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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology characterized by mononuclear cell infiltration and fibrosis. Vascular injury occurs early in the course of disease, and previous in vitro studies suggest a primary role for anti–endothelial cell antibodies (AECAs) in mediating endothelial cell apoptosis. The aim of the present study was to analyze the apoptosis-inducing effect of AECAs in vivo.

Methods

The optimum animal model for transfer experiments was the University of California at Davis line 200 (UCD-200) chickens that spontaneously develop a hereditary disease with features closely resembling those of scleroderma in humans. AECA-positive serum samples from UCD-200 chickens were used for intravenous injection into normal CC chicken embryos on embryonic day (ED) 13 as well as for application onto chorionallantoic membranes (CAMs) of healthy control lines on ED 10. CAMs of ED 16 embryos and combs of 1-week-old CC chickens that had received the injected serum samples were analyzed for apoptotic endothelial cells by TUNEL.

Results

Staining of frozen CAM sections by immunofluorescence showed evidence of in vivo binding of AECAs to the microvascular endothelium. In most groups, transfer of AECA-positive sera resulted in a significant increase in endothelial cell apoptosis as compared with controls.

Conclusion

This study is the first to demonstrate the in vivo apoptosis-inducing effects of AECAs. The findings support our hypothesis of a primary pathogenetic role of AECAs in SSc.

Systemic sclerosis (SSc) is a generalized connective tissue disorder of unknown etiology characterized by vascular lesions, immune dysregulation, and an increased deposition of extracellular matrix in the skin and in internal organs. These 3 pathogenetic processes are thought to be closely related, since endothelium, mononuclear cells, and fibroblasts interact through direct contact and via cytokines, which finally leads to fibrosis (1, 2). The initiating event that starts the pathogenetic cascade remains to be elucidated. The search for the etiology requires animal models.

University of California at Davis (UCD) line 200 chickens spontaneously develop the entire spectrum of SSc, i.e., vascular lesions, mononuclear infiltrates, fibrosis of skin and internal organs, as well as serologic abnormalities, including anti–endothelial cell antibodies (AECAs), antinuclear antibodies, anticardiolipin antibodies, and rheumatoid factors (3–6). This inherited disease begins at 1–2 weeks of age with an inflammatory stage, occurring most prominently in the comb, which becomes erythematous and swollen and is finally lost to necrosis (so-called “self-dubbing”). These early skin lesions, which resemble those of Raynaud's phenomenon in humans, proceed to a chronic stage that is characterized by excessive collagen deposition 3–4 weeks after the chicks hatch. At 6 months of age, most of the animals have internal organ involvement (7). Microvascular injury appears to be one of the earliest features of both the human and avian disease (8, 6). Recently, we demonstrated that endothelial cell apoptosis is the primary pathogenetic event underlying the occurrence of skin lesions in avian and human scleroderma, becoming apparent before any other alteration is observed (9). The programmed cell death of the endothelium appears to be induced by AECAs, as revealed by anti-immunoglobulin staining.

Autoantibodies directed against the vascular endothelium have been found to be a common serologic feature in several diseases characterized by immune-mediated damage of the vessels (10). In SSc, the proportion of AECA-positive sera ranges from 28% to 71%, depending on patient selection criteria and the laboratory method used (11). AECAs represent a heterogeneous family of autoantibodies that react with a variety of antigens on endothelial and other cells (10). AECAs have been reported to fix complement in some autoimmune diseases, such as systemic lupus erythematosus, but they fail to exhibit this effect in SSc (12). Clinically, AECAs are associated with digital infarcts and pulmonary arterial hypertension, indicating a correlation of these antibodies with the extent of the vascular involvement (13). In vitro assays have revealed that AECAs are capable not only of inducing the expression of adhesion molecules and sustaining leukocyte adhesion in SSc (14), but also of initiating apoptosis, when the endothelium was additionally exposed to mononuclear cells (15). Using human dermal microvascular endothelial cells (HDMECs) as substrate, AECAs were shown to produce apoptosis by antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro (16). In contrast to these in vitro studies, there are no in vivo data concerning the cytotoxicity of AECAs in human SSc.

Based on our previous in vitro findings, the aim of the present study was to determine if transfer of AECAs into healthy chickens induces endothelial cell apoptosis and results in the development of scleroderma-like features. To consider a possible role of ADCC, additional groups of animals received activated peripheral blood lymphocytes (PBLs). We chose 2 different approaches for the transfer of AECAs: the chorionallantoic membrane (CAM) assay and the intravenous (IV) injection of AECA-positive sera into normal chicken embryos. Our findings are presented herein.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Animals.

Strains of chickens used in the experiments included the UCD-200 line (MHC haplotype B17/B17), which spontaneously develops a hereditary SSc-like disease. These birds served as donors of AECA-positive sera. Control sera negative for AECA were obtained from healthy UCD-058 chickens (haplotype B15/B15), outbred normal white Leghorn (NWL) chickens, and obese strain (OS) subline B5 chickens. OS chickens represent a model of spontaneous autoimmune thyroiditis with autoantibodies against thyroglobulin and provide a control group with an unrelated autoimmune disease (17, 18).

For CAM-transfer experiments, 10-day-old healthy UCD-058, NWL, and inbred CC chicken embryos were used, the latter also served as recipients for the injected AECA-positive sera. Highly inbred CC chickens (haplotype B4/B4) represent an animal group with a homogeneous genetic background.

NWL chickens were purchased from a local breeder (F. Moser, Polling, Austria). The other strains were maintained in the Central Laboratory Animal Facilities of the University of Innsbruck Medical School (19).

Antibodies and conjugates.

All antibodies and conjugates were used at optimum dilutions, which were determined in pilot experiments. Fluorescein isothiocyanate (FITC)–conjugated goat anti-chicken Ig was prepared in our laboratory and diluted 1:64 in a blocking reagent mixture consisting of 90% blocking reagent (Roche, Basel, Switzerland) and 10% fetal calf serum (FCS; PAA Laboratories, Linz, Austria). Rabbit anti–von Willebrand factor (anti-vWF; Dakopatts, Glostrup, Denmark), as an endothelial marker, and rabbit antikeratin (Dakopatts, Glostrup, Denmark), as a marker for the CAM epithelium, were diluted 1:50 in phosphate buffered saline (PBS), pH 7.2, 1% bovine serum albumin (BSA; Sigma, St. Louis, MO). Tetramethylrhodamine isothiocyanate (TRITC)–conjugated swine anti-rabbit IgG was diluted 1:30 in the blocking reagent mixture (Dakopatts).

Detection of AECAs and purification of sera.

Sera from UCD-200, UCD-058, NWL, and OS chickens were tested for AECAs by indirect immunofluorescence (IIF). For this purpose, 4-μm frozen unfixed comb sections of 4–12-week-old NWL chickens were incubated for 90 minutes at room temperature with serum diluted 1:5 in blocking reagent mixture. After washing for 60 minutes in PBS, FITC-conjugated goat anti-chicken antibodies were added at room temperature for 60 minutes. The slides were then washed again for 60 minutes in PBS and mounted with Mowiol (Hoechst, Frankfurt, Germany). In addition, serum samples from the injected 1-week-old CC chickens were tested for transferred AECAs by IIF on NWL comb sections.

Slides were analyzed using a Leitz Ortholux epiillumination microscope (Leitz, Wetzlar, Germany) equipped for multiple color immunofluorescence. AECA-positive UCD-200 serum samples and AECA-negative UCD-058, NWL, and OS serum samples were selected for the transfer experiments. In order to exclude cytotoxicity due to pyrogenic factors and complement determinants, serum samples underwent complement inactivation by heat treatment (56°C for 30 minutes in a water bath), purification of endotoxins by affinity chromatography (Detoxi-Gel AffinityPak columns; Pierce, Rockford, IL), dialysis against PBS at 4°C (Slide-A-Lyser 10K dialysis cassettes with a 10,000 MW cutoff; Pierce), and sterile filtration (Filtropur 0.2 μm; Sarstedt, Leicester, UK).

Preparation of PBLs for ADCC.

Heparinized blood was drawn from healthy inbred CC chickens. Blood was centrifuged at 60g for 7 minutes at room temperature, and the lymphocytes were separated and washed twice with RPMI 1640 (PAA Laboratories) supplemented with penicillin–streptomycin (10,000 IU/ml; Gibco BRL, Carlsbad, CA) and L-glutamine (200 mM; Gibco BRL). The PBL suspension was adjusted to a concentration of 1 × 107 cells/ml in X-Vivo Medium 20 (BioWhittaker, Walkersville, MD) and stimulated with an optimum concentration of chicken interleukin-2 (IL-2; 330 ng/ml, as previously determined [20]). After incubation for 24 hours at 40°C, PBLs were pelleted at 160g for 5 minutes and resuspended in the serum samples to a final concentration of 1 × 107 cells/ml for use in the transfer experiments.

Transfer experiments.

To analyze apoptosis induction in vivo, CAM assays were performed under sterile conditions on 10-day-old chicken embryos of either the UCD-058, NWL, or CC lines. As described previously (21), a 1 × 1–cm2 window was carefully drilled on the broad side of the egg over embryonic blood vessels, as identified by candling. This area was moistened with PBS to facilitate removal of the section of shell. A needle was used to produce a small hole at the blunt end of the egg. Negative pressure was applied to the hole by suction with a rubber bulb, which resulted in a dropping of the CAM. The stratum papillaceum was cut out carefully through the square window to avoid injuring the CAM below. Then, 100 μl of AECA-positive serum was dropped onto the CAM. The window was closed with Parafilm. Control groups were treated with serum from UCD-058, NWL, or OS chickens (the latter containing autoantibodies to thyroglobulin), with PBS, or remained untreated. To assess the effect of ADCC, groups of inbred CC embryos were treated with 106 IL-2–stimulated PBLs per 100 μl of serum.

To determine whether AECAs cause a disease similar to SSc when applied to healthy chicken embryos, 100 μl of serum containing 106 IL-2–stimulated PBLs was injected into the CAM veins of healthy inbred CC chickens on embryonic day (ED) 13 after removal of the shell as described above. Control groups were prepared in the same way with AECA-negative sera or with PBS. It should be emphasized that IV-injected AECAs were “diluted” in vivo in an intra- and extravascular space of ∼70 ml (corresponding to the weight of a fertilized ED 13 egg). In contrast to the CAM assay, all CC chicken embryos in the IV injection experiments received IL-2–stimulated PBLs in addition to serum samples. A further division of recipients into subgroups with and without PBL treatment was not accomplished because of the high mortality rate of the injected CC embryos and the consecutively limited number of surviving animals that could be analyzed.

Eggs for both experiments (i.e., AECAs dropped onto the CAM surface and AECAs injected IV) were then incubated at 37°C and candled for visible changes during the following days. Dead embryos were discarded. In the CAM assays, surviving embryos were killed on ED 16, and the CAM was removed and prepared. IV-injected embryos were observed until 1 week after hatching and then killed by cardiac bleeding under pentobarbital anesthesia. Combs were obtained for histopathologic and immunofluorescence examination, and sera were collected to determine if the transferred AECAs could be detected in the recipients. Tissue samples from animals of both experiments were immediately frozen in liquid nitrogen and, together with the serum samples, were maintained at −80°C until the microscopic evaluation was performed.

Detection of antibodies bound in vivo.

To determine if AECAs from the transferred sera had bound in vivo, CAMs and comb sections were analyzed for AECAs by immunofluorescence. For additional microscopic orientation on the sections, double staining for Ig bound to keratin-positive CAM epithelial cells was also performed. Frozen unfixed tissue sections (4 μm) were incubated with FITC-conjugated goat anti-chicken Ig antibodies at room temperature for 60 minutes, and the slides were washed in PBS for 60 minutes. Endothelial and epithelial cells were detected by IIF using rabbit anti-vWF and antikeratin as primary antibodies. After incubation, the sections were washed for 30 minutes at room temperature, and TRITC-conjugated swine anti-rabbit Ig secondary antibodies in PBS were added for 30 minutes at room temperature. Slides were washed again for 30 minutes in PBS and mounted with Mowiol. Comb sections from UCD-200 chickens, which showed positive staining for Ig bound to the endothelium in vivo, served as positive controls.

Simultaneous detection and characterization of apoptotic cells.

Endothelial cells were stained by IIF as described above, and apoptosis was detected by the TUNEL technique (9, 22). For this purpose, sections were fixed for 20 minutes in 4% paraformaldehyde, washed for 30 minutes in PBS, permeabilized with 0.1% Triton X-100/0.1% sodium citrate for 2 minutes on ice, washed in Tris buffered saline (TBS; pH 7.4) for 5 minutes, dehydrated with 50%, 75%, and 100% ethanol, and then rinsed in chloroform. After a 60-minute incubation in a moist chamber at 37.8C, the TUNEL reaction was carried out by incubating sections with 15 μl of 0.6 μM FITC-labeled 12-dUTP (Roche, Basel, Switzerland), 60 μM dATP, 1 mM CaCl2, terminal deoxynucleotidyl transferase buffer (30 mM Tris, pH 7.2, 140 mM sodium cacodylate), and 25 units of terminal deoxynucleotidyl transferase (Roche, Basel, Switzerland) for 1 hour at 37°C and covered with a plastic coverslip. The reaction was stopped by the addition of 10 mM Tris/1 mM EDTA, pH 8, for 5 minutes. Sections were washed twice in TBS and mounted in Mowiol. Tissue sections from the bursa of Fabricius of UCD-058 chickens served as positive controls for the TUNEL reaction and the conjugation.

Microscopic evaluation.

IIF staining of AECAs in UCD-200 sera was evaluated and documented using a Leitz Ortholux epiillumination fluorescence microscope. All other slides were analyzed using a Zeiss LSM 10 laser scanning microscope (Zeiss, Wetzlar, Germany). Digital images of the fluorescence after excitation with a helium neon laser (543 nm; filter setting BP 575–640) for cells stained with TRITC, and with the argon laser (488 nm; filter setting BP 530/30) for FITC-stained cells, were collected at a scan rate of 8 seconds/image. To determine the size of tissue sections stained by TUNEL/IIF, we used the Leitz Ortholux epiillumination fluorescence microscope. All samples were blinded before evaluation and were examined by 2 investigators (MW and RS).

Statistical analysis.

Apoptotic endothelial cells were counted on whole sections, and the numbers were related to the size of the section. Statistical evaluation of the calculated number of apoptotic endothelial cells per mm2 was performed by Mann-Whitney U test.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Survival rates after CAM assay and macroscopic evaluation.

Ten-day-old chicken embryos of the UCD-058, NWL, and CC strains served as recipients for the transferred serum samples in separate experiments and, after this manipulation, were candled daily up to ED 16. In comparison with controls, transfer of AECA-positive serum (Figure 1, which shows reactivity of AECAs with endothelial cells before being selected for passive transfer) did not result in alterations that were observable by candling. Macroscopically, CAMs from all groups were unremarkable. Compared with the controls, treatment with UCD-200 serum did not lead to an increased mortality rate. Mortality rates in all groups were highly influenced by vessel injury and bleeding that occurred during performance of the CAM treatment. No correlation could be found between the transferred serum samples and the mortality rate (Table 1).

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Figure 1. Indirect immunofluorescence of A, frozen unfixed comb sections from a normal white Leghorn (NWL) chicken treated with an anti–endothelial cell antibody (AECA)–containing serum (diluted 1:5) from a 4-month-old University of California at Davis line 200 chicken and B, an age-matched AECA-negative NWL serum. (Original magnification × 500.)

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Table 1. Survival rates and in vivo binding of AECAs after transfer of serum, with or without IL-2–stimulated PBLs, onto CAMs of chicken embryos on embryonic day 10*
Recipient chicken strainTransfer of serum or PBSTransfer of stimulated PBLsSurvival rateIn vivo binding of AECAs
  • *

    Data for animals that were not treated (NT) are summarized together with data for animals that received phosphate buffered saline (PBS). Peripheral blood lymphocytes (PBLs) were stimulated with interleukin-2 (IL-2).

  • Values are the number of animals surviving up to embryonic day (ED) 16/number of animals treated on ED 10.

  • Values are the number of animals with positive anti–endothelial cell antibody (AECA) binding to chorionallantoic membranes (CAMs) harvested on ED 16/number of animals examined.

UCD-058PBS1/30/1
 UCD-058 serum4/90/4
 UCD-200 serum6/115/6
NWLNT or PBS3/70/3
 UCD-058 serum5/80/5
 OS-B5 serum4/80/4
 UCD-200 serum9/143/9
CCNT or PBS5/90/5
 UCD-058 serum1/70/1
 UCD-200 serum2/72/2
 PBS+2/50/2
 UCD-058 serum+4/70/4
 UCD-200 serum+2/71/2

Microscopic evaluation of CAMs.

Detection of antibodies bound in vivo.

Frozen tissue sections revealed binding of antibodies to the endothelium, predominantly in the area of small vessels, only on CAMs that had been treated with UCD-200 serum, and not on any of the controls (Figure 2). Staining of Ig bound to keratin-positive cells was shown on CAMs from all groups, regardless of treatment. These data are summarized in Table 1.

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Figure 2. In vivo binding of anti–endothelial cell antibodies (AECAs) after transfer of serum onto the chorionallantoic membranes (CAMs) of chicken embryos. Frozen sections of CAMs were double stained by immunofluorescence using a fluorescein isothiocyanate–labeled anti-chicken Ig (green) to detect antibodies bound in vivo and tetramethylrhodamine-labeled anti–von Willebrand factor (red) to detect endothelial cells. AECAs were identified only after transfer of A, serum from a University of California at Davis line 200 chicken, but not after transfer of B, serum from an obese strain subline B5 chicken or C, phosphate buffered saline. (Original magnification × 400.)

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Simultaneous detection and characterization of apoptotic cells on CAM sections.

Sections of CAMs from 16-day-old UCD-058, NWL, and CC chicken embryos were analyzed by the combined IIF and TUNEL technique. In all recipient strains, transfer of UCD-200 serum led to an increased number of apoptotic endothelial cells compared with controls (Figure 3). In addition, there was apoptosis of non–endothelial cells, which appeared to be unrelated to the serum sample that was transferred. This apoptosis was observed on control CAMs as well.

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Figure 3. Simultaneous visualization of endothelial cells and apoptosis on 4-μm frozen sections of chorionallantoic membranes from A and B, normal white Leghorn and C and D, University of California at Davis (UCD)-058 chicken embryos. Apoptotic endothelial cells are indicated by a yellow-green–stained nucleus, as revealed by TUNEL staining. Endothelial cells are indicated by the red staining, as revealed by indirect immunofluorescence using a rabbit anti–von Willebrand factor antibody and an anti-rabbit tetramethylrhodamine conjugate. Apoptotic endothelial cells are seen after transfer of A and C, serum from a UCD-200 chicken. No endothelial cell apoptosis is seen after transfer of B, phosphate buffered saline or D, serum from a UCD-058 chicken.

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In UCD-058 recipients, CAMs revealed a 7-fold increase in the number of apoptotic endothelial cells after treatment with UCD-200 serum, as compared with controls that had received UCD-058 serum (P < 0.05) (Figure 4A). Statistically significant increases in apoptotic endothelial cells were also found after administration of UCD-200 serum onto the CAMs of NWL recipients (P < 0.01) (Figure 4B).

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Figure 4. Number of apoptotic endothelial cells on chorionallantoic membranes (CAMs) from 16-day-old embryos of A, University of California at Davis (UCD)-058, B, normal white Leghorn, and C, CC chickens. Phosphate buffered saline (PBS) or serum from UCD-058, obese strain subline B5, or UCD-200 chickens, with or without interleukin-2–stimulated peripheral blood lymphocytes (PBLs), was transferred to the recipients on embryonic day 10. Data for embryos that were not treated (n.t.) are summarized together with data for those that received PBS. Values are the mean and SEM; n = number of animals. Transfer of UCD-200 serum significantly increased endothelial cell apoptosis ( = P < 0.05; ∗∗ = P < 0.01, by Mann-Whitney U test).

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Subgroups of embryos of the inbred CC chicken strain were treated with serum samples as well as with IL-2–stimulated PBLs as a source of activated natural killer cells. Application of UCD-200 serum again led to an increase in the number of apoptotic endothelial cells (Figure 4C). This tendency was not further enhanced by the addition of IL-2–stimulated PBLs.

The use of a histocompatible donor–recipient combination was a prerequisite for the transfer of cells, but as a result of the inbreeding, CC chickens showed higher mortality rates than did the outbred animals. Consequently, a statistical evaluation of the data from this experiment was not possible.

Survival rate and macroscopic evaluation after IV serum injection.

The use of inbred CC recipients and the sophisticated technique of embryonic IV injection resulted in low survival of all animals. However, injection of AECA-positive sera did not lead to an increase in the mortality rate in comparison with that of the controls.

By diaphanoscopy (candling the egg with a bright lamp in the dark), no between-group differences were observed during the embryonic period. The hatched chickens did not develop any symptoms of disease (data not shown).

Microscopic evaluation after IV serum injection.

Detection of AECAs bound in vivo and in the serum of CC recipients.

AECAs were not detected on frozen comb sections from 1-week-old CC recipients, regardless of the source of the injected serum. Serum samples from CC recipients were analyzed for AECAs. IIF revealed negative antibody patterns in all animals. This finding is perhaps due to the low sensitivity of the IIF method, since the 100-μl injected volume (and antibodies within) is diluted in the intra- and extravascular spaces of chicken embryos.

Detection of apoptotic endothelial cells in comb sections.

Using the combined IIF and TUNEL technique, only the combs of CC chickens that had been treated with UCD-200 serum samples plus IL-2–stimulated PBLs revealed apoptotic endothelial cells (Figure 5). Apoptotic endothelial cells could not be found in comb sections from any of the controls.

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Figure 5. Number of apoptotic endothelial cells on frozen comb sections from 1-week-old CC chickens after injection of phosphate buffered saline (PBS) or serum from normal white Leghorn or University of California at Davis line 200 chickens, with or without interleukin-2–stimulated peripheral blood lymphocytes (PBLs), on embryonic day 13. Data for embryos that were not treated (n.t.) are summarized together with data for those that received PBS. Values are the mean and SEM; n = number of animals.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Vascular alterations in SSc, as verified by (ultra)microscopic changes and increased levels of soluble endothelial cell markers, including thrombomodulin, endothelin, or vWF, represent a very early marker in the course of disease (8, 23–25). Several in vitro studies suggest a possible role of AECAs in mediating endothelial cell damage (15, 16, 26–28), but evidence for the in vivo effects of these autoantibodies is lacking. We therefore sought to determine whether transfer of AECA-positive serum samples into healthy recipient chickens led to endothelial cell apoptosis and/or induction of SSc-like symptoms. UCD-200 chickens represented the appropriate animal model required for the acquisition of AECA-positive serum samples. Sera were transferred onto the CAMs of healthy breeds of chickens on ED 10 or, for a different approach, were injected intravenously into 13-day-old normal CC chicken embryos.

CAMs from UCD-058, NWL, and CC chicken embryos were obtained on ED 16 for microscopic evaluation. Immunofluorescence double staining revealed the presence of AECA only after transfer of UCD-200 sera, which were placed predominantly in the area of small-sized vessels. We selected the microvessels because of microscopic studies localizing the early vascular injury of SSc to the small arterioles and capillaries (8). In general, endothelial cells show tissue-dependent characteristics with regard to the expression of surface molecules, the production of prostaglandins, and in vitro growth requirements (29–31). These differences are reflected by the heterogeneity of AECAs that react with various structures on endothelial cells and fibroblasts (32). Preliminary data from studies of SSc in humans indicate a significantly higher binding activity of AECAs to the microvasculature than to the large arteries and veins (11, 16). We demonstrated that sera from all patients with early acute SSc contained IgG AECAs to HDMECs, whereas only 50% of these patients were also positive for IgG AECAs to human umbilical vein endothelial cells (HUVECs) (16). Interestingly, apoptosis induction by AECAs via ADCC was shown only in HDMECs.

Not all CAM sections examined revealed antibodies bound in vivo, since this effect depends on the topography of vascularization and the distribution of AECAs over the CAM surface after application of 0.1 ml of serum. The 0.1-ml volume of serum dropped onto the CAM is diluted ∼70 ml (calculated from the total weight of the egg), which leads to AECA concentrations below the detection limit of the IIF technique when evaluated by fluorescence microscopy.

None of the controls showed antibodies bound to the endothelium, but Ig staining of epithelial keratin-positive structures could be detected on CAM sections from all study groups, regardless of the serum transferred. This is probably due to remaining egg yolk (containing maternal Ig) on the outside of the membrane that was not entirely eliminated during dissection.

Importantly, after application of AECA-positive sera, CAMs from all strains revealed increased numbers of apoptotic endothelial cells as compared with the controls. Mainly the microvessels were affected by the cytotoxic effects of the transferred serum samples, as was expected. Nearly all CAM sections also showed apoptosis of other cell types, which were not further characterized. The latter finding is typical for the period of embryogenesis and explains the appearance of some apoptotic endothelial cells on control CAMs. Since it is not possible to discriminate microscopically between physiologically occurring cell death and exogenously induced apoptosis, evaluations were performed statistically by comparison with controls.

There are several possible mechanisms for the induction of endothelial cell apoptosis found in our experiments. The effect of a complement-mediated cytotoxicity can be excluded, since this would lead to necrosis (33). In previous experiments, we were able to show that AECA-induced endothelial cell apoptosis in vitro is accomplished by ADCC via the Fas pathway (16).

The fact that the rate of apoptosis was not further increased by the addition of IL-2–stimulated PBLs may be explained by the availability of natural killer cells from the embryos themselves. Bordron et al (34) triggered apoptosis of HUVECs by the application of purified, highly concentrated AECAs from SSc patients. Other groups of investigators have postulated the presence of peripheral blood mononuclear cells (PBMCs) as a prerequisite for the cytotoxic effect of AECAs. Penning et al (15) showed that sera from ∼20% of SSc patients were capable of causing cytotoxicity of HUVECs when cocultured with PBMCs. The mechanism was thought to be ADCC, since the activity resided in IgG fractions and the responsible effector cells were Fc receptor positive. Marks et al (27) and Holt et al (28) reported similar results with studies of HDMECs and HUVECs, respectively. In previous experiments (16), we showed that sera alone had no effect and that apoptosis induction could be observed only with microvascular endothelial cells, but not with HUVECs, as targets. We also demonstrated that IgG AECAs from SSc patients bound to both HDMECs and HUVECs, whereas apoptosis induction occurred only with AECAs to HDMECs.

These in vitro studies indicate that ADCC might be operative in the in vivo induction of apoptosis. Mature natural killer cells have been detected in ED 14 chicken embryos (35). Since the induction of AECA-dependent cellular cytotoxicity requires just 4–20 hours in vitro (Sgonc R: unpublished observations) and since harvesting of CAMs was not performed before ED 16 (i.e., 6 days after AECA transfer), this mechanism might be responsible for mediating the endothelial cell apoptosis seen in our experiments.

Similar to the results of the CAM assays, we also found an apoptosis-inducing effect of AECAs after IV injection of UCD-200 serum samples containing IL-2–stimulated PBLs into healthy CC embryos. Results of examinations for circulating AECAs in blood samples from these chickens as well as staining for AECAs bound in vivo were negative in all animals. These findings do not, however, exclude the induction of ADCC by the transferred AECAs, since concentrations of antibodies required for this process (≤50 pg/ml) (16, 36) are clearly below the detection limit of the IIF (1–100 ng/ml) when evaluated by fluorescence microscopy. An enzyme-linked immunosorbent assay for the evaluation of chicken AECAs is not available yet.

In neither the CAM assays nor the IV transfer experiments did the application of UCD-200 serum samples result in an increase in the mortality rates as compared with the controls. IV injection of AECAs was not followed by the development of SSc-like features in CC chickens.

What is the reason for the limited effect of transferred AECAs, resulting in the induction of apoptosis but not the production of a macroscopic pattern of disease? As discussed above, we hypothesize that endothelial cell apoptosis represents only a first step in the course of disease. Comparative studies of skin biopsy tissues from UCD-200 chickens and humans with SSc identified endothelial cell apoptosis as the primary pathogenetic event in the development of SSc, occurring before any other alterations, such as the formation of perivascular infiltrates (9). Endothelial cell apoptosis is not localized to the skin; it is the first visible alteration in other affected organs of UCD-200 chickens (37). Thus, we suggest that further development of disease requires factors in addition to a single transfer of 100 μl of AECA-positive serum. Whether these prerequisites consist of constant exposure to higher concentrations of AECAs, a genetically determined susceptibility of target organs to autoimmune attack (38), or other as-yet-unknown components remains to be elucidated. The single transfer of 100 μl of AECA-positive serum, which was diluted in the intra- and extravascular space, was probably not sufficient to induce an SSc-like disease in the recipients. However, chicken embryos can hardly tolerate the IV injection of larger volumes.

Although the present experiments were performed with whole sera rather than the purified IgG fraction, our study provides the first evidence in SSc that AECA-positive sera are capable of inducing endothelial cell apoptosis in vivo. In conclusion, our findings support the hypothesis that AECAs might play an important role in triggering the cascade of pathogenesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Dr. Karel Hala for providing the CC chickens.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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