomenon in the skin or is also present in internalorgans, particularly whether apoptotic endothelialcells in the affected internal organs occur simul-taneously with, or later than in the skin, which wouldpoint to endothelial heterogeneity. Since internalorgans are not accessible in human patients, especiallyin the initial disease stage, the studies were performedin UCD 200 chickens. In parallel, we also analysed thechronological appearance of endothelial cell apop-tosis, perivascular mononuclear cell infiltration andcollagen deposition in the skin and viscera.

An animal model for systemic sclerosis (Scleroderma) 145

and detection of apoptotic cells was accomplishedby terminal deoxynucleotidyl transferase-mediatedFITC-dUTP nick end labeling (TUNEL) technique [25,27]. For this purpose, 4-�m frozen tissue sections wereincubated for 30 min at room temperature (RT) withrabbit anti-vWf primary antibodies. After washing for30 min in PBS, TRITC-conjugated swine anti-rabbit Igsecondary antibodies were added to the tissue for30 min at RT. After additional washing for 30 min inPBS, sections were fixed for 20 min in 4% paraformal-dehyde, washed as previously described, permeabil-ized with 0.1% Triton X-100/0.1% sodium citrate for2 min on ice, dehydrated by 50%, 75%, and 100%ethanol, rinsed in chloroform and incubated for60 min in a moist chamber at 37°C. Thereafter, theTUNEL reaction was carried out by incubating tissuesections with 15 �l 0.6 �M FITC-12-dUTP (BoehringerMannheim, Germany), 60 �M dATP, 1 mM CoCl2,TdT buffer (30 mM Tris, pH 7.2, 140 mM sodiumcacodylate) and 25 units terminal deoxynucleotidyltransferase (Boehringer Mannheim) covered witha plastic cover slip (1 h at 37°C). The reactionwas stopped with Tris/EDTA, pH 8 (5 min). Afterthree washes of 5 min with TBS, pH 7.4, slideswere mounted with Mowiol (Hoechst, Frankfurt,Germany). Tissue sections from bursa of Fabricius ofUCD 058 chickens served as positive control for theTUNEL reaction, and as control for the conjugateas well.

Characterization of mononuclear cell infiltrates

Mononuclear cell infiltrates were stained with hema-toxylin and eosin [28]. Prior to staining, tissue sectionswere fixed for 5 min in 4% formaldehyde (Merck,Darmstadt, Germany) and washed for 10 min inwater. Sections were then stained for 30 s in Mayer’shaemalum (Merck) and washed as previouslydescribed. After staining for 20–30 s in 1% eosin(Merck), sections were dehydrated by 50%, 75%, 100%ethanol and mounted with Eukitt (Kindler, Freiburg,Germany).

Detection of increased collagen deposition

Increased collagen deposition was demonstrated byGoldner staining [28]. Briefly, tissue sections wereairdried for 30 min at room temperature, fixed for30 min in Zenker’s solution and stained for 1 min inWeigert’s ferric hematoxylin. After washes with water,sections were stained for 5 min in azophloxin andrinsed briefly in 1% acetic acid (Merck). Subsequently,sections were differentiated for 3 min in phosphor-molybdene acid-orange (Merck) and rinsed briefly in1% acetic acid. Finally, slides were dehydrated by50%, 75%, 100% ethanol and mounted with Eukitt(Kindler, Freiburg).

Microscopic analysis

IIF and TUNEL double stainings were analysed anddocumented using a Leitz Ortholux epi-illuminationfluorescence microscope. Histologic preparationswere read on a Nikon Optiphot microscope (Nikon,Tokyo, Japan).

Results and Discussion

Table 1. Apoptotic endothelial cells in internal organs of UCD 200 chickens

Chickens AgeApoptotic endothelial cells

Oesophagus Lung Kidney Heart Liver

UCD 200 1 weeka 1c/5d 4/5 0/5 0/5 0/51 weekb 0/5 0/5 1/5 0/5 0/52 weeks 1/5 3/5 0/5 0/5 0/55 weeks 1/5 0/5 0/5 0/5 0/52 month 0/5 1/5 0/5 0/5 0/511 month 0/5 1/5 0/5 0/5 0/5

UCD 058 1 week–11 month 0/25 0/25 0/25 0/25 0/25

a Comb showing no macroscopic alterations compared to healthy chickens.b Comb with erythema and edema.c Number of animals showing apoptotic endothelial cells.d Number of animals analysed.

Simultaneous detection and characterization ofapoptotic cells

Using our modified TUNEL technique [27], apoptoticendothelial cells were detected in oesophagus, lungand kidney. Thirty percent of the UCD 200 chickensexhibited endothelial cell apoptosis in the lung, 10% inthe oesophagus and 3% in the kidney. Additionally,increased numbers of apoptotic cells were observed inthe lung of UCD 200 chickens, scattered diffusely ininterstitium and concentrated primarily around smallblood vessels. In contrast, these findings were notnoted in other visceral organs. No apoptotic endo-thelial cells were found in heart and liver of UCD200 chickens. As expected, endothelial cells did notundergo apoptosis in any of the healthy control line058 birds (Table 1, Figure 2).


Figure 2. Simultaneous determination of endothelial cells and apoptosis on 4-�m frozen sections using vWF-IIF and TUNELtechniques: vWF-positive endothelial cells in red, and TUNEL-positive apoptotic nuclei in bright green (arrows) on (A)oesophagus, (C) lung and (E) kidney sections of 1–2-week-old UCD 200 chickens. No apoptotic endothelial cells were foundon (B) oesophagus, (D) lung or (F) kidney sections of healthy UCD 058 control chickens (original magnification 500×).


Time course analyses revealed that, similar to theskin, endothelial cell apoptosis in internal organs wasvisible at the initial and early inflammatory stage ofthe disease, i.e. 1–2 weeks after hatching. Sporadicapoptotic endothelial cells were seen in oesophagealsections of UCD 200 chickens at 1, 2 and 5 weeks ofage. In lung, endothelial cell apoptosis was evident at1 and 2 weeks, and at 2 months post-hatching,whereas apoptotic endothelial cells in kidney wereonly detected in 1-week-old UCD 200 chickens, whichby that age have already developed erythema andoedema of the comb.

The data described herein are generally consistentwith those previously demonstrated in the skinshowing apoptotic endothelial cells by the TUNEL

technique in the deeper dermis of UCD 200 chickensand human SSc patients at the initial and earlyinflammatory disease stages.

Figure 3. HE staining on oesophagus sections of 2-month-old (A) UCD 200 and (B) UCD 058 chickens showing perivascularmononuclear cell infiltrates (arrows) only in the UCD 200 strain. Goldner staining on oesophagus sections of 2-month-old (C)UCD 200 and (D) UCD 058 chickens with collagen in green and muscle in red. Note the increased collagen deposition in UCD200 chicken (original magnification 200×).

Mononuclear cell infiltrates and collagendeposition

Intense accumulation of mononuclear cells was shownby HE-staining in the vicinity of small- to medium-sized blood vessels in the oesophagus. The perivascu-lar inflammatory infiltrates were evident at 5 weeks,and at 2 and 11 months in approximately 10% of UCD200 chickens, thus appearing later than endothelialcell apoptosis.


Collagen deposition was analysed in affected andunaffected organs by Goldner staining. Comparedwith healthy controls, oesophagus sections of UCD200 chickens showed thickening and hyalinization ofconnective tissue in the lamina propria and the sub-mucosa by densely packed collagen extending intoand through the muscular layer. Fibrotic lesions inoesophagus started 2 months post-hatching and wereseen in 40% of UCD 200 chickens at 1 year of age, thusfollowing endothelial cell apoptosis and perivascularinflammatory infiltrates (Figure 3).

Although endothelial cell apoptosis was found inoesophagus as well as in lung and kidney of UCD 200chickens, perivascular mononuclear cell infiltrates andfibrotic lesions were not seen in the latter organs. Thismight be due to the selective breeding conditions.Resulting from collaboration with M. E. Gershwin,UCD line 200 chickens have been maintained in theCentral Laboratory Animal Facilities of the Universityof Innsbruck Medical School since 1988. Thesechickens are selected according to the phenotypicalalterations of the comb and the neck, and accordingto their reproduction rate. The breeding of UCD 200birds is very difficult, since involvement of lung, heartor kidney results in high mortality rates and theprocreative capacity of male chickens is impaired byfibrotic destruction of the testes. Thus, animals withless visceral involvement were bred to accomodateour selection criteria. This also explains the higherpercentages of visceral involvement in UCD 200chickens originally described by Gershwin andcoworkers [11, 29].

As in previous investigations on the skin [25], thefindings in the present study clearly show that bothmononuclear cell infiltrations and excessive collagendeposition appear in the disease progression afterendothelial cell apoptosis. Therefore, we conclude thatperivascular accumulation of mononuclear cells is notresponsible for endothelial cell apoptosis, but is rathera sequel of endothelial damage. The role of inflamma-tory cells in induction and/or perpetuation of apop-totic processes remains unclear, and requires furtherinvestigation.

What triggers apoptosis in endothelial cells, sinceendothelial cell apoptosis is obviously not induced byperivascular infiltrating mononuclear cells? It isimportant to emphasize that the lack of perivascularlymphocytes in the acute stage of the disease does notexclude a possible immunologic attack againstendothelial cells by inflammatory cells located in thelumen of the vessels. In fact, NK cells have beenconsidered as possible effector cells [30, 31]. Recentin vitro studies have shown that endothelial cell apop-tosis in SSc is induced by ADCC via the fas-FasLpathway [26].

We observed that, similar to the comb, endothelialcells in internal organs of UCD 200 chickens undergoapoptosis, but in a smaller percentage of the animals.Accordingly, we questioned why endothelial cellapoptosis seems to occur preferentially in the skin,since all endothelial cells are exposed equally toAECA and NK cells. One possible explanation for thisphenomenon would be heterogeneity of endothelial

cells. There have been reports indicating significantdifferences between endothelial cells from large andsmall arteries and veins from the skin and internalorgans [32–35], including differences in prostaglandinproduction, in vitro growth requirements, and cellsurface protein expression. Based on these findings, itis conceivable that AECA and NK cells located in thevessel lumen can induce apoptosis only in thoseendothelial cells which are, based on still unknownproperties, susceptible targets for anti-endothelialcell-antibody dependent cellular cytotoxicity.

In summary, the results obtained in this studyrepresent the first report that endothelial cell apopto-sis is not a localized phenomenon in the skin, but alsothe initial pathogenetic event demonstrable in otheraffected organs of UCD 200 chickens. This findingstrongly confirms the hypothesis that endothelial cellapoptosis plays an important role in the initiation ofSSc followed by accumulation of perivascular mono-nuclear cells and fibrosis. However, an unsolved prob-lem in understanding the mechanisms underlying theapoptosis-inducing effect of AECA on endothelium isthe lack of definition of the respective target struc-tures. Our further studies will focus on the identifi-cation of the cell surface epitope(s) recognized byAECA with apoptosis-inducing activity.

Acknowledgements

We thank Ruth Resch and Ilona Atzinger for thehistologic stainings and for figure production, respect-ively. This work was supported by the AustrianScience Fund (project No. 12715).

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Endothelial Injury in Internal Organs of University of California at Davis Line 200 (UCD 200) Chickens, an Animal Model for Systemic Sclerosis (Scleroderma)