April 2000
Volume 41, Issue 5
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Physiology and Pharmacology  |   April 2000
Integrin-Mediated Neutrophil Adhesion and Retinal Leukostasis in Diabetes
Author Affiliations
  • Fina Cañas Barouch
    From the Laboratory for Surgical Research, Children’s Hospital, Boston, Massachusetts;
  • Kazuaki Miyamoto
    From the Laboratory for Surgical Research, Children’s Hospital, Boston, Massachusetts;
    Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts;
    Department of Ophthalmology and Visual Science, Kyoto University Graduate School of Medicine, Kyoto, Japan;
  • Jennifer R. Allport
    Vascular Research Division, Departments of Pathology, Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts;
  • Kenji Fujita
    From the Laboratory for Surgical Research, Children’s Hospital, Boston, Massachusetts;
  • Sven–Erik Bursell
    Department of Ophthalmology, Harvard Medical School, Joslin Diabetes Center, Boston, Massachusetts.
  • Lloyd Paul Aiello
    Department of Ophthalmology, Harvard Medical School, Joslin Diabetes Center, Boston, Massachusetts.
  • Francis William Luscinskas
    Vascular Research Division, Departments of Pathology, Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts;
  • Anthony P. Adamis
    From the Laboratory for Surgical Research, Children’s Hospital, Boston, Massachusetts;
    Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts;
Investigative Ophthalmology & Visual Science April 2000, Vol.41, 1153-1158. doi:
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      Fina Cañas Barouch, Kazuaki Miyamoto, Jennifer R. Allport, Kenji Fujita, Sven–Erik Bursell, Lloyd Paul Aiello, Francis William Luscinskas, Anthony P. Adamis; Integrin-Mediated Neutrophil Adhesion and Retinal Leukostasis in Diabetes. Invest. Ophthalmol. Vis. Sci. 2000;41(5):1153-1158.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. A critical early event in the pathogenesis of diabetic retinopathy is leukocyte adhesion to the diabetic retinal vasculature. The process is mediated, in part, by intercellular adhesion molecule-1 (ICAM-1) and results in blood–retinal barrier breakdown and capillary nonperfusion. This study evaluated the expression and function of the corresponding ICAM-1–binding leukocyte β2-integrins in experimental diabetes.

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.

Leukocyte–endothelial cell interactions in tissues are mediated by adhesion molecules expressed on the surface of leukocytes and endothelial cells. 1 2 Immunoglobulin superfamily molecules such as intercellular adhesion molecule-1 (ICAM-1) are expressed on endothelial cells and bind to β2-integrins expressed on leukocytes. 1 3 The integrins are transmembrane receptors that consist of noncovalently bound heterodimers composed of α- and β-chains. 4 5 Theβ 2-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 β2-integrins has a common β-chain in combination with a unique α-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 coworkers 8 demonstrated increased numbers of neutrophils in the choroid and retina of diabetic individuals. Expression of ICAM-1, the best characterized endothelial receptor for leukocyteβ 2-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 coworkers 10 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. 
Methods
Animals
Male Long–Evans rats weighing approximately 200 g were used for these experiments. All protocols followed the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committees of Children’s Hospital and Joslin Diabetes Center. The rats were fed standard laboratory chow and allowed free access to water in an air-conditioned room with a 12-hour–12-hour, light–dark cycle. 
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 × 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-α; Genzyme, Cambridge, MA), and incubated for 15 minutes with RPMI-5%. TNF-α stimulation of ICAM-1 surface expression was used for all experiments. Neutrophils were resuspended at 2 × 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 (30μ g/ml) to CD11a, CD11b, or CD18 for 10 minutes at room temperature. The neutrophils were washed and then incubated (2 × 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 × 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 (×10−5 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. 
Results
Increased Surface Integrin Expression on Diabetic Neutrophils
Integrin expression was measured on the surface of neutrophils from normal and diabetic rats. As shown in Table 1 , the flow cytometric analyses demonstrated statistically significant increases in the diabetic leukocyte CD11a, CD11b, and CD18 levels, as evidenced by the increases in mean channel fluorescence. Neutrophil CD11a, CD11b, and CD18 levels were 62% (n = 5, P = 0.006), 54% (n = 5, P = 0.045), and 38% (n = 5, P = 0.009) greater, respectively, on the 1-week diabetic leukocytes versus the nondiabetic leukocytes. Integrin expression was similarly increased on 2-week diabetic neutrophils with CD11a, CD11b, and CD18 levels being 53%, 24%, and 38% greater, respectively (data not shown). 
Increased Adhesion of Diabetic Neutrophils to TNFα-Activated Endothelial Cell Monolayers
The functional adhesion of purified neutrophils to cultured endothelial cell monolayers was investigated. Adhesion assays were performed by adding diabetic or nondiabetic neutrophils to TNF-α–stimulated rat endothelial cell monolayers under static conditions. TNF-α 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-α (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). 
The β2-integrin molecules mediating neutrophil adhesion in vitro were examined. Neutrophils were pretreated with a panel of anti-CD18 (β2-integrin) mAbs. In a representative experiment shown in Figure 2 , untreated diabetic neutrophils exhibited increased adhesion to TNFα-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). 
Decreased Leukostasis in Diabetic Rat Retinas by In Vivo CD18 Blockade
Retinal leukostasis in living animals was measured with AOLF. Intravenous injection of acridine orange causes leukocytes and endothelial cells to fluoresce through the noncovalent binding of the molecule to double-stranded DNA. 11 16 17 When a scanning laser ophthalmoscope is used, retinal leukocytes within blood vessels can be visualized in vivo. Twenty minutes after acridine orange injection, static leukocytes in the capillary bed can be observed as fluorescent dots. 11 These labeled cells are leukocytes because blocking CD18, expressed on leukocytes but not on endothelial cells, causes them to disappear (see below). 
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. 
Discussion
In this study, CD11a, CD11b, and CD18β 2-integrin levels were increased on the surface of neutrophils from diabetic rats. The increases correlated with the enhanced functional adhesiveness of diabetic neutrophils to rat endothelial cell monolayers. Similarly, in an in vivo model of experimentally induced diabetes, use of anti-CD18 F(ab′)2 fragments significantly decreased diabetic retinal leukostasis by 62%, confirming the relevance of the in vitro findings. Taken together, the data suggest that the Mac-1–integrin complex is operative in the adhesion of diabetic neutrophils to the retinal capillary endothelium. Because a major ligand for Mac-1 is ICAM-1, 18 these results are consistent with our previous work showing that ICAM-1 blockade prevents diabetic retinal leukostasis and blood–retinal barrier breakdown. 12  
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 TNFα 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-1 21 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. 
 
Table 1.
 
Flow-Cytometric Analysis of Integrin Molecule Expression on Neutrophils
Table 1.
 
Flow-Cytometric Analysis of Integrin Molecule Expression on Neutrophils
Control Diabetes P Value n
CD11a 115.0 ± 12.8 185.9 ± 18.5 0.006 5
CD11b 182.6 ± 39.2 281.9 ± 84.9 0.045 5
CD18 193.2 ± 34.2 267.1 ± 34.3 0.009 5
Figure 1.
 
Adhesion of control and diabetic rat neutrophils to confluent TNF-activated rat endothelial cell monolayers under static conditions. Neutrophils isolated from diabetic rats demonstrated significantly increased adhesion to rat endothelial cell monolayers. All data shown are means ± SD.
Figure 1.
 
Adhesion of control and diabetic rat neutrophils to confluent TNF-activated rat endothelial cell monolayers under static conditions. Neutrophils isolated from diabetic rats demonstrated significantly increased adhesion to rat endothelial cell monolayers. All data shown are means ± SD.
Figure 2.
 
The effect of anti-integrin antibodies on neutrophil adhesion in vitro. Neutrophils were preincubated with anti-CD11a, anti-CD11b, anti-CD18 (30 μg/ml of each mAb), or an equimolar mixture of anti-CD11a/CD11b/CD18 antibodies before their use in the adhesion studies. Diabetic neutrophils demonstrated increased adhesion to TNFα-activated endothelial cell monolayers under all treatment conditions. Diabetic neutrophil adhesion was significantly reduced by pretreatment with anti-CD11b, anti-CD18, or the mixture anti-CD11a/CD11b/CD18 antibodies (P < 0.050 for each condition vs. untreated diabetic neutrophils). Pretreatment with anti-CD11a did not reduce the adhesion of diabetic neutrophils (P = 0.5). Control nondiabetic neutrophil adhesion was also significantly reduced with all antibody treatments (P < 0.05). All data shown are means ± SD.
Figure 2.
 
The effect of anti-integrin antibodies on neutrophil adhesion in vitro. Neutrophils were preincubated with anti-CD11a, anti-CD11b, anti-CD18 (30 μg/ml of each mAb), or an equimolar mixture of anti-CD11a/CD11b/CD18 antibodies before their use in the adhesion studies. Diabetic neutrophils demonstrated increased adhesion to TNFα-activated endothelial cell monolayers under all treatment conditions. Diabetic neutrophil adhesion was significantly reduced by pretreatment with anti-CD11b, anti-CD18, or the mixture anti-CD11a/CD11b/CD18 antibodies (P < 0.050 for each condition vs. untreated diabetic neutrophils). Pretreatment with anti-CD11a did not reduce the adhesion of diabetic neutrophils (P = 0.5). Control nondiabetic neutrophil adhesion was also significantly reduced with all antibody treatments (P < 0.05). All data shown are means ± SD.
Figure 3.
 
Retinal leukostasis after CD18 blockade. Representative photographs from acridine orange leukocyte fluorography revealed static fluorescent leukocytes in the retinas of control and diabetic rats. The leukostasis in nondiabetic rat retina (A) was increased in diabetic rat retina (B) and unchanged after treatment with the control F(ab′)2 (C); however, retinal leukostasis was reduced by 62% in diabetic rats treated with anti-CD18 F(ab′)2 fragments (D).
Figure 3.
 
Retinal leukostasis after CD18 blockade. Representative photographs from acridine orange leukocyte fluorography revealed static fluorescent leukocytes in the retinas of control and diabetic rats. The leukostasis in nondiabetic rat retina (A) was increased in diabetic rat retina (B) and unchanged after treatment with the control F(ab′)2 (C); however, retinal leukostasis was reduced by 62% in diabetic rats treated with anti-CD18 F(ab′)2 fragments (D).
Figure 4.
 
Quantitation of retinal leukostasis after CD18 blockade. When CD18 bioactivity was inhibited via systemic administration of 5 mg/kg of the anti-CD18 neutralizing F(ab′)2 (clone WT.3), retinal leukostasis was inhibited by 62% in diabetic rat retinas. All data shown are means ± SD.
Figure 4.
 
Quantitation of retinal leukostasis after CD18 blockade. When CD18 bioactivity was inhibited via systemic administration of 5 mg/kg of the anti-CD18 neutralizing F(ab′)2 (clone WT.3), retinal leukostasis was inhibited by 62% in diabetic rat retinas. All data shown are means ± SD.
Table 2.
 
Characteristics of Control, Diabetic, Control mAb–Treated Diabetic, and Anti-CD18 F(ab′)2–Treated Diabetic Rats
Table 2.
 
Characteristics of Control, Diabetic, Control mAb–Treated Diabetic, and Anti-CD18 F(ab′)2–Treated Diabetic Rats
Control Diabetes Diabetes + Control F(ab′)2 Diabetes + anti-CD18 F(ab′)2
n 5 5 5 5
Body weight (g) 268 ± 10 236 ± 4* 233 ± 7* 237 ± 17*
Plasma glucose (mg/dl) 122 ± 21 327 ± 40* 357 ± 60* 351 ± 28*
Blood pressure (mm Hg) 110 ± 7 106 ± 13 103 ± 8 103 ± 7
Leukocyte count (×103 μl) 6.4 ± 1.4 4.9 ± 1.6, † 5.0 ± 1.3 6.7 ± 0.9
Luscinskas FW, Lawler J. Integrins and dynamic regulators of vascular function. FASEB J. 1994;8:929–938. [PubMed]
Rosales C, Juliano RL. Signal transduction by cell adhesion receptors in leukocytes. Leukocyte Biol. 1995;57:189–198.
Hynes RO. Integrins: a family of cell surface receptors. Cell. 1987;48:549–554. [CrossRef] [PubMed]
Anderson DC, Springer TA. Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1 and P150,95 glycoprotein. Annu Rev Med. 1987;38:175–194. [CrossRef] [PubMed]
Buck CA, Horwitz AF. Cell surface receptors for extracellular matrix molecules. Annu Rev Cell Biol. 1987;3:179–205. [CrossRef] [PubMed]
Lawrence MB, Smith CW, Eskin SG, McIntire LV. Effect of venous shear stress on CD18-mediated neutrophil adhesion to cultured endothelium. Blood. 1990;75:227–237. [PubMed]
Schroder S, Palinski W, Schmid-Schonbein GW. Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy. Am J Pathol. 1991;139:81–100. [PubMed]
McLeod DS, Lefer DJ, Merges C, Lutty GA. Enhanced expression of intracellular adhesion molecule-1 and P-selectin in the diabetic human retina and choroid. Am J Pathol. 1995;147:642–653. [PubMed]
Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994;76:301–314. [CrossRef] [PubMed]
Braun RD, Fisher TC, Meiselman HJ, Hatchell DL. Decreased deformability of polymorphonuclear leukocytes in diabetic cats. Microcirculation. 1996;3:271–278. [CrossRef] [PubMed]
Miyamoto K, Hiroshiba N, Tsujikawa A, Ogura Y. In vivo demonstration of increased leukocyte entrapment in retinal microcirculation of diabetic rats. Invest Opthalmol Vis Sci. 1998;39:2190–2194.
Miyamoto K, Khosrof S, Bursell S–E, et al. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition. Proc Natl Acad Sci USA. 1999;96:10836–10841. [CrossRef] [PubMed]
Allport JR, Ding HT, Ager A, Steeber DA, Tedder TF, Luscinskas FW. L-selectin shedding does not regulate human neutrophil attachment, rolling or transmigration across human vascular endothelium in vitro. J Immunol. 1997;158:4365–4372. [PubMed]
Luscinskas FW, Kiely JM, Ding H, et al. In vitro inhibitory effect of IL-8 and other chemoattractants on neutrophil-endothelial adhesive interactions. J Immunol. 1992;149:2163–2171. [PubMed]
Kiely JM, Luscinskas FW, Gimbrone MA., Dejana E, Corada M. Leukocyte-endothelial monolayer adhesion assay (static conditions). Methods in Molecular Biology, Adhesion Protein Protocols. 1999;131–136. Humana Press Totowa, NJ.
Nishiwaki H, Ogura Y, Kimura H, Kiryu J, Honda Y. Quantitative evaluation of leukocyte dynamics in retinal microcirculation. Invest Ophthalmol Vis Sci. 1995;36:123–130. [PubMed]
Nishiwaki H, Ogura Y, Kimura H, Kiryu J, Miyamoto K, Matsuda N. Visualization and quantitative analysis of leukocyte dynamics in retinal microcirculation of rats. Invest Ophthalmol Vis Sci. 1996;37:1341–1347. [PubMed]
Diamond DS, Springer TA. A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen. J Cell Biol. 1993;120:545–556. [CrossRef] [PubMed]
Davenpeck KL, Sterbinsky SA, Bochner BS. Rat neutrophils express alpha4 and beta1 integrins and bind to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Blood. 1998;91:2341–2346. [PubMed]
Morohoshi M, Fujisawa K, Uchimura I, Numano F. Glucose-dependent interleukin 6 and tumor necrosis factor production by human peripheral blood monocytes in vitro. Diabetes. 1996;45:954–959. [CrossRef] [PubMed]
Kim JA, Berliner JA, Nataraja RD, Nadler J. Evidence that glucose increases monocyte binding to human aortic endothelial cells. Diabetes. 1994;43:1103–1107. [CrossRef] [PubMed]
Manduteanu I, Voinea M, Serban G, Simionescu M. High glucose induces enhanced monocyte adhesion to valvular endothelial cells via a mechanism involving ICAM-1, VCAM-1 and CD18. Endothelium. 1999;6:315–324. [CrossRef] [PubMed]
Figure 1.
 
Adhesion of control and diabetic rat neutrophils to confluent TNF-activated rat endothelial cell monolayers under static conditions. Neutrophils isolated from diabetic rats demonstrated significantly increased adhesion to rat endothelial cell monolayers. All data shown are means ± SD.
Figure 1.
 
Adhesion of control and diabetic rat neutrophils to confluent TNF-activated rat endothelial cell monolayers under static conditions. Neutrophils isolated from diabetic rats demonstrated significantly increased adhesion to rat endothelial cell monolayers. All data shown are means ± SD.
Figure 2.
 
The effect of anti-integrin antibodies on neutrophil adhesion in vitro. Neutrophils were preincubated with anti-CD11a, anti-CD11b, anti-CD18 (30 μg/ml of each mAb), or an equimolar mixture of anti-CD11a/CD11b/CD18 antibodies before their use in the adhesion studies. Diabetic neutrophils demonstrated increased adhesion to TNFα-activated endothelial cell monolayers under all treatment conditions. Diabetic neutrophil adhesion was significantly reduced by pretreatment with anti-CD11b, anti-CD18, or the mixture anti-CD11a/CD11b/CD18 antibodies (P < 0.050 for each condition vs. untreated diabetic neutrophils). Pretreatment with anti-CD11a did not reduce the adhesion of diabetic neutrophils (P = 0.5). Control nondiabetic neutrophil adhesion was also significantly reduced with all antibody treatments (P < 0.05). All data shown are means ± SD.
Figure 2.
 
The effect of anti-integrin antibodies on neutrophil adhesion in vitro. Neutrophils were preincubated with anti-CD11a, anti-CD11b, anti-CD18 (30 μg/ml of each mAb), or an equimolar mixture of anti-CD11a/CD11b/CD18 antibodies before their use in the adhesion studies. Diabetic neutrophils demonstrated increased adhesion to TNFα-activated endothelial cell monolayers under all treatment conditions. Diabetic neutrophil adhesion was significantly reduced by pretreatment with anti-CD11b, anti-CD18, or the mixture anti-CD11a/CD11b/CD18 antibodies (P < 0.050 for each condition vs. untreated diabetic neutrophils). Pretreatment with anti-CD11a did not reduce the adhesion of diabetic neutrophils (P = 0.5). Control nondiabetic neutrophil adhesion was also significantly reduced with all antibody treatments (P < 0.05). All data shown are means ± SD.
Figure 3.
 
Retinal leukostasis after CD18 blockade. Representative photographs from acridine orange leukocyte fluorography revealed static fluorescent leukocytes in the retinas of control and diabetic rats. The leukostasis in nondiabetic rat retina (A) was increased in diabetic rat retina (B) and unchanged after treatment with the control F(ab′)2 (C); however, retinal leukostasis was reduced by 62% in diabetic rats treated with anti-CD18 F(ab′)2 fragments (D).
Figure 3.
 
Retinal leukostasis after CD18 blockade. Representative photographs from acridine orange leukocyte fluorography revealed static fluorescent leukocytes in the retinas of control and diabetic rats. The leukostasis in nondiabetic rat retina (A) was increased in diabetic rat retina (B) and unchanged after treatment with the control F(ab′)2 (C); however, retinal leukostasis was reduced by 62% in diabetic rats treated with anti-CD18 F(ab′)2 fragments (D).
Figure 4.
 
Quantitation of retinal leukostasis after CD18 blockade. When CD18 bioactivity was inhibited via systemic administration of 5 mg/kg of the anti-CD18 neutralizing F(ab′)2 (clone WT.3), retinal leukostasis was inhibited by 62% in diabetic rat retinas. All data shown are means ± SD.
Figure 4.
 
Quantitation of retinal leukostasis after CD18 blockade. When CD18 bioactivity was inhibited via systemic administration of 5 mg/kg of the anti-CD18 neutralizing F(ab′)2 (clone WT.3), retinal leukostasis was inhibited by 62% in diabetic rat retinas. All data shown are means ± SD.
Table 1.
 
Flow-Cytometric Analysis of Integrin Molecule Expression on Neutrophils
Table 1.
 
Flow-Cytometric Analysis of Integrin Molecule Expression on Neutrophils
Control Diabetes P Value n
CD11a 115.0 ± 12.8 185.9 ± 18.5 0.006 5
CD11b 182.6 ± 39.2 281.9 ± 84.9 0.045 5
CD18 193.2 ± 34.2 267.1 ± 34.3 0.009 5
Table 2.
 
Characteristics of Control, Diabetic, Control mAb–Treated Diabetic, and Anti-CD18 F(ab′)2–Treated Diabetic Rats
Table 2.
 
Characteristics of Control, Diabetic, Control mAb–Treated Diabetic, and Anti-CD18 F(ab′)2–Treated Diabetic Rats
Control Diabetes Diabetes + Control F(ab′)2 Diabetes + anti-CD18 F(ab′)2
n 5 5 5 5
Body weight (g) 268 ± 10 236 ± 4* 233 ± 7* 237 ± 17*
Plasma glucose (mg/dl) 122 ± 21 327 ± 40* 357 ± 60* 351 ± 28*
Blood pressure (mm Hg) 110 ± 7 106 ± 13 103 ± 8 103 ± 7
Leukocyte count (×103 μl) 6.4 ± 1.4 4.9 ± 1.6, † 5.0 ± 1.3 6.7 ± 0.9
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