Anti-Fibroblast Antibodies in Systemic Sclerosis

Clinical Immunology and Allergy Anti-Fibroblast Antibodies in Systemic Sclerosis

1, Elena Raschi PhD3, Cinzia Testoni PhD3, Maria O. Borghi PhD2, Rita Gatti MD2, Nicoletta Ronda MD 4 Jean M. Dayer MD , Guido Orlandini MD2, Carlo Chizzolini MD4 and Pier L. Meroni MD3 1 Department

Parma, Italy

of Clinical Medicine, Nephrology and Health Sciences, and 2Department of Experimental Medicine, University of Parma,

3 Allergy and Clinical Immunology Unit, Department of Internal Medicine, University of Milan, and IRCCS Istituto Auxologico Italiano, Milan,

Italy

4 Division

of Immunology and Allergy, Hans Wildorf Laboratory, Geneva University Hospital, Geneva, Switzerland

Key words:

systemic sclerosis, anti-fibroblast antibodies, adhesion molecules, cytokines, antibody internalization IMAJ 2002;4(Suppl):858±864

Systemic sclerosis is a disease characterized by fibrosis of skin and internal organs. Although at present the classical criteria defining an autoimmune disease are not entirely fulfilled for SSc, several autoimmune abnormalities, both cellular and humoral, have been described in this disease [1]. In particular, autoantibodies to nuclear components are characteristically associated with SSc and segregate distinct clinical subsets [2]. In addition, several other autoantibodies against cytoplasmic structures have been described: released products such as fibrillin-1, immunoglobulin E and surface antigens on fibroblasts, lymphocytes and endothelial cells [3±8]. Besides the cytolytic capacity of IgM directed against CD4+ T cells [9], no information was available until recently on the functional role of SSc antibodies to the cell surface. Fibroblasts play a key role in SSc pathogenesis by secreting soluble pro-inflammatory and fibrogenic mediators, by remodeling of connective tissue [10±13] and by expressing adhesion molecules for leucocytes, such as intracytoplasmic adhesion molecule, whose serum levels of the soluble isoform appear to correlate with disease activity [14±16]. The present paper reviews our studies on the prevalence of autoantibodies against fibroblasts in SSc, on the mechanisms of the interaction with living cells, and on their capacity to induce a pro-inflammatory and a pro-adhesive phenotype on fibroblasts. AFA activity in the serum and in IgG fractions of SSc patients

Sera from SSc patients, healthy individuals, and patients with pulmonary sarcoidosis were tested for the presence of AFA using a cell-based enzyme-linked immunosorbent assay on normal fibroblasts [16]. IgG AFA were detected above normal threshold in 30 of 69 SSc sera (43.5%) and IgM AFA in 25 (36.2 %) [Figure 1A]. A significant (P < 0.05) higher prevalence of AFA was found in SSc patients with diffuse (72% for IgG and 53% for IgM) versus limited skin involvement (37% for IgG and 30% for IgM). When samples were tested against skin and lung fibroblasts, comparable reactivity was found. Only 2 of 30 sarcoidosis sera were positive for AFA ± one for IgG and one for IgM. SSc = systemic sclerosis Ig = immunoglobulin AFA = anti-fibroblast antigens

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IgG fractions were affinity-purified by protein-G column chromatography, as described [16], from a subset of AFA-positive and AFA-negative SSc sera and from normal sera. All of the five AFA-positive IgG preparations showed a dose-dependent antifibroblast binding activity, while IgG from five AFA-negative SSc and normal controls did not display any significant binding [Figure 1B]. The same results were obtained using SSc skin fibroblasts. Pre-treatment of cell monolayers with high amounts of rabbit IgG did not affect AFA-positive SSc IgG binding, suggesting that the Fc gamma receptor is not involved in the reaction. Binding of AFA-positive IgG was confirmed by conventional immunofluorescence on unfixed human skin fibroblasts, showing a membrane binding (data not shown), and by flow cytometry [Figure 2]. In this case, binding above background was observed with all preparations tested, but AFA-negative SSc IgG and normal human serum IgG gave comparable results, while AFA-positive SSc IgG resulted in a higher mean fluorescence intensity and greater percentage of positive cells. Overall, these studies demonstrate that fibroblast binding of SSc sera is due to IgG reacting with components expressed at the surface of skin fibroblasts. Mechanisms of AFA binding to living fibroblasts

The mechanisms of AFA binding to living fibroblasts were investigated by confocal laser scanning microscopy of cell monolayers mounted on an incubation chamber installed in the microscope [17] and treated with fluoresceinated IgG preparations. AFA-positive IgG, incubated with living fibroblasts at 400 mg/ml in standard culture conditions, bound to fibroblast cell membrane within 10 minutes. The antibodies were then internalized within 30 minutes, localizing in tubular-like structures originating from the cell membrane and protruding in the cytoplasm [Figure 3A]. After 1 hour a network of fluorescent cytoplasmic filaments became evident and was followed by the appearance of a granular pattern [Figure 3B] that moved to the perinuclear area during the subsequent 2 hours [Figure 3C]. Similar results were obtained with all five distinct AFA-positive IgG from SSc individuals. No differences were observed when AFA-positive IgG were tested using normal skin fibroblasts from two donors or fibroblasts derived from one SSc patient.

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Clinical Immunology and Allergy A)

% binding activity

100 80 60 40 20 0 LSSc

dSSC

Sarcoidosis

NHS

Flow cytometric analysis on fibroblasts incubated with AFA positive and negative fractions; IgG were used at the final protein concentration of 100 mg/ml. Data are presented as positive events versus fluorescence intensity. Representative data from one of three distinct experiments are shown. Figure 2.

IgM

% binding activity

100 80

We ruled out that intracellular fluorescence was due to molecules contaminating IgG preparations, revealing intracellular IgG by conventional immunofluorescence (with both a mouse 40 monoclonal anti-human IgG and an anti-human IgG polyclonal antibody) in fibroblasts incubated with purified non-fluoresceinated 20 AFA-positive IgG for 1 hour before fixation. The fluorescence pattern observed with both reagents was similar to the pattern obtained 0 LSSc dSSC Sarcoidosis NHS after 1 hour using fluoresceinated AFA-positive IgG. To verify whether the dynamic interactions with fibroblasts were specific for AFA-positive IgG, we applied CLSM also to test binding B) to fibroblasts of purified IgG from normal serum, from pooled commercial IgG for intravenous therapeutic use, as well as from SSc 2000 sera negative for AFA by cell-ELISA. These fluoresceinated IgG preparations, incubated with living human fibroblasts at 400 mg/ml 1600 in standard culture conditions, showed membrane binding within 1200 30 minutes [Figure 3D]. However, after prolonged examination (4 hours), neither internalization nor morphologic modification of 800 the fluorescence pattern was observed. Similar results were 400 obtained using skin fibroblasts from two healthy donors and from one SSc patient. 0 200 100 50 25 12.5 6.2 3.1 1.5 To further test the specificity of AFA-positive IgG interaction with IgG ( g/ml) m fibroblasts, we verified whether normal human IgG could inhibit AFA binding and internalization. Living fibroblasts were incubated Anti-fibroblast antibodies in SSc. Prevalence of fibroblast cell overnight with 20-fold excess of unlabeled normal pooled IgG (8 binding activity in SSc, sarcoidosis and normal sera. The ELISA was performed mg/ml) before being exposed to fluoresceinated AFA-positive IgG. on living, unfixed fibroblasts as described previously [24], with minor Alternatively, fluoresceinated AFA-positive IgG were added simulmodifications. The optical density value of a positive reference serum at taneously with 20-fold excess of normal pooled IgG to fibroblast standard 1:25 dilution was arbitrarily defined as 100% of fibroblast binding cultures and then examined by CLSM. No inhibition of AFA-positive activity. To adjust for inter-assays variability, samples were expressed as a IgG binding and internalization was observed in either case. percentage of the positive reference serum; values greater than the mean + 3 Altogether, these results indicate that AFA from SSc sera, SD of 50 healthy individuals were considered positive, i.e., 22.2% for IgG and 47.3% for IgM AFA. The horizontal line represents the cut-off calculated as the selected on the basis of an abnormal binding on cell-ELISA, have mean percentage of binding activity plus 3 SD of 50 normal sera. Binding the capacity to recognize specific targets on the surface of normal activity to unfixed human dermal fibroblasts of SSc AFA-positive (-.-) (n = 5), AFA-negative (-~-) (n = 1), NHS (-*-) (n = 1), IgG or medium alone (-o-). Values are expressed as mean OD SD of triplicate experiments. LSSc = limited SSc, DSSc = diffuse SSc. OD = optical density, NHS = normal human CLSM = confocal laser scanning microscopy sera. ELISA = enzyme-linked immunosorbent assay O.D. Values x 10 -3

60

Figure 1.

[A]

[B]

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Anti-Fibroblast Antibodies in Systemic Sclerosis

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Figure 3. [A] Internalization of fluoresceinated IgG purified from an AFA-positive SSc serum in a living fibroblast. Picture taken 30 minutes after IgG-free medium

mm. [B] Internalization of fluoresceinated IgG purified from an AFA-positive SSc serum in a living fibroblast. Picture taken 1 hour after IgG-free mm. [C] The same cell depicted in [A] and [B] after a further 2 hours from IgG-free medium replacement. Bar = 5 mm. [D] Binding of fluoresceinated intravenous immunoglobulin to the membrane of a living fibroblast. Picture taken 30 minutes after IgG-free medium replacement. Bar = 10 mm. [E] Fibroblast caveolin visualized by immunofluorescence after cell fixation. Bar = 10 mm. [F] Detail of a fibroblast membrane after AFA-positive fluoresceinated IgG replacement. Bar = 5

medium replacement. Bar = 5

internalization (green signal), fixation and caveolin staining, with a specific monoclonal antibody and a Texas red secondary antibody (red signal). Caveolin signal appears to surround the IgG one. Bar = 2

mm.

and SSc fibroblasts. This specific recognition results in internaliza-

the two molecules in resting cells and possible differences in their

tion and perinuclear localization.

distribution upon stimulation with AFA. In both cases clathrin could be

visualized,

while

fibroblasts suggest the involvement of receptor-like cell membrane

fibrillar

structures,

mostly

molecules,

membrane, sometimes with a tubular appearance [Figure 3E]. No

Since the characteristics of AFA-positive IgG internalization in

we

investigated

the

possible

role

of

caveolin-

or

not

clathrin-associated microdomains, which are particularly rich in

differences

receptors.

caveolin-bearing

We

performed

direct

immunofluorescence

with

anti-

caveolin and anti-clathrin antibodies on fixed fibroblasts, previously incubated or not with AFA-positive IgG, to show the localization of

860

N. Ronda et al.

were

noted

caveolin close

when

structures

in

staining

to

showed

certain

comparing fibroblasts

zones

the

interlacing of

the

morphology

exposed

to

cell

of

AFA with

those not exposed. We then performed a double staining to verify the possible co-

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localization of internalized AFA and caveolin. We incubated living

Incubation of dermal fibroblast monolayers with three AFA-positive

fibroblasts with AFA-positive fluoresceinated IgG for 30 minutes,

SSc IgG, but not with controls, resulted in enhanced adhesion of

waited for the internalization to progress (usually within another 30

U937 monocytic cells in all cases [Figure 4A]. The enhanced

minutes), then fixed the cells and performed direct immunofluor-

adhesion was comparable in magnitude to that induced by

escence with a monoclonal anti-caveolin antibody and the relevant

saturating amounts of interleukin 1 [Figure 4A].

Texas red coupled secondary antibody. Indeed, a precise corre-

The expression of ICAM-1, a ligand for beta2 integrins expressed

spondence in localization of IgG and caveolin was found. In

at the surface of leukocytes, is increased on SSc fibroblasts [15]. We

particular, in images collected at high magnification, the IgG signal

therefore tested skin fibroblasts for ICAM-1 expression and its

appeared surrounded by that from caveolin [Figure 3F]. In addition,

modulation by SSc IgG. A dose-dependent up-regulation of ICAM-1

we obtained a complete inhibition of AFA-positive IgG membrane

was observed in the presence of AFA-positive, but not AFA-negative

m

binding and internalization in fibroblasts treated with a caveolae-

or control IgG [Figure 4B]. Similar experiments performed in the

disassembling agent (2

presence of polymyxin B yielded comparable results, ruling out

g/ml filipin for 1 hour before and

throughout the experiment) (data not shown).

a

endotoxin contamination of the IgG preparations. In addition, IL-

These data firmly indicate that AFA-positive IgG, but not normal

1 , tumor necrosis factor-alpha and IL-6 ± known to enhance ICAM-

IgG or SSc AFA-negative IgG, specifically bind surface antigens

1 expression on fibroblasts ± were not detected by ELISA in the IgG

associated with cellular structures that elicit caveolin-dependent

preparations used (data not shown). Thus, these data indicate that

internalization.

AFA-positive IgG specifically induce ICAM-1 expression on fibroblasts and enhance mononuclear cell adhesion to fibroblasts.

Activation of fibroblasts by AFA Induction of pro-adhesive phenotype

Induction of pro-inflammatory cytokine secretion

Previous studies by our group and by others showed that antibodies

Fibroblast monolayers were incubated in the presence of SSc AFA-

reacting with cell membrane structures of different cell types,

positive or control IgG, and mRNA levels of IL-1 , IL-1

namely endothelial cells, might modulate cell functions [18].

were determined by RNase protection assay. Up-regulation of IL-1 ,

a b

a

and IL-6

Therefore, we evaluated whether mononuclear cell adhesion to fibroblasts was enhanced by exposure to AFA-positive SSc IgG.

A)

ICAM = intracytoplasmic adhesion molecule

B) 100

1200

60

O.D. Values x 10 -3

%U937 adhesion

80

40

800

400

20

0 100

0

IgG

m

1

25

12.5

6.2

3.1

1.5

IgG (ug/ml)

AFA- NHS IL-1 b medium

AFA+IgG

50

IgG

b

&

Figure 4. Induction of a pro-adhesive fibroblast phenotype: [A] Percentage of U937 adhering to fibroblast monolayers incubated with different IgG preparations (100

g/ml) or the controls (

~

b

SSc AFA-positive, n = 3),

monolayers incubated with 50 U/ml of hrIL-1

+

SSc AFA-negative,

gave OD values of 1.058

NHS,

50 U/ml of hrIL-1 , and

medium). As a positive control, fibroblast

.

167 for ICAM-1 expression; monolayers incubated with medium alone served as the

negative control and gave background values. [B] Dose-dependent ICAM-1 up-regulation induced by serial protein concentrations of SSc AFA-positive (- -) (n = 3), AFA-negative (-

as mean (OD values)

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-) (n = 1), and NHS (- -) (n = 1) IgG fractions. ICAM-1 expression was evaluated by the cell solid ELISA as described [24]; results are expressed

*

SD of triplicate experiments.

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B

A

m

Figure 5. [A] Up-regulation of steady-state mRNA levels of pro-inflammatory cytokines induced in fibroblasts by AFA-positive SSc IgG. Lung fibroblasts were

cultured in the presence of 100

~

.

g/ml of IgG or medium alone for 4 hours. The number in brackets after AFA+SSc IgG denotes IgG from two distinct individuals.

b

+

[B] Induction of a pro-inflammatory phenotype (IL-6 production) induced in fibroblasts by AFA-positive SSc IgG (- -) (n = 3). Negative controls are represented by

AFA-negative (-

*

ml of IL-6, while cells in medium alone gave background values (2

b

IL-1

+

+

-) (n = 1) and NHS (- -) (n = 1) IgG fractions. As a positive control, fibroblast monolayers incubated with 50 U/ml of hrIL-1 0.3 pg/ml). Values are expressed as mean

secreted 815

SD of triplicate experiments.

12 pg/

and IL-6 mRNA was observed when fibroblasts were treated

IgG binding to the surface of fibroblasts induces the production of

with AFA-positive SSc IgG, but not with control IgG [Figure 5A].

cytokines that up-regulate ICAM-1 in an autocrine manner. In this

Maximal up-regulation was observed when fibroblasts were treated

autocrine up-regulation IL-1 appears to play a major role.

with IgG for 4 hours, returning to baseline at 24 hours. The upregulation was selective since macrophage colony-stimulating factor mRNA, detectable at baseline, was unaffected after both 4

a b

and 24 hours of treatment with SSc IgG. Furthermore, we tested the antibody effect on IL-1 , IL-1

and IL-6 production. When cultured

in the presence of AFA-positive SSc IgG, human dermal fibroblasts released IL-6 in a dose-dependent manner [Figure 5B]. Halfmaximal activity was observed at SSc IgG concentrations ranging

a

b

from 12.5 to 3.1 g/ml for the three IgG preparations tested. Under the same culture conditions, IL-1

and IL-1

protein were not

detected consistently above the sensitivity threshold (15.6 pg/ml).

Discussion

Our studies demonstrate that antibodies reacting with the cell membrane of dermal and lung fibroblasts can be detected in a large proportion of SSc sera. Upon binding, these autoantibodies undergo cell internalization and modulate fibroblast functions by inducing a pro-adhesive and pro-inflammatory phenotype. AFA interactions with fibroblasts were comparable by using normal skin fibroblasts, PP2 fibroblast cell line or fibroblasts from scleroderma, suggesting that recognized molecules are constitutively expressed in all cell types tested.

Up-regulation of ICAM-1 is inhibited by IL-1Ra and not by anti-IL-6

Anti-fibroblast antibodies have been previously described in SSc

Finally, we tested whether the autocrine effects of cytokines

[6,7,9]; the higher AFA prevalence in our series (43.5% for IgG AFA)

produced by SSc IgG-activated fibroblasts would account for

may be explained by differences in patient selection and/or in the

ICAM-1 up-regulation or whether this was due to a direct

techniques used. In fact, it is quite possible that our cell-based

mechanism. When IL-1Ra was added to fibroblast cultures, ICAM-

ELISA may provide a better sensitivity and specificity. Indeed, in

1 up-regulation induced by SSc AFA-positive IgG was completely

agreement with a previous report, flow cytometry revealed positive

reversed in a dose-dependent manner (data not shown). In

binding of AFA-negative or NHS IgG, although with an intensity

contrast, the use of anti-IL-6 monoclonal antibodies as a

lower than AFA-positive SSc [16]. Such background reactivity with

neutralizing reagent in similar experiments resulted in a marginal inhibition (27%, data not shown). These results indicate that SSc

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NHS = normal human serum

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normal IgG has also been reported in studies carried out with the CLSM [19].

g

g

ing of caveolin-rich invaginations, unclustering of receptors and loss of caveolae-mediated endocytosis [23]. These effects of filipin

This finding may be due to the binding of normal IgG to Fc R

may also explain why not only internalization, but also membrane

expressed on fibroblast membrane, but Fc R have not been

binding of AFA-positive IgG was inhibited in filipin-treated

reported on resting human fibroblasts [20]. An alternative explana-

fibroblasts.

tion may be the presence of natural autoantibodies reacting with

The internalization of AFA-positive IgG in living fibroblasts

fibroblast antigens and revealed by flow cytometry or CLSM.

correlates with the ability of the same IgG fractions to activate

Natural autoantibodies constitute the majority of circulating

fibroblasts. Indeed, antigen recognition by AFA resulted in the up-

immunoglobulins and have been found to react with several human

regulation of mRNA of several cytokines as well as ICAM-1

autoantigens both soluble and cellular [21]. Quantitative measure-

expression, and IL-6 production ± all these events requiring

ment of natural autoantibody binding to autoantigens (e.g., by

metabolically active fibroblasts.

ELISA) shows values lower than those obtained with the

AFA-positive IgG up-regulated ICAM-1 expression and IL-6

corresponding pathologic autoantibodies, and usually epitope

production at concentrations lower than those resulting in maximal

specificity is also different. These differences justify the indication

antibody binding to fibroblasts. This suggests that fibroblast

of certain pathologic autoantibodies as markers of autoimmune

activation may require amounts of agonistic antibody lower than

diseases even in the presence of a natural autoantibody physiologic

those detectable by ELISA. The concentration of IgG found able to

counterpart [21]. Accordingly, SSc sera might be classified as AFA-

induce fibroblast activation could fall within the physiologic range

negative or AFA-positive, depending on the results of the cell-based

of IgG diffusing in the interstitium, if it is true that in dynamic

ELISA.

conditions interstitial IgG may reach concentrations of 50% or

All IgG preparations from AFA-positive SSc patients, as defined

higher than those present in the intravascular compartment [24].

by the cell-based ELISA, not only bound to cell membrane but were

AFA-positive SSc IgG, but not control or AFA-negative SSc IgG,

also internalized. In contrast, such a phenomenon has not been

induce a pro-adhesive phenotype in fibroblasts. Such a phenom-

displayed by AFA-negative SSc or NHS IgG fractions.

enon might play a role, particularly in the early inflammatory

g

The possibility that internalization of AFA-positive IgG is

phases of the disease, when mononuclear cells (T cells and

mediated by Fc R is excluded by the lack of internalization of

monocyte/macrophages) are found in perivascular areas and mixed

AFA-negative IgG. In addition, both the pre- and co-incubation with

with fibroblasts [25]. This finding is consistent with the hypothesis

large amounts of normal IgG did not affect AFA IgG internalization.

that immune cells may be crucial for the initial activation of

This last finding also indicates that normal IgG do not interact

connective tissue metabolism in fibrosis.

idiotypically with AFA, as reported for other pathologic autoanti-

a

b

In addition, AFA-positive SSc IgG induced up-regulation of the steady-state mRNA levels of IL-1 , IL-1

bodies [22].

and IL-6, while mRNA

In agreement with the lack of any correlation between the

levels of M-CSF were unaffected. This indicates that the signaling

presence of anti-nuclear reactivity and fibroblast binding, IgG

pathways triggered in fibroblasts by AFA recognition have the

internalization was also not dependent on the presence of any of

capacity for broad, although selective, mRNA up-regulation. This is

the known anti-nuclear antibodies typical of the disease.

also consistent with the possibility that a single or limited number

The characteristics (time, kinetics, morphology) of AFA interac-

of mediators may be induced, which then activate secondary

tion with fibroblasts are consistent with a receptor-ligand inter-

pathways. Indeed, IL-1Ra (but not anti-IL-6) inhibited in a dose-

nalization process, in which receptor-ligand complex induces

dependent manner the capacity of AFA-positive SSc IgG to up-

folding of the cell membrane and formation of endosomes. We

regulate ICAM-1 expression. Thus, in analogy with a recent report

found that a network of filaments and tubules jutting out from the

showing that IL-1 acts in an autocrine loop to mediate human

cell membrane into the cytoplasm did contain caveolin, whereas

fibroblast activation induced by monocyte chemotactic protein-1

clathrin could not be visualized. Caveolin distribution was the same

[26], our findings indicate that IL-1 is involved in ICAM-1 up-

in resting fibroblasts or following incubation with AFA-positive IgG.

regulation induced by AFA-positive SSc IgG.

The pattern of AFA-positive IgG distribution in fibroblasts was very

Overall, our results indicate that AFA, upon binding to the cell

similar to that of caveolin, and double staining of fibroblasts to

surface, penetrate the cells and induce fibroblast activation, leading

visualize both internalized IgG and caveolin demonstrated a co-

to the enhanced production of pro-inflammatory cytokines and up-

localization of the two molecules. Altogether, our findings strongly

regulation of ICAM-1. We speculate that these events may

suggest that AFA IgG internalization in fibroblasts occurs through

contribute to triggering or maintaining the recruitment of inflam-

caveolae-associated receptors, as also supported by the complete

matory cells in tissues undergoing fibrosis and to promoting a pro-

inhibition of internalization obtained when treating fibroblasts with

fibrotic phenotype on fibroblasts.

filipin. This sterol-binding molecule, widely used to disassemble caveolae, removes plasma membrane cholesterol, inducing flatten-

Acknowledgment.

This study was supported in part by Progetto

Biennale MURST 1998 (to P.L.M. and G.O.), by Ricerca Corrente 2000 IRCCS Istituto Auxologico Italiano (to P.L.M.), by the Swiss National

g

Fc R = Fc gamma receptor

Science Foundation grant # 3100-058558.99/1 (to C.C.) and #

M-CSF = macrophage colony-stimulating factor

31.50930.97 (to J.M.D.), and by the Hans Wilsdorf Foundation.

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Dr. P.L. Meroni, Allergy & Clinical Immunology Unit;

Dept. of Internal Medicine, University of Milan, IRCCS Istituto

involvement in systemic sclerosis and other examples. Semin Immuno-

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Fax: (39-2) 58211-559

15. Gruschwitz M, von den Driesch P, Kellner I, Hornstein OP, Sterry W.

email: [email protected]

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leads to increased expression of vascular endothelial growth

of such an inhibitor are now in progress to evaluate the efficacy of

factor, a mitogen that increases proliferation of vascular

this approach to prevent the progression, or induce regression, of

endothelial cells leading to neovascularization and enhances

"nonclinically significant" diabetic macular edema and of severe

breakdown of the blood-retinal barrier, perhaps resulting in

non-proliferative diabetic retinopathy,

Am J Ophthalmol 2002;133:693

macular edema. By interfering with the above biochemical

864

N. Ronda et al.

IMAJ

.

Vol 4, Supplement

.

November 2002