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1 From the Laboratory for Surgical Research, Children’s Hospital, Boston, Massachusetts; 2 Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts; 3 Department of Ophthalmology and Visual Science, Kyoto University Graduate School of Medicine, Kyoto, Japan; 4 Vascular Research Division, Departments of Pathology, Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts; 5 Department of Ophthalmology, Harvard Medical School, Joslin Diabetes Center, Boston, Massachusetts.
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Abstract |
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METHODS. Diabetes was induced in Long Evans rats with streptozotocin. The expression of the surface integrin subunits CD11a, CD11b, and CD18 on rat neutrophils isolated from peripheral blood was quantitated with flow cytometry. In vitro neutrophil adhesion was studied using quantitative endothelial cell–neutrophil adhesion assays. The adhesive role of the integrin subunits CD11a, CD11b, and CD18 was tested using specific neutralizing monoclonal antibodies. CD18 bioactivity was blocked in vivo with anti-CD18 F(ab')2 fragments, and the effect on retinal leukocyte adhesion was quantitated with acridine orange leukocyte fluorography.
RESULTS. Neutrophil CD11a, CD11b, and CD18 surface integrin levels were 62% (n = 5, P = 0.006), 54% (n = 5, P = 0.045), and 38% (n = 5, P = 0.009) greater in diabetic versus nondiabetic animals, respectively. Seventy-five percent more neutrophils from diabetic versus nondiabetic animals adhered to rat endothelial cell monolayers (n = 6, P = 0.02). Pretreatment of leukocytes with either anti-CD11b or anti-CD18 antibodies lowered the proportion of adherent diabetic neutrophils by 41% (n = 6, P = 0.01 for each treatment), whereas anti-CD11a antibodies had no significant effect (n = 6, P = 0.5). In vivo, systemic administration of anti-CD18 F(ab')2 fragments decreased diabetic retinal leukostasis by 62% (n = 5, P = 0.001).
CONCLUSIONS. Neutrophils from diabetic animals exhibit higher levels of surface integrin expression and integrin-mediated adhesion. In vivo, CD18 blockade significantly decreases leukostasis in the diabetic retinal microvasculature. Integrin adhesion molecules may serve as therapeutic targets for the treatment and/or prevention of early diabetic retinopathy.
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Introduction |
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- and ß-chains.4
5
The
ß2-integrins are operative in leukocyte
adhesion and include LFA-1 (lymphocyte function–associated antigen,
CD11a/CD18), Mac-1 (leukocyte adhesion receptor, CD11b/CD18), and
p150/95 (CD11c/CD18). Each of the ß2-integrins
has a common ß-chain in combination with a unique -chain. Lawrence
et al.6
have previously shown that CD18 is required for
the firm attachment of healthy human neutrophils to human umbilical
vein endothelial cells. There is growing evidence that leukocyte adhesion plays an important role in the pathogenesis of diabetic retinopathy. In a rat model of experimental diabetic retinopathy, Schroeder et al.7 demonstrated retinal capillary occlusion by neutrophils and monocytes in histologic sections. Adjacent to the static leukocytes were areas of endothelial cell damage, capillary loss, and leukocyte extravasation. In a postmortem study of human subjects, Lutty and coworkers8 demonstrated increased numbers of neutrophils in the choroid and retina of diabetic individuals. Expression of ICAM-1, the best characterized endothelial receptor for leukocyte ß2-integrins (CD11/CD18),9 was elevated in the diabetic retinal vasculature as assessed by immunohistochemistry, a result consistent with an adhesive mechanism mediating the retinal leukocytosis. In a separate study, Hatchell and coworkers10 showed that leukocytes from diabetic cats are less deformable than those from nondiabetic cats. Because leukocytes must deform to traverse the smallest retinal capillaries,7 stiff leukocytes may contribute to the development of capillary nonperfusion.
In vivo studies from our laboratory have further investigated the role of leukocytes in diabetic retinopathy. Using acridine orange leukocyte fluorography, the density of static leukocytes in the retinas of streptozotocin-induced diabetic rats was demonstrated to be increased.11 12 Retinal leukocyte stasis (leukostasis) was observed within 3 days of diabetes induction and was temporally and spatially correlated with capillary nonperfusion and blood–retinal barrier breakdown. The onset of retinal leukostasis coincided with the upregulation of retinal ICAM-1 expression. Causality was demonstrated when an anti–ICAM-1 antibody prevented the diabetes-associated increases in retinal leukostasis and vascular leakage by 48.5% and 85.6%, respectively.12 However, the identities and bioactivities of the neutrophil adhesion molecules mediating diabetic retinal leukostasis are less well understood.
The aim of the present study was to investigate in greater detail the role of neutrophils in early diabetic retinal leukostasis. A time point of 1 week of diabetes was chosen in this study because steady state increases in diabetic retinal leukostasis and ICAM-1 expression are achieved in 1 week.12 Because adhesion can occur in the absence of increased adhesion molecule expression, both adhesion molecule expression and bioactivity were examined. Finally, the role of CD18 in the development of diabetic retinal leukostasis was examined in vivo using acridine orange leukocyte fluorography and neutralizing anti-CD18 F(ab')2 fragments.
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Methods |
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Induction of Diabetes
Rats received a single 60 mg/kg intraperitoneal injection of
streptozotocin (Sigma, St. Louis, MO) in 10 mM sodium citrate buffer,
pH 4.5, after an overnight fast. Control nondiabetic animals received
citrate buffer alone. Animals with blood glucose levels greater than
250 mg/dl 24 hours after injection were considered diabetic. All
experiments were performed 1 week after the induction of diabetes.
Monoclonal Antibodies and F(ab')2 Fragments
The monoclonal antibodies (mAbs) were murine in origin and were
used as purified IgG. For the in vitro studies, mAbs WT.1 (anti-rat
CD11a), 6G2 (anti-rat CD18), and MRC OX-42 (anti-rat CD11b) were
obtained from Serotec Inc. (Raleigh, NC). FITC-conjugated mouse
IgG1 mAb isotype control was obtained from
PharMingen (San Diego, CA). Fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse IgG1 Ab was
obtained from Caltag Laboratories (Burlingame, CA). For the in vivo
studies, WT.3 anti-rat LFA-1 beta chain (CD18)
F(ab')2 fragments were obtained from Seikagaku
America (Division of Associates of Cape Cod, Inc., Falmouth, MA).
Purified mouse anti-human IgG F(ab')2 fragments
were obtained from Jackson ImmunoResearch Laboratories (West Grove,
PA).
Flow Cytometry
The surface expression of CD11a, CD11b, and CD18 on rat
neutrophils was determined using flow cytometry as previously
described.13
Briefly, whole blood anticoagulated with EDTA
was obtained from the hearts of rats anesthetized with inhaled
isofluorane. Leukocytes were isolated by dextran sedimentation and
hypotonic lysis of contaminating erythrocytes. Aliquots of 5 x
105 cells in 100 µl RPMI 1640 medium
(BioWhittaker, Walkersville, MD) containing 5% fetal bovine serum
(FBS; RPMI-5%) were incubated on ice for 10 minutes. The tubes were
centrifuged at 400g for 5 minutes at 4°C. The cell pellets
were resuspended in 100 µl RPMI-5% containing 20 µg/ml primary mAb
to CD11a, CD11b, CD18 or isotype control and incubated for 45 minutes
on ice. Primary mAb were detected with FITC-conjugated goat anti-mouse
IgG1 Ab as previously detailed.14
The fluorescence of 104 cells was measured on a
FACScan (Becton Dickinson, San Jose, CA). Neutrophils were manually
gated on the basis of their characteristic forward and side
light-scattering properties. The surface expression is presented as the
mean channel fluorescence on a logarithmic scale.
Endothelial Cell–Neutrophil Adhesion Assays
Peripheral blood was obtained from rats anesthetized with inhaled
isofluorane via heart puncture with a 16-gauge EDTA-flushed needle.
Neutrophils were isolated from whole blood by density gradient
centrifugation with NIM · 2 (Neutrophil Isolation Media; Cardinal
Associates, Santa Fe, NM) according to the manufacturer’s
instructions. Preparations contained >94% neutrophils as determined
by eosin and methylene blue staining (Leukostat staining system;
Fischer Scientific, Pittsburgh, PA). There was no red blood cell
contamination. The cells were used immediately after collection.
The adhesion of unstimulated neutrophils to confluent monolayers of rat
prostate endothelial cells (RPECs) was determined under static
conditions as previously described.14
15
RPECs were
obtained from the American Type Culture Collection (ATCC; Manassas, VA)
and cultured in Eagle’s minimum essential media (ATCC) supplemented
with 5% FBS (GIBCO, Gaithersburg, MD) and 0.3 ng/ml porcine intestinal
heparin (Sigma). RPECs were grown to confluence on tissue
culture–treated plastic microtiter 96-well plates, stimulated for 24
hours with 30 ng/ml recombinant human tumor necrosis factor-alpha
(TNF-; Genzyme, Cambridge, MA), and incubated for 15 minutes with
RPMI-5%. TNF- stimulation of ICAM-1 surface expression was used for
all experiments. Neutrophils were resuspended at 2 x
106 cells/ml in RPMI-5% and incubated for 10
minutes at 37°C with 1 µM of the fluorescent marker,
2',7'-bis-(2-carboxyethyl)-5 (and 6) carboxyfluorescein, acetoxymethyl
ester (Molecular Probes, Eugene, OR) in dimethyl sulfoxide (vehicle).
Fluorescent labeled neutrophils were washed once and then incubated in
RPMI-5% alone or RPMI-5% with a saturating concentration of mAbs (30
µg/ml) to CD11a, CD11b, or CD18 for 10 minutes at room temperature.
The neutrophils were washed and then incubated (2 x
106 neutrophils/ml, 50 µl per well) with RPEC
for 10 minutes at 37°C. Nonadherent cells were removed, and the
content of the wells was lysed with 10 mM Tris-HCl, pH 8.4, containing
0.1% SDS. Fluorescence was determined in a microtiter plate
fluorometer (excitation 485 nm, emission 530–540 nm), and the adhesion
was reported as the number of adherent
neutrophils/mm2.
Acridine Orange Leukocyte Fluorography
Leukocyte dynamics in the retina were studied with acridine orange
leukocyte fluorography (AOLF).11
12
16
17
Rats were
anesthetized with 4 mg/kg xylazine hydrochloride (Phoenix
Pharmaceutical, St. Joseph, MO) and 25 mg/kg ketamine hydrochloride
(Parke-Davis, Morris Plains, NJ). The day before leukocyte dynamics
were observed, a heparin-lock catheter was surgically implanted in the
right jugular vein of each rat. The catheter was subcutaneously
externalized to the back of the neck. Rats received intravenous
injections of 5 mg/kg anti-rat beta chain (CD18, WT.3)
F(ab')2 fragments or 5 mg/kg anti-human IgG
isotype control F(ab')2 fragments in sterile
phosphate-buffered saline 24 hours before AOLF was performed. The
experiments were carried out in a masked fashion.
Immediately before AOLF, each rat was again anesthetized, and the pupil of the left eye was dilated with 1% tropicamide (Alcon, Humancao, Puerto Rico) to observe leukocyte dynamics. A focused image of the peripapillary fundus of the left eye was obtained with a scanning laser ophthalmoscope (Rodenstock Instruments, Munich, Germany). Acridine orange (Sigma) was dissolved in sterile saline (1.0 mg/ml), and 3 mg/kg was injected through the jugular vein catheter at a rate of 1 ml/min. The fundus was observed with the scanning laser ophthalmoscope using the argon blue laser as the illumination source and the standard fluorescein angiography filter in the 40° field setting for 1 minute. Twenty minutes later, the fundus again was observed to evaluate leukostasis in the retina. The images were recorded on videotape at the rate of 30 frames/sec. The video recordings were analyzed on a computer equipped with a video digitizer (Radius, San Jose, CA) that digitizes the video image in real time (30 frames/sec) to 640 x 480 pixels with an intensity resolution of 256 steps. For evaluating retinal leukostasis, an observation area around the optic disc measuring five disc diameters in radius was determined by drawing a polygon bordered by the adjacent major retinal vessels. The density of trapped leukocytes was calculated by dividing the number of static leukocytes (recognized as fluorescent dots) by the area of the observation region (in pixels). The density of static leukocytes was calculated in 8 to 10 peripapillary observation areas and an average density (x10-5 cells/pixel2) was obtained.
Blood pressures and heart rates were measured using a noninvasive cuff sensor and monitoring system (Ueda Electronics, Tokyo, Japan). Blood anticoagulated with EDTA was drawn from the abdominal aorta of each rat after the experiment to determine the leukocyte count using a hematology analyzer.
Statistical Analysis
All results are expressed as means ± SD. The data were
compared by analysis of variance with post hoc comparisons tested using
Fisher’s protected least significant difference procedure. Differences
were considered statistically significant when P values were
<0.05.
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Results |
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-Activated
Endothelial Cell Monolayers–stimulated rat endothelial cell monolayers under static
conditions. TNF- was added to maximize endothelial cell ICAM-1
expression. Preliminary experiments demonstrated a 2.7-fold increase in
endothelial cell ICAM-1 expression with TNF- (n = 4,
P < 0.0001; data not shown). As shown in Figure 1
, 75% more neutrophils from the diabetic rats adhered to the
endothelial cell monolayers than neutrophils isolated from nondiabetic
rats (n = 6, P = 0.02).
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-activated endothelial cell monolayers. Pretreatment with
anti-CD11b or anti-CD18 antibodies each decreased diabetic neutrophil
adhesion by 41% (n = 6, P = 0.01 for
each treatment). In contrast, pretreatment with the anti-CD11a antibody
did not significantly affect diabetic neutrophil adhesion
(n = 6, P = 0.5 vs. untreated diabetic
neutrophils). Moreover, treatment with an equimolar mixture of
anti-CD11a, anti-CD11b, and anti-CD18 monoclonal antibodies reduced
diabetic neutrophil adhesion by 72% (n = 6,
P < 0.0001 vs. untreated diabetic neutrophils).
Nondiabetic neutrophil adhesion also was reduced with the anti-CD11a,
anti-CD11b, and anti-CD18 antibodies, as well as with the
anti-CD11a/CD11b/CD18 antibody cocktail. The decreases were 39%, 49%,
53%, and 52%, respectively (n = 6, P < 0.05 for each treatment vs. untreated nondiabetic neutrophils).
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Leukocyte dynamics in the retina were observed after CD18 F(ab')2 blockade as shown in the representative photographs of Figure 3 . As expected, retinal leukostasis was increased in the diabetic versus nondiabetic rat retinas (Fig. 3B vs. 3A ). Treatment of the diabetic rats with the isotype control F(ab')2 fragments did not lead to detectable changes in the degree of leukostasis (Fig. 3C vs. 3B ). However, treatment with the anti-CD18 F(ab')2 fragments led to a striking decrease in retinal leukostasis (Fig. 3D vs. 3C ). Measurements of leukostasis were obtained throughout the entire retinas to avoid any potential sampling error and the means and SDs from independent experiments were compared (Fig. 4) . This confirmed that anti-CD18 blockade significantly decreased leukostasis in diabetic rats by 62% (n = 5, P = 0.001 vs. animals receiving control F(ab')2) (Fig. 4) . The body weight, plasma glucose level, blood pressure, and leukocyte counts for the control and diabetic animals are shown in Table 2 . The diabetic animals all had significantly elevated blood glucose levels and decreased body weight compared with the normal rats, as is the norm. Blood pressure was similar among groups. The peripheral leukocyte counts in the diabetic anti-CD18, F(ab')2-treated animals were increased compared to the untreated diabetic animals, a result consistent with successful CD18 blockade.
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Discussion |
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The results of the in vitro blocking adhesion studies suggest that Mac-1 is the predominant CD18 integrin involved in diabetic neutrophil adhesion to activated RPEC monolayers. At present, the reason for a lack of a CD11a-dependent component in diabetic versus nondiabetic neutrophil adhesion is not known. We speculate that the residual non–CD18-dependent neutrophil adhesion may be due to the VLA4–VCAM adhesion pathway because rat neutrophils constitutively express VLA4 on their surface.19
These data strongly support the concept that the leukocyte adhesive
changes in this model of diabetes are of a systemic nature. The assayed
neutrophils were isolated from the peripheral blood and therefore
reflected systemic neutrophil adhesion molecule expression and
bioactivity. The causes of the surface integrin changes remain unknown;
however, they are likely to be linked to hyperglycemia. For example,
hyperglycemia has a direct impact on TNF expression, a cytokine
known to activate integrin adhesion molecules on
leukocytes.20
In vitro work also has shown that
hyperglycemia promotes increased leukocyte adhesion to endothelium via
ICAM-121
22
and CD18.22
Thus, hyperglycemia,
either directly or indirectly, is likely a proximal stimulus for the
ICAM-1 and CD18 upregulation seen in diabetes.
Another observation in these studies is that a low-level retinal leukostasis occurs in the normal state.11 The same molecules that are operative in the diabetic state also mediate this presumably normal phenomenon. If the low-level leukostasis in the nondiabetic state is physiologic, then the specificity of an anti-integrin therapy may be compromised.
Finally, these results provide additional evidence of leukocyte involvement in the pathogenesis of diabetic retinopathy. The aggregate data suggest that diabetic retinopathy should be, in one sense, redefined as an inflammatory disease. Very early in diabetes, leukocytes adhere to the vascular endothelium, trigger breakdown of the blood–retinal barrier, impede flow, and in some instances, extravasate into the retinal parenchyma.7 12
In summary, the identification of Mac-1 as a functional adhesive molecule in diabetic retinopathy may provide a potential target for the prevention and/or treatment of the disease. The effect of ICAM-1 and Mac-1 inhibition on the development of diabetic retinopathy deserves further investigation.
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Footnotes |
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Submitted for publication August 30, 1999; revised November 17, 1999; accepted December 1, 1999.
Commercial relationships policy: N.
Corresponding author: Anthony P. Adamis, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114. adamis{at}a1.tch.harvard.edu
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