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Neutrophils in Galactose-Fed Dogs
Neutrophils in Galactose-Fed Dogs: Suppressed Apoptosis and Increased Adhesion to Retinal Capillary Endothelial Cells Nobuo Ohta Jen-Yue Tsai E. Filippo Secchi Peter F. Kador Sanai Sato
ABSTRACT Dogs fed a diet containing 30% galactose develop diabetes-like retinal capillary changes. As retinal capillary occlusion is commonly observed in diabetic retinopathy, neutrophil apoptosis and the interaction of neutrophils with retinal capillary endothelial cells were investigated. Neutrophils were isolated with Ficoll-Hypaque centrifugation from dogs fed a 30% galactose diet and dogs fed a normal, control diet containing 30% non-nutrient filler. Apoptosis of neutrophils was microscopically examined after incubation at 378C for 3 hours with either 100 U/mL tumor necrosis factor a (TNF-a), 2 mg/mL cycloheximide or 50 ng/mL phorbol 12-myristate 13-acetate (PMA). Neutrophil adhesion to dog retinal capillary endothelial cells was examined by counting the cells attached to the surface of endothelial cells after the incubation in the presence of either 100 U/mL TNF-a or 5 mg/mL lipopolysaccharides (LPS) at 378C for 3 hours. With all three stimulants TNF-a,
cycloheximide and PMA, the rate of apoptosis was significantly lower for neutrophils isolated from galactose-fed dogs compared to control dogs fed a normal diet. Preincubation of neutrophils from control dogs in medium containing 30% galactose for 3 hours did not affect the rate of apoptosis. Neutrophil adhesion to retinal capillary endothelial cells induced by incubation in the presence of either 100 U/mL TNF-a or 5 mg/ml LPS was significantly higher with neutrophils isolated from galactose-fed dogs than those from control dogs. The data indicate that long-term galactose feeding is essential with development of various neutrophil dysfunctions. These neutrophil changes may contribute to the development of retinal microangiopathy associated with diabetes and galactosemia. (Journal of Diabetes and Its Complications 1313; 3: 151–158, 1999.) 1999 Elsevier Science Inc.
INTRODUCTION Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, Maryland, U.S.A. Reprint requests to be sent to: Dr. Sanai Sato, LOT, NEI, NIH, Bldg 10, Rm 10B09, 10 Center Drive, MCS 1850, Bethesda, MD 208921850, U.S.A. Journal of Diabetes and Its Complications 1999; 13:151–158 1999 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
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apillary occlusions constitute a characteristic pathologic feature of diabetic retinopathy. The formation of nonperfusion and/or avascular areas resulting from capillary occlusions eventually initiate clinically apparent retinal 1056-8727/99/$–see front matter PII S1056-8727(99)00040-9
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capillary changes, which include microaneurysms, intraretinal microvascular abnormality (IRMA), and advanced stages of neovascularization. In the development of microangiopathies, leukocytes have been suggested to play an important role. Both neutrophils and monocytes naturally tend to adhere to the vascular endothelium.1,2 Because these cells release various proteases and oxygen-derived free radicals,3 the activation of monocytes and/or neutrophils causes the endothelial cell injury that eventually initiates the occlusion of capillaries. It is also well-established that, in some extreme conditions, such as severe ischemia or shock, capillary occlusions by leukocytes commonly occur in various organs.4–6 There are a number of reports that leukocytes may contribute in the development of diabetic retinopathy. Diabetic leukocytes are less deformative7,8 and tend to plug in microcirculation.9,10 Moreover, the interaction between leukocytes and endothelial cells appears to increase in diabetes and/or hyperglycemia.11,12 The levels of soluble leukocyte adhesion molecules are elevated in patients with diabetic retinopathy.13 Diabetic neutrophils not only increase their interaction with endothelial cells but also increase superoxide production.14,15 it has also been reported that bovine retinal capillary endothelial cells when exposed to high glucose have been increased adhesion to neutrophils.16 The expression of adhesion molecules is also higher in diabetic human retina and choroid.17 Capillary occlusions and endothelial cells damages by leukocytes have also been observed in experimental diabetic rats.18 Using acridine orange digital fluorography, it has been observed that leukocyte entrapment in retinal microcirculation is significantly increased in vivo in diabetic rats.19 It is known that diabetic dogs develop early retinal changes that are similar to human diabetic retinopathy.20–22 Dogs fed a diet containing 30% galactose also develop similar retinal capillary changes with the selective loss of pericytes as the initial lesion.23–25 This galactose-fed dog model even develops the more advanced stages of pre-proliferative and proliferative retinopathy.26,27 Previously, we have reported that galactosefed dogs develop both neutrophil and lymphocyte dysfunctions similar to those observed with human diabetics. These include decreased lymphocyte blastformation by lectins28 and impaired induction of NADPH oxidase, a key enzyme for superoxide production.29 Here, we report additional evidences of neutrophil dysfunctions in galactose-fed dogs. The induction of neutrophil apoptosis by tumor necrosis factor a (TNF-a), cycloheximide, and protein kinase C activation is significantly lower in galactose-fed dogs, while at the same time, the interaction of neutrophils with retinal capillary endothelial cells is significantly increased.
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FIGURE 1 Microscopic appearances of normal dog neutrophils (A) and apoptotic cells induced by the incubation with 100 U/mL of TNF-a at 378C for 3 h (B). Note the nuclear fragmentation and condensation of apoptotic neutrophils. May-Giemsa stain 3400.
METHODS Reagents and Media. All chemicals utilized were of reagent grade. RPMI 1640 culture medium and fetal calf serum (FCS) were purchased from GibcoBRL Life Technologies, Inc. (Grand Island, NY) and Biofluids, Inc. (Rockville, MD), respectively. Recombinant human tumor necrosis factor a (TNF-a) was purchased from Genzyme Diagnostics (Cambridge, MA). Cycloheximide, phorbol 12-myristate 13-acetate (PMA), 1(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and lipoplysaccharides (LPS) were purchased from Sigma Chemical Co. (St. Louis, MO). Ficoll-Hypaque (specific gravity 1.0777) was a product of Pharmacia Biotech, Inc. (Uppsala, Sweden). Galactose-Fed Dogs. Thirty-six 9-year-old male beagles, obtained from Marshall Farms USA, Inc. (North Rose, NY), were randomly divided into two groups. Dogs were individually housed in 0.9 3 2.7 m runs and fed once a day a diet consisting 450 g of standard dog chow containing either 30% galactose or 30% nonnutrient filler as previously described.24,25 Diets were prepared by Bioserve (Frenchtown, NJ). All dogs were monitored through quarterly ocular examinations and blood chemistry evaluations. All experiments in this study were conducted with dogs placed on galactose diet for 3 years. Isolation of Neutrophils. Blood was drawn in early morning prior to feeding. The heparinized venous blood was mixed with an equal volume of 3% dextran, and, after the red blood cells had settled down in the bottom of the tubes, the neutrophils were collected from the buffy-coat by the centrifugation at 400 3 g for 30 min with Ficoll-Hypaque. Contaminating erythrocytes were removed by hypotonic shock with 0.2% sodium chloride. Neutrophil purity of all final preparations, determined by differential counting, were greater than 95%. Examination of Apoptosis. Isolated dog neutrophils were suspended in RPMI medium containing 100
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U/mL TNF-a, 2 mg/mL cycloheximide or 50 ng/mL PMA and incubated at 378C for 3 h. After incubation, the cells were collected by the centrifugation at 400 3 g for 5 min and stained with May-Giemsa. Apoptotic cell counts were based on their microscopic morphological appearance (Figure 1). A series of experiments were conducted with neutrophils isolated from randomly selected five dogs. The results were validated by repeating the same experiments at least three times with another randomly selected five dogs. Statistical significance (P , 0.05) was determined by either the Wilcoxon test or Student’s t test. Agarose Gel Electrophoresis of DNA. Neutrophils were suspended in 10 mM Tris-HCl buffer, pH 8.0 containing 1 mM EDTA. The cells were lysed by incubation for 2 h at 558C in 10 mM Tris-HCl buffer, pH 8.0 containing 0.1 M EDTA, 20 mg/mL RNase, 100 mg/mL proteinase K and 0.5% (W/V) sodium dodecyl sulfate (SDS). DNA was extracted with phenol and precipitated with 70% ethanol. Electrophoresis was conducted on 0.8% agarose gels containing 0.5 mg/mL ethidium bromide at 15 V for 12 h. DNA size markers utilized include a 100 pb molecular ruler and a 1 kb DNA ladder (Bio-Lad Laboratories, Hercules, CA). Preparation of Retinal Capillary Endothelial Cells. Retinal capillary endothelial cells were cultured from normal beagle dog eyes. The neural retina, isolated under the dissecting microscope, was minced with two razor blades, digested in PBS containing 0.1% collagenase at 378C for 20 min, and then passed through two stainless sieves (mesh 150 and mesh 50). The retainments from the second sieve (mesh 50) were collected, washed with PBS, resuspended in DMEM supplemented with 10% dog serum and cultured in fibronectin-coated plastic plates at 378C under a 5% carbon dioxide atmosphere. The identity of the cultured endothelial cells was confirmed by their typical morphological appearance and by the uptake of acetylated-low density lipoprotein.30 Interaction of Neutrophils to Retinal Capillary Endothelial Cells. At the fifth passage, dog retinal capillary endothelial cells were transferred to 24-well culture plates. When the cells reached confluence, the medium was replaced with a dog neutrophil suspension (5 3 106/mL) in the RPMI medium containing either 100 U/mL TNF-a or 5 mg/mL LPS. After the incubation at 378C for 3 hours, the plates were washed with PBS and the attached cells were examined with May-Giemsa staining. The adhesion of neutrophils was expressed as the average of cell numbers attached to the surface of plate of randomly selected five microscopic fields.
FIGURE 2 Time course of TNF-a induced apoptosis of dog neutrophils. Neutrophils isolated from non-galactosemic control (open circles) and 30% galactose-fed dogs (gray circles) were incubated in RPMI medium containing 100 U/mL of TNF-a up to 6 hours. Specific apoptosis induced by TNF-a was expressed as the percentage of apoptotic cells corrected with the naturally occurred apoptosis during the incubation in the absence of TNF-a. Mean 6 S.D. (n 5 5).
RESULTS Dogs receiving diets containing either 30% galactose or 30% non-nutrient filler for up to 3 years revealed no abnormalities in their periodic evaluations of blood chemistries. Similarly, no significant difference in blood counts was observed between the galactose-fed and non-nutrient filler-fed control dogs. When incubated in medium containing 100 U/mL of TNF-a, dog neutrophils underwent apoptosis. The apoptotic cells were easily distinguished by their morphological characteristics such an nuclear fragmentation and condensation (Figure 1). The number of apoptotic cells increased with incubation time (Figure 2). Although TNF-a induced apoptosis of neutrophils isolated from both 30% galactose-fed and 30% non-nutrient filler-fed control dogs, the percentage of apoptotic cells was significantly lower with neutrophils isolated from galactose-fed dogs. Because difference between control and galactose-fed dogs was most clearly observed after 3 h of incubation, subsequent studies were all conducted with 3 h incubations. Although the nonspecific protein synthesis inhibitor cycloheximide is often utilized to protect cells from apoptosis, higher concentrations of cycloheximide induced apoptosis of neutrophils isolated from both control and galactose-fed dogs (Figure 3A). As apoptosis by TNF-a, the rate of apoptosis induced by cycloheximide was significantly lower with neutrophils isolated from galactose-fed dogs than control dogs. Combining
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FIGURE 4 The effect of galactose on dog neutrophil apoptosis. Neutrophils isolated from non-galactosemic control dogs were preincubated in either the absence (white bars) or presence of 30 mM galactose at 378C for 3 h before apoptosis was induced by the incubation with a mixture of 100 U/mL of TNF-a and 2 mg/mL of cycloheximide. Mean 6 standard deviation (n 5 5).
To examine the possibility that galactose directly interferes with neutrophil apoptosis in galactose-fed dogs, neutrophils isolated from non-galactosemic, fiber-fed control dogs were preincubated for 3 hours in medium containing 30 mM D-galactose prior to the
FIGURE 3 Apoptosis of dog neutrophils induced by TNF-a, cycloheximide and a PKC activator phorbol 12-myristate 13-acetate (PMA). Neutrophils isolated from non-galactosemic control (white bars) and galactose-fed dogs (gray bars) were incubation at 378C for 3 h in the medium (A) containing either 100 U/mL of TNF-a, 2 mg/mL of cycloheximide, or both 100 U/mL of TNF-a and 2 mg/ mL of cycloheximide and the medium (B) containing either 1-(5isoquinolinesulphonyl)-2-methylpiperazine (H7) (100 mM), PMA (50 ng/ml), or both H7 (100 mM) and PMA (50 ng/mL). Mean 6 S.D. (n 5 5).
the two stimulants TNF-a and cycloheximide remarkably enhanced the rate of apoptosis. However, the rate of apoptosis remained significantly lower with neutrophils from galactose-fed dogs compared to non-galactosemic control dogs. The addition of a PKC activator, phorbol 12-myristate 13-acetate (PMA), into the incubation medium also increased the rate of apoptosis of dog neutrophils (Figure 3B). Apoptosis induced by PMA was almost completely prevented by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), confirming that PMA-induced apoptosis is initiated through the activation of PKC. The percentage of apoptotic cells induced by PMA was again significantly lower with neutrophils from galactose-fed dogs than non-galactosemic control dogs.
FIGURE 5 Agarose gel electrophoresis of DNA isolated from non-galactosemic control dog neutrophils incubated at 378C for 3 h in either the absence (lane 1) or presence of 100 U/mL of TNF-a (lane 2) and a mixture of 100 U/mL of TNF-a and 2 mg/ mL of cycloheximide (lane 3). Each lane contains 5 mg of DNA. Electrophoresis was performed on 0.8% agarose gel containing 0.5 mg/mL ethidium bromide at 15 volts for 12 h. Lane M1 and M2 represented 1 kb DNA ladder and 100 bp molecular ruler, respectively.
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induction of apoptosis by a mixture of TNF-a and cycloheximide. This preincubation did not result in a significant decrease in the induction of apoptosis by the mixture of TNF-a and cycloheximide (Figure 4). DNA fragmentation, referred to as a ladder formation, is characteristically observed in apoptotic cells. To verify the presence of DNA fragmentation in apoptotic neutrophils, DNAs isolated from neutrophils of nongalactosemic control dogs incubated with TNF-a and a mixture of TNF-a and cycloheximide were analyzed by agarose gel electrophoresis (Figure 5). No significant fragmentation was observed in DNA isolated from dog neutrophils incubated without stimulants and the isolated DNA remained at the origin of the agarose gel. In contrast, neutrophils incubated with TNF-a and a mixture of both TNF-a and cycloheximide displayed obvious DNA fragmentation. DNA fragmentation was especially severe in neutrophils incubated with a mixture of TNF-a and cycloheximide and DNA was essentially absent at the origin of the gel. Because it is known that the stimulation by cytokines, such as TNF-a, not only induce apoptosis but also increase the adhesion of neutrophils, the interaction of neutrophils to dog retinal capillary endothelial cells was also examined by incubating isolated neutrophils in culture plates containing confluent dog retinal capillary endothelial cell (Figure 6). In the absence of stimulants in the incubation medium, only a slight attachment of neutrophils to the surface of endothelial cells could be observed. The average numbers of cells attached were 4.7 6 3.1 and 6.6 6 3.9 cells/microscopic field (n 5 5) with neutrophils isolated from non-galactosemic control dogs and galactose-fed dogs, respectively. Addition of TNF-a to the incubation medium resulted in a significant increase in the number of neutrophils attached. Although TNF-a increased the adhesion of neutrophils isolated
FIGURE 6 Adhesion of neutrophils isolated from control (B) and galactose-fed dogs (C) to the surface of retinal capillary endothelial cells. Neutrophils (5 3 106 cells/mL) were incubated in the culture plate covered with dog retinal capillary endothelial cells in the presence of 100 U/mL of TNF-a at 378C for 3 h. Neutrophils are observed as small round cells (indicated by arrows). Note some area (indicated by circle with broken line in picture C) was almost completely covered with neutrophils. A; control endothelial cells incubated without neutrophils. Stained with May-Giemsa. The graph below represents the average numbers of neutrophils attached to the surface of plate of randomly selected five microscopic fields after the incubation in the presence of either 100 U/mL of TNF-a or 5 mg/mL of lipopolysaccharides (LPS). White and gray bars represent neutrophils isolated from control and galactose-fed dogs, respectively. For this calculation, the area where neutrophils are attached with extremely high density (see picture C) was excluded. Mean 6 S.D. (n 5 5).
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from both galactose-fed and control dogs, a significantly higher attachment (151.6 6 42.2 cells/field, n 5 5) of neutrophils from galactose-fed dogs was observed compared to those (64.8 6 20.3 cells/field, n 5 5) of control dogs. The same increase of neutrophil adhesion was observed with an another stimulant LPS. The adhesion of neutrophils isolated from galactose-fed dogs was 120.3 6 41.4 cells/field (n 5 5). This was significantly higher than that (67.2 6 19.5 cells/field, n 5 5) of non-galactosemic control dogs. DISCUSSION We have previously reported that neutrophils isolated from dogs fed a 30% galactose diet for up to 3 years demonstrate a significantly impaired induction of NADPH oxidase by zymosan despite the normal appearance of routine blood chemistry profiles.29 The present study provides additional evidence that longterm galactose-fed dogs develop fundamental neutrophil dysfunctions. Neutrophil apoptosis induced by various stimulants was significantly suppressed in the long-term galactose-fed dogs. Moreover, when activated by TNF-a and LPS, galactosemic neutrophils strongly interacted with dog retinal capillary endothelial cells. Clinically, it is well known that diabetic individuals have a higher incidence of bacterial infections compared to nondiabetic individuals. Persistent inflammation and delayed wound healing are additional problems for diabetic patients. In inflammatory response, vast numbers of leukocytes migrate into the perturbed tissues. Obviously, the efficient clearance of aging neutrophils is crucial for the successful resolution of inflammation and the promotion of tissue repair. Failure of aging neutrophils to be quickly and completely cleared results in further unwanted tissue damage and results in a prolongation of inflammation.31,32 To date, no report has been published on delayed neutrophil clearance in diabetic patients. However, it has been demonstrated that apoptotic cells are increased throughout the process of wound healing of diabetic mice.33 Since the major mechanism for clearance of aging neutrophils has been established to be apoptosis followed by phagocytosis by macrophages,31,32 the decreased response for neutrophils to TNF-a and/or many other cytokines may contribute to the delayed resolution of inflammation associated with diabetes or galactosemia. As galactosemic neutrophils failed to quickly respond to TNF-a stimulus, one may question whether the incidence of infections and/or inflammations is surely increased in galactose-fed dogs. We have observed that the incidence of thyroiditis, similar to that observed in human diabetic patients, is increased in long-term galactose-fed dogs.34 This suggests that neutrophil changes may be linked to increased incidence
of infections or prolonged inflammations. However, because the study was conducted with only a limited number of dogs and no overt clinical increase in infection of galactose-fed dogs was observed, further studies are needed to determine the relevance between neutrophil dysfunctions and the higher incidence of infections and/or inflammations in galactose-fed dogs. Capillary occlusion and the resulting tissue hypoxia/anoxia are underlying pathologic lesions associated with diabetic microangiopathies. In diabetes, the interaction of leukocytes to capillary endothelial cells is increased.11–13 Because activated neutrophils can cause endothelial cell injury, the increased adhesion of neutrophils can initiate capillary occlusion.35 Indeed, retinal capillary occlusions and endothelial cell damages by leukocytes has been reported to occur in experimental diabetic rats.18 Moreover, a recent study has also clearly demonstrated that leukocyte entrapment in retinal microcirculation is significantly increased in diabetic rats.19 The present results are consistent with these observations. Neutrophils from galactose-fed dogs more strongly interact with retinal capillary endothelial cells compared to those from non-galactosemic control dogs. Because these galactose-fed dogs develop diabetes-like retinal capillary changes, the observed neutrophil changes that increase the interaction with retinal capillary endothelial cells may be important in developing and/or accelerating the process of retinal microangiopathy. Interestingly, apparent differences in neutrophil adhesion to retinal capillary endothelial cells were observed only when neutrophils were activated. In diabetes, infections occur more frequently and their associated inflammation tends to be prolonged. If the activated neutrophils interact more strongly in diabetes, repeated infections with prolonged inflammation can be a risk factor that enhances the process of microangiopathy. TNF-a is an inflammatory cytokine released by macrophages that requires specific cell surface receptors to initiate signals inducing apoptosis.36 Two other stimulants PMA and cycloheximide initiate apoptosis through different pathways. PMA directly activates PKC while cycloheximide is a non-specific protein synthesis inhibitor. Since the responses of neutrophils to all three different stimulants were similarly affected in galactose-fed dogs, a single event such as the different level of PKC37,38 can not explain this observation. It has been reported that aldose reductase inhibitors delay retinal capillary changes39,40 and prevent cataract formation in galactose-fed dogs in a dose-dependent manner.41,42 We have also reported that galactitol accumulation occurs in neutrophils of galactose-fed dogs despite the extremely low level of aldose reductase in dog neutrophils.29 However, although polyol accumulation has been shown to be a common biochemical mechanism that initiates the onset of these complications, the
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importance of polyol accumulation in the development of neutrophil dysfunctions has not been established. REFERENCES 1. Atherton A, Born GV: Quantitative investigations of the adhesiveness of circulating polymorphonuclear leukocytes to blood vessel walls. J Physiol 222:447–474, 1972. 2. Schmid-Schonbein GW, Usami S, Skalak R, Chien S: The interaction of leukocytes in capillary and postcapillary vessels. Microvasc Res 19:45–70, 1980. 3. Fantone JC, Ward PA: Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Am J Physiol 107:397–418, 1982. 4. Bagge U, Amundson B, Lauritzen C: White blood cell deformability and plugging of skeletal muscle capillaries in hemorrhagic shock. Acta Physiol Scand 108:159– 163, 1980. 5. Barroso-Aranda J, Schmid-Schonbein GW, Zweifach BW, Engler RL: Granulocytes and no-reflow phenomenon in irreversible hemorrhagic shock. Circ Res 63:437– 447, 1988. 6. Kaminski PM, Proctor KG: Attenuation of no-reflow phenomenon, neutrophil activation, and reperfusion injury in intestinal microcirculation by topical adenosine. Circ Res 65:426–435, 1989. 7. Pecsvarady Z, Fisher TC, Darwin CH, Fabok A, Maqueda TS, Saad MF, Meiselman HJ: Decreased polymorphonuclear leukocyte deformability in NIDDM. Diabetes Care 17:57–63, 1994. 8. Braun RD, Fisher TC, Meiselman HJ, Hatchell DL: Decreased deformability of polymorphonuclear leukocytes in diabetic cats. Microcirculation 3:271–278, 1996. 9. Harris AG, Skalak TC, Hatchell DL: Leukocyte-capillary plugging and network resistance are increased in skeletal muscle of rats with streptozotocin-induced hyperglycemia. Int J Microcirc Clin Exp 14:159–166, 1994. 10. Miyamoato K, Ogura Y, Kenmochi S, Honda Y: Role of leukocytes in diabetic microcirculatory disturbances. Microvasc Res 54:43–48, 1997. 11. Kim JA, Berliner JA, Natarajan RD, Nadler JL: Evidence that glucose increases monocyte binding to human aortic endothelial cells. Diabetes 43:1103–1107, 1994.
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cells exposed to hyperosmolarity. Exp Eye Res 58:641– 647, 1994. 17. McLeod DS, Lefer DJ, Merges C, Lutty GA: Enhanced expression of intracellular adhesion molecule-1 and Pselectin in the diabetic human retina and choroid. Am J Pathol 147:642–653, 1995. 18. Schroder S, Palinski W, Schmid-Schonbein GW: Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy. Am J Pathol 139:81–100, 1991. 19. Miyamoto K, Hiroshiba N, Tsujikawa A, Ogura Y: In vitro demonstration of increased leukocyte entrapment in retinal microcirculation of diabetic rats. Invest Ophthalmol Vis Sci 39:2190–2194, 1998. 20. Patz A, Maumenee AE: Studies on diabetic retinopathy. I. Retinopathy in a dog with spontaneous diabetes mellitus. Am J Ophthalmol 54:532–541, 1962. 21. Hausler HR, Sibay TM, Campbell J: Retinopathy in a dog following diabetes induced by growth hormone. Diabetes 13:122–126, 1964. 22. Engerman RL, Kramer JW: Dogs with induced or spontaneous diabetes as models for the study of human diabetes mellitus. Diabetes 31 (Suppl 1 Pt 2):26–29, 1982. 23. Engerman RL, Kern TS: Experimental galactosemia produces diabetic-like retinopathy in dogs. Diabetes 33:97– 100, 1984. 24. Kador PF, Akagi Y, Terubayashi H, Wyman M, Kinoshita JH: Prevention of pericyte ghost formation in retinal capillaries of galactose-fed dogs by aldose reductase inhibitors. Arch Ophthalmol 106:1099–1102, 1988. 25. Kador PF, Akagi Y, Takahashi Y, Ibeke H, Wyman M, Kinoshita JH: Prevention of retinal vessel changes associated with diabetic retinopathy in galactose-fed dogs by aldose reductase inhibitors. Arch Ophthalmol 108: 1301–1309, 1990. 26. Takahashi Y, Wyman M, Frederick F III, Kador PF: Diabeteslike proliferative retinal changes in galactose-fed dogs. Arch Ophthalmol 110:1295–1302, 1992. 27. Kador PF, Takahashi Y, Myman M, Ferris F III: Diabeteslike proliferative retinal changes in galactose-fed dogs. Arch Ophthalmol 113:352–354, 1995. 28. Fukase S, Sato S, Secchi EF, Gery I, Kador PF: Decreased lymphocyte blastformation in long term galactose-fed dogs. Int J Diabetes 3:91–97, 1995.
12. Wierusz-Wysocka B, Wykretowicz A, Byks H, Sadurska K, Wysocki H: Polymorphonuclear neutrophils adherence, superoxide anion (O2-) production and HBA1 level in diabetic patients. Diabetes Res Clin Pract 21:109– 114, 1993.
29. Fukase S, Sato S, Mori K, Secchi EF, Kador PF: Polyol pathway and NADPH-dependent reductases in dog leukocytes. J Diabet Complications 10:304–313, 1996.
13. Olsen JA, Whitelaw CM, McHardy KC, Pearson DWM, Forrester JV: Soluble leukocyte adhesion molecules in diabetic retinopathy stimulate retinal capillary endothelial cell migration. Diabetologia 40:1166–1171, 1997.
30. Voyta JC, Via DP, Butterfield CE, Zetter BR: Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 99:2034–2040, 1984.
14. Shah SV, Wallin JD, Eilen SD: Chemiluminescence and superoxide anion production by leukocytes from diabetic patients. J Clin Endocrinol Metab 57:402–409, 1983.
31. Savill J, Haslett C: Granulocyte clearance by apoptosis in the resolution of inflammation. Semin Cell Biol 6:385– 393, 1995.
15. Freedman SF, Hatchell DL: Enhanced superoxide radical production by stimulated polymorphonuclear leukocytes in a cat model of diabetes. Exp Eye Res 55:767– 773, 1992.
32. Savill J: Apoptosis in resolution of inflammation. J Leukoc Biol 61:375–380, 1997.
16. Bullard SR, Hatchell DL, Cohen HJ, Rao KMK: Increased adhesion of neutrophils to retinal vascular endothelial
33. Darby IA, Bisucci T, Hewitson TD, MacLellan DG: Apoptosis is increased in a model of diabetes-impaired wound healing in genetically diabetic mice. Int J Biochem Cell Biol 29:191–200, 1997.
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OHTA ET AL.
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34. Takahashi Y, Kador PF: Lymphocytic thyroiditis in galactose-fed dogs. US-Japan Aldose Reductase Workshop, Feb. 12–16 Kona, Hawaii, p. 78.
39.
35. Schmid-Schonbein GW: The damaging potential of leukocyte activation in the microcirculation. Angiology 44:45–46, 1993.
40.
36. Darnay BG, Aggarwal BB: Early events in TNF signalling: A story of associations and dissociations. J Leukoc Biol 61:559–566, 1997. 37. Xia P, Inoguchi T, Kern T, Engerman RL, Oates PJ, King GL: Characterization of the mechanism for the chronic activation of diacylglycerol-protein kinase C pathway in diabetes and hyperglycemia. Diabetes 43:1122–1129, 1994. 38. Keogh RJ, Dunlop ME, Larkins RG: Effect of inhibition of aldose reductase on glucose flux, diacylglycerol for-
41.
42.
mation, protein kinase C, and phospholipase A2 activation. Metabolism 46:41–47, 1997. Kador PF, Takahashi Y, Sato S, Wyman M: Amelioration of diabetes-like retinal changes in galactose-fed dogs. Prevent Med 23:1925–1991, 1994. Neuenschwander H, Takahashi Y, Kador PF: Dosedependent reduction of retinal vessel changes associated with diabetic retinopathy in galactose-fed dogs by the aldose reductase inhibitor M79175. J Ocul Pharmacol Ther 13:517–528, 1997. Sato S, Takahashi Y, Wyman M, Kador PF: Progression of sugar cataract in the dog. Invest Ophthalmol Vis Sci 32:1925–1931, 1991. Sato S, Mori K, Wyman M, Kador PF: Dose-dependent prevention of sugar cataracts in galactose-fed dogs by the aldose reductase inhibitor M79175. Exp Eye Res 66:217–222, 1998.
ABSTRACT Dogs fed a diet containing 30% galactose develop diabetes-like retinal capillary changes. As retinal capillary occlusion is commonly observed in diabetic retinopathy, neutrophil apoptosis and the interaction of neutrophils with retinal capillary endothelial cells were investigated. Neutrophils were isolated with Ficoll-Hypaque centrifugation from dogs fed a 30% galactose diet and dogs fed a normal, control diet containing 30% non-nutrient filler. Apoptosis of neutrophils was microscopically examined after incubation at 378C for 3 hours with either 100 U/mL tumor necrosis factor a (TNF-a), 2 mg/mL cycloheximide or 50 ng/mL phorbol 12-myristate 13-acetate (PMA). Neutrophil adhesion to dog retinal capillary endothelial cells was examined by counting the cells attached to the surface of endothelial cells after the incubation in the presence of either 100 U/mL TNF-a or 5 mg/mL lipopolysaccharides (LPS) at 378C for 3 hours. With all three stimulants TNF-a,
cycloheximide and PMA, the rate of apoptosis was significantly lower for neutrophils isolated from galactose-fed dogs compared to control dogs fed a normal diet. Preincubation of neutrophils from control dogs in medium containing 30% galactose for 3 hours did not affect the rate of apoptosis. Neutrophil adhesion to retinal capillary endothelial cells induced by incubation in the presence of either 100 U/mL TNF-a or 5 mg/ml LPS was significantly higher with neutrophils isolated from galactose-fed dogs than those from control dogs. The data indicate that long-term galactose feeding is essential with development of various neutrophil dysfunctions. These neutrophil changes may contribute to the development of retinal microangiopathy associated with diabetes and galactosemia. (Journal of Diabetes and Its Complications 1313; 3: 151–158, 1999.) 1999 Elsevier Science Inc.
INTRODUCTION Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, Maryland, U.S.A. Reprint requests to be sent to: Dr. Sanai Sato, LOT, NEI, NIH, Bldg 10, Rm 10B09, 10 Center Drive, MCS 1850, Bethesda, MD 208921850, U.S.A. Journal of Diabetes and Its Complications 1999; 13:151–158 1999 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
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apillary occlusions constitute a characteristic pathologic feature of diabetic retinopathy. The formation of nonperfusion and/or avascular areas resulting from capillary occlusions eventually initiate clinically apparent retinal 1056-8727/99/$–see front matter PII S1056-8727(99)00040-9
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capillary changes, which include microaneurysms, intraretinal microvascular abnormality (IRMA), and advanced stages of neovascularization. In the development of microangiopathies, leukocytes have been suggested to play an important role. Both neutrophils and monocytes naturally tend to adhere to the vascular endothelium.1,2 Because these cells release various proteases and oxygen-derived free radicals,3 the activation of monocytes and/or neutrophils causes the endothelial cell injury that eventually initiates the occlusion of capillaries. It is also well-established that, in some extreme conditions, such as severe ischemia or shock, capillary occlusions by leukocytes commonly occur in various organs.4–6 There are a number of reports that leukocytes may contribute in the development of diabetic retinopathy. Diabetic leukocytes are less deformative7,8 and tend to plug in microcirculation.9,10 Moreover, the interaction between leukocytes and endothelial cells appears to increase in diabetes and/or hyperglycemia.11,12 The levels of soluble leukocyte adhesion molecules are elevated in patients with diabetic retinopathy.13 Diabetic neutrophils not only increase their interaction with endothelial cells but also increase superoxide production.14,15 it has also been reported that bovine retinal capillary endothelial cells when exposed to high glucose have been increased adhesion to neutrophils.16 The expression of adhesion molecules is also higher in diabetic human retina and choroid.17 Capillary occlusions and endothelial cells damages by leukocytes have also been observed in experimental diabetic rats.18 Using acridine orange digital fluorography, it has been observed that leukocyte entrapment in retinal microcirculation is significantly increased in vivo in diabetic rats.19 It is known that diabetic dogs develop early retinal changes that are similar to human diabetic retinopathy.20–22 Dogs fed a diet containing 30% galactose also develop similar retinal capillary changes with the selective loss of pericytes as the initial lesion.23–25 This galactose-fed dog model even develops the more advanced stages of pre-proliferative and proliferative retinopathy.26,27 Previously, we have reported that galactosefed dogs develop both neutrophil and lymphocyte dysfunctions similar to those observed with human diabetics. These include decreased lymphocyte blastformation by lectins28 and impaired induction of NADPH oxidase, a key enzyme for superoxide production.29 Here, we report additional evidences of neutrophil dysfunctions in galactose-fed dogs. The induction of neutrophil apoptosis by tumor necrosis factor a (TNF-a), cycloheximide, and protein kinase C activation is significantly lower in galactose-fed dogs, while at the same time, the interaction of neutrophils with retinal capillary endothelial cells is significantly increased.
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FIGURE 1 Microscopic appearances of normal dog neutrophils (A) and apoptotic cells induced by the incubation with 100 U/mL of TNF-a at 378C for 3 h (B). Note the nuclear fragmentation and condensation of apoptotic neutrophils. May-Giemsa stain 3400.
METHODS Reagents and Media. All chemicals utilized were of reagent grade. RPMI 1640 culture medium and fetal calf serum (FCS) were purchased from GibcoBRL Life Technologies, Inc. (Grand Island, NY) and Biofluids, Inc. (Rockville, MD), respectively. Recombinant human tumor necrosis factor a (TNF-a) was purchased from Genzyme Diagnostics (Cambridge, MA). Cycloheximide, phorbol 12-myristate 13-acetate (PMA), 1(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and lipoplysaccharides (LPS) were purchased from Sigma Chemical Co. (St. Louis, MO). Ficoll-Hypaque (specific gravity 1.0777) was a product of Pharmacia Biotech, Inc. (Uppsala, Sweden). Galactose-Fed Dogs. Thirty-six 9-year-old male beagles, obtained from Marshall Farms USA, Inc. (North Rose, NY), were randomly divided into two groups. Dogs were individually housed in 0.9 3 2.7 m runs and fed once a day a diet consisting 450 g of standard dog chow containing either 30% galactose or 30% nonnutrient filler as previously described.24,25 Diets were prepared by Bioserve (Frenchtown, NJ). All dogs were monitored through quarterly ocular examinations and blood chemistry evaluations. All experiments in this study were conducted with dogs placed on galactose diet for 3 years. Isolation of Neutrophils. Blood was drawn in early morning prior to feeding. The heparinized venous blood was mixed with an equal volume of 3% dextran, and, after the red blood cells had settled down in the bottom of the tubes, the neutrophils were collected from the buffy-coat by the centrifugation at 400 3 g for 30 min with Ficoll-Hypaque. Contaminating erythrocytes were removed by hypotonic shock with 0.2% sodium chloride. Neutrophil purity of all final preparations, determined by differential counting, were greater than 95%. Examination of Apoptosis. Isolated dog neutrophils were suspended in RPMI medium containing 100
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U/mL TNF-a, 2 mg/mL cycloheximide or 50 ng/mL PMA and incubated at 378C for 3 h. After incubation, the cells were collected by the centrifugation at 400 3 g for 5 min and stained with May-Giemsa. Apoptotic cell counts were based on their microscopic morphological appearance (Figure 1). A series of experiments were conducted with neutrophils isolated from randomly selected five dogs. The results were validated by repeating the same experiments at least three times with another randomly selected five dogs. Statistical significance (P , 0.05) was determined by either the Wilcoxon test or Student’s t test. Agarose Gel Electrophoresis of DNA. Neutrophils were suspended in 10 mM Tris-HCl buffer, pH 8.0 containing 1 mM EDTA. The cells were lysed by incubation for 2 h at 558C in 10 mM Tris-HCl buffer, pH 8.0 containing 0.1 M EDTA, 20 mg/mL RNase, 100 mg/mL proteinase K and 0.5% (W/V) sodium dodecyl sulfate (SDS). DNA was extracted with phenol and precipitated with 70% ethanol. Electrophoresis was conducted on 0.8% agarose gels containing 0.5 mg/mL ethidium bromide at 15 V for 12 h. DNA size markers utilized include a 100 pb molecular ruler and a 1 kb DNA ladder (Bio-Lad Laboratories, Hercules, CA). Preparation of Retinal Capillary Endothelial Cells. Retinal capillary endothelial cells were cultured from normal beagle dog eyes. The neural retina, isolated under the dissecting microscope, was minced with two razor blades, digested in PBS containing 0.1% collagenase at 378C for 20 min, and then passed through two stainless sieves (mesh 150 and mesh 50). The retainments from the second sieve (mesh 50) were collected, washed with PBS, resuspended in DMEM supplemented with 10% dog serum and cultured in fibronectin-coated plastic plates at 378C under a 5% carbon dioxide atmosphere. The identity of the cultured endothelial cells was confirmed by their typical morphological appearance and by the uptake of acetylated-low density lipoprotein.30 Interaction of Neutrophils to Retinal Capillary Endothelial Cells. At the fifth passage, dog retinal capillary endothelial cells were transferred to 24-well culture plates. When the cells reached confluence, the medium was replaced with a dog neutrophil suspension (5 3 106/mL) in the RPMI medium containing either 100 U/mL TNF-a or 5 mg/mL LPS. After the incubation at 378C for 3 hours, the plates were washed with PBS and the attached cells were examined with May-Giemsa staining. The adhesion of neutrophils was expressed as the average of cell numbers attached to the surface of plate of randomly selected five microscopic fields.
FIGURE 2 Time course of TNF-a induced apoptosis of dog neutrophils. Neutrophils isolated from non-galactosemic control (open circles) and 30% galactose-fed dogs (gray circles) were incubated in RPMI medium containing 100 U/mL of TNF-a up to 6 hours. Specific apoptosis induced by TNF-a was expressed as the percentage of apoptotic cells corrected with the naturally occurred apoptosis during the incubation in the absence of TNF-a. Mean 6 S.D. (n 5 5).
RESULTS Dogs receiving diets containing either 30% galactose or 30% non-nutrient filler for up to 3 years revealed no abnormalities in their periodic evaluations of blood chemistries. Similarly, no significant difference in blood counts was observed between the galactose-fed and non-nutrient filler-fed control dogs. When incubated in medium containing 100 U/mL of TNF-a, dog neutrophils underwent apoptosis. The apoptotic cells were easily distinguished by their morphological characteristics such an nuclear fragmentation and condensation (Figure 1). The number of apoptotic cells increased with incubation time (Figure 2). Although TNF-a induced apoptosis of neutrophils isolated from both 30% galactose-fed and 30% non-nutrient filler-fed control dogs, the percentage of apoptotic cells was significantly lower with neutrophils isolated from galactose-fed dogs. Because difference between control and galactose-fed dogs was most clearly observed after 3 h of incubation, subsequent studies were all conducted with 3 h incubations. Although the nonspecific protein synthesis inhibitor cycloheximide is often utilized to protect cells from apoptosis, higher concentrations of cycloheximide induced apoptosis of neutrophils isolated from both control and galactose-fed dogs (Figure 3A). As apoptosis by TNF-a, the rate of apoptosis induced by cycloheximide was significantly lower with neutrophils isolated from galactose-fed dogs than control dogs. Combining
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FIGURE 4 The effect of galactose on dog neutrophil apoptosis. Neutrophils isolated from non-galactosemic control dogs were preincubated in either the absence (white bars) or presence of 30 mM galactose at 378C for 3 h before apoptosis was induced by the incubation with a mixture of 100 U/mL of TNF-a and 2 mg/mL of cycloheximide. Mean 6 standard deviation (n 5 5).
To examine the possibility that galactose directly interferes with neutrophil apoptosis in galactose-fed dogs, neutrophils isolated from non-galactosemic, fiber-fed control dogs were preincubated for 3 hours in medium containing 30 mM D-galactose prior to the
FIGURE 3 Apoptosis of dog neutrophils induced by TNF-a, cycloheximide and a PKC activator phorbol 12-myristate 13-acetate (PMA). Neutrophils isolated from non-galactosemic control (white bars) and galactose-fed dogs (gray bars) were incubation at 378C for 3 h in the medium (A) containing either 100 U/mL of TNF-a, 2 mg/mL of cycloheximide, or both 100 U/mL of TNF-a and 2 mg/ mL of cycloheximide and the medium (B) containing either 1-(5isoquinolinesulphonyl)-2-methylpiperazine (H7) (100 mM), PMA (50 ng/ml), or both H7 (100 mM) and PMA (50 ng/mL). Mean 6 S.D. (n 5 5).
the two stimulants TNF-a and cycloheximide remarkably enhanced the rate of apoptosis. However, the rate of apoptosis remained significantly lower with neutrophils from galactose-fed dogs compared to non-galactosemic control dogs. The addition of a PKC activator, phorbol 12-myristate 13-acetate (PMA), into the incubation medium also increased the rate of apoptosis of dog neutrophils (Figure 3B). Apoptosis induced by PMA was almost completely prevented by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), confirming that PMA-induced apoptosis is initiated through the activation of PKC. The percentage of apoptotic cells induced by PMA was again significantly lower with neutrophils from galactose-fed dogs than non-galactosemic control dogs.
FIGURE 5 Agarose gel electrophoresis of DNA isolated from non-galactosemic control dog neutrophils incubated at 378C for 3 h in either the absence (lane 1) or presence of 100 U/mL of TNF-a (lane 2) and a mixture of 100 U/mL of TNF-a and 2 mg/ mL of cycloheximide (lane 3). Each lane contains 5 mg of DNA. Electrophoresis was performed on 0.8% agarose gel containing 0.5 mg/mL ethidium bromide at 15 volts for 12 h. Lane M1 and M2 represented 1 kb DNA ladder and 100 bp molecular ruler, respectively.
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induction of apoptosis by a mixture of TNF-a and cycloheximide. This preincubation did not result in a significant decrease in the induction of apoptosis by the mixture of TNF-a and cycloheximide (Figure 4). DNA fragmentation, referred to as a ladder formation, is characteristically observed in apoptotic cells. To verify the presence of DNA fragmentation in apoptotic neutrophils, DNAs isolated from neutrophils of nongalactosemic control dogs incubated with TNF-a and a mixture of TNF-a and cycloheximide were analyzed by agarose gel electrophoresis (Figure 5). No significant fragmentation was observed in DNA isolated from dog neutrophils incubated without stimulants and the isolated DNA remained at the origin of the agarose gel. In contrast, neutrophils incubated with TNF-a and a mixture of both TNF-a and cycloheximide displayed obvious DNA fragmentation. DNA fragmentation was especially severe in neutrophils incubated with a mixture of TNF-a and cycloheximide and DNA was essentially absent at the origin of the gel. Because it is known that the stimulation by cytokines, such as TNF-a, not only induce apoptosis but also increase the adhesion of neutrophils, the interaction of neutrophils to dog retinal capillary endothelial cells was also examined by incubating isolated neutrophils in culture plates containing confluent dog retinal capillary endothelial cell (Figure 6). In the absence of stimulants in the incubation medium, only a slight attachment of neutrophils to the surface of endothelial cells could be observed. The average numbers of cells attached were 4.7 6 3.1 and 6.6 6 3.9 cells/microscopic field (n 5 5) with neutrophils isolated from non-galactosemic control dogs and galactose-fed dogs, respectively. Addition of TNF-a to the incubation medium resulted in a significant increase in the number of neutrophils attached. Although TNF-a increased the adhesion of neutrophils isolated
FIGURE 6 Adhesion of neutrophils isolated from control (B) and galactose-fed dogs (C) to the surface of retinal capillary endothelial cells. Neutrophils (5 3 106 cells/mL) were incubated in the culture plate covered with dog retinal capillary endothelial cells in the presence of 100 U/mL of TNF-a at 378C for 3 h. Neutrophils are observed as small round cells (indicated by arrows). Note some area (indicated by circle with broken line in picture C) was almost completely covered with neutrophils. A; control endothelial cells incubated without neutrophils. Stained with May-Giemsa. The graph below represents the average numbers of neutrophils attached to the surface of plate of randomly selected five microscopic fields after the incubation in the presence of either 100 U/mL of TNF-a or 5 mg/mL of lipopolysaccharides (LPS). White and gray bars represent neutrophils isolated from control and galactose-fed dogs, respectively. For this calculation, the area where neutrophils are attached with extremely high density (see picture C) was excluded. Mean 6 S.D. (n 5 5).
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from both galactose-fed and control dogs, a significantly higher attachment (151.6 6 42.2 cells/field, n 5 5) of neutrophils from galactose-fed dogs was observed compared to those (64.8 6 20.3 cells/field, n 5 5) of control dogs. The same increase of neutrophil adhesion was observed with an another stimulant LPS. The adhesion of neutrophils isolated from galactose-fed dogs was 120.3 6 41.4 cells/field (n 5 5). This was significantly higher than that (67.2 6 19.5 cells/field, n 5 5) of non-galactosemic control dogs. DISCUSSION We have previously reported that neutrophils isolated from dogs fed a 30% galactose diet for up to 3 years demonstrate a significantly impaired induction of NADPH oxidase by zymosan despite the normal appearance of routine blood chemistry profiles.29 The present study provides additional evidence that longterm galactose-fed dogs develop fundamental neutrophil dysfunctions. Neutrophil apoptosis induced by various stimulants was significantly suppressed in the long-term galactose-fed dogs. Moreover, when activated by TNF-a and LPS, galactosemic neutrophils strongly interacted with dog retinal capillary endothelial cells. Clinically, it is well known that diabetic individuals have a higher incidence of bacterial infections compared to nondiabetic individuals. Persistent inflammation and delayed wound healing are additional problems for diabetic patients. In inflammatory response, vast numbers of leukocytes migrate into the perturbed tissues. Obviously, the efficient clearance of aging neutrophils is crucial for the successful resolution of inflammation and the promotion of tissue repair. Failure of aging neutrophils to be quickly and completely cleared results in further unwanted tissue damage and results in a prolongation of inflammation.31,32 To date, no report has been published on delayed neutrophil clearance in diabetic patients. However, it has been demonstrated that apoptotic cells are increased throughout the process of wound healing of diabetic mice.33 Since the major mechanism for clearance of aging neutrophils has been established to be apoptosis followed by phagocytosis by macrophages,31,32 the decreased response for neutrophils to TNF-a and/or many other cytokines may contribute to the delayed resolution of inflammation associated with diabetes or galactosemia. As galactosemic neutrophils failed to quickly respond to TNF-a stimulus, one may question whether the incidence of infections and/or inflammations is surely increased in galactose-fed dogs. We have observed that the incidence of thyroiditis, similar to that observed in human diabetic patients, is increased in long-term galactose-fed dogs.34 This suggests that neutrophil changes may be linked to increased incidence
of infections or prolonged inflammations. However, because the study was conducted with only a limited number of dogs and no overt clinical increase in infection of galactose-fed dogs was observed, further studies are needed to determine the relevance between neutrophil dysfunctions and the higher incidence of infections and/or inflammations in galactose-fed dogs. Capillary occlusion and the resulting tissue hypoxia/anoxia are underlying pathologic lesions associated with diabetic microangiopathies. In diabetes, the interaction of leukocytes to capillary endothelial cells is increased.11–13 Because activated neutrophils can cause endothelial cell injury, the increased adhesion of neutrophils can initiate capillary occlusion.35 Indeed, retinal capillary occlusions and endothelial cell damages by leukocytes has been reported to occur in experimental diabetic rats.18 Moreover, a recent study has also clearly demonstrated that leukocyte entrapment in retinal microcirculation is significantly increased in diabetic rats.19 The present results are consistent with these observations. Neutrophils from galactose-fed dogs more strongly interact with retinal capillary endothelial cells compared to those from non-galactosemic control dogs. Because these galactose-fed dogs develop diabetes-like retinal capillary changes, the observed neutrophil changes that increase the interaction with retinal capillary endothelial cells may be important in developing and/or accelerating the process of retinal microangiopathy. Interestingly, apparent differences in neutrophil adhesion to retinal capillary endothelial cells were observed only when neutrophils were activated. In diabetes, infections occur more frequently and their associated inflammation tends to be prolonged. If the activated neutrophils interact more strongly in diabetes, repeated infections with prolonged inflammation can be a risk factor that enhances the process of microangiopathy. TNF-a is an inflammatory cytokine released by macrophages that requires specific cell surface receptors to initiate signals inducing apoptosis.36 Two other stimulants PMA and cycloheximide initiate apoptosis through different pathways. PMA directly activates PKC while cycloheximide is a non-specific protein synthesis inhibitor. Since the responses of neutrophils to all three different stimulants were similarly affected in galactose-fed dogs, a single event such as the different level of PKC37,38 can not explain this observation. It has been reported that aldose reductase inhibitors delay retinal capillary changes39,40 and prevent cataract formation in galactose-fed dogs in a dose-dependent manner.41,42 We have also reported that galactitol accumulation occurs in neutrophils of galactose-fed dogs despite the extremely low level of aldose reductase in dog neutrophils.29 However, although polyol accumulation has been shown to be a common biochemical mechanism that initiates the onset of these complications, the
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importance of polyol accumulation in the development of neutrophil dysfunctions has not been established. REFERENCES 1. Atherton A, Born GV: Quantitative investigations of the adhesiveness of circulating polymorphonuclear leukocytes to blood vessel walls. J Physiol 222:447–474, 1972. 2. Schmid-Schonbein GW, Usami S, Skalak R, Chien S: The interaction of leukocytes in capillary and postcapillary vessels. Microvasc Res 19:45–70, 1980. 3. Fantone JC, Ward PA: Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Am J Physiol 107:397–418, 1982. 4. Bagge U, Amundson B, Lauritzen C: White blood cell deformability and plugging of skeletal muscle capillaries in hemorrhagic shock. Acta Physiol Scand 108:159– 163, 1980. 5. Barroso-Aranda J, Schmid-Schonbein GW, Zweifach BW, Engler RL: Granulocytes and no-reflow phenomenon in irreversible hemorrhagic shock. Circ Res 63:437– 447, 1988. 6. Kaminski PM, Proctor KG: Attenuation of no-reflow phenomenon, neutrophil activation, and reperfusion injury in intestinal microcirculation by topical adenosine. Circ Res 65:426–435, 1989. 7. Pecsvarady Z, Fisher TC, Darwin CH, Fabok A, Maqueda TS, Saad MF, Meiselman HJ: Decreased polymorphonuclear leukocyte deformability in NIDDM. Diabetes Care 17:57–63, 1994. 8. Braun RD, Fisher TC, Meiselman HJ, Hatchell DL: Decreased deformability of polymorphonuclear leukocytes in diabetic cats. Microcirculation 3:271–278, 1996. 9. Harris AG, Skalak TC, Hatchell DL: Leukocyte-capillary plugging and network resistance are increased in skeletal muscle of rats with streptozotocin-induced hyperglycemia. Int J Microcirc Clin Exp 14:159–166, 1994. 10. Miyamoato K, Ogura Y, Kenmochi S, Honda Y: Role of leukocytes in diabetic microcirculatory disturbances. Microvasc Res 54:43–48, 1997. 11. Kim JA, Berliner JA, Natarajan RD, Nadler JL: Evidence that glucose increases monocyte binding to human aortic endothelial cells. Diabetes 43:1103–1107, 1994.
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cells exposed to hyperosmolarity. Exp Eye Res 58:641– 647, 1994. 17. McLeod DS, Lefer DJ, Merges C, Lutty GA: Enhanced expression of intracellular adhesion molecule-1 and Pselectin in the diabetic human retina and choroid. Am J Pathol 147:642–653, 1995. 18. Schroder S, Palinski W, Schmid-Schonbein GW: Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy. Am J Pathol 139:81–100, 1991. 19. Miyamoto K, Hiroshiba N, Tsujikawa A, Ogura Y: In vitro demonstration of increased leukocyte entrapment in retinal microcirculation of diabetic rats. Invest Ophthalmol Vis Sci 39:2190–2194, 1998. 20. Patz A, Maumenee AE: Studies on diabetic retinopathy. I. Retinopathy in a dog with spontaneous diabetes mellitus. Am J Ophthalmol 54:532–541, 1962. 21. Hausler HR, Sibay TM, Campbell J: Retinopathy in a dog following diabetes induced by growth hormone. Diabetes 13:122–126, 1964. 22. Engerman RL, Kramer JW: Dogs with induced or spontaneous diabetes as models for the study of human diabetes mellitus. Diabetes 31 (Suppl 1 Pt 2):26–29, 1982. 23. Engerman RL, Kern TS: Experimental galactosemia produces diabetic-like retinopathy in dogs. Diabetes 33:97– 100, 1984. 24. Kador PF, Akagi Y, Terubayashi H, Wyman M, Kinoshita JH: Prevention of pericyte ghost formation in retinal capillaries of galactose-fed dogs by aldose reductase inhibitors. Arch Ophthalmol 106:1099–1102, 1988. 25. Kador PF, Akagi Y, Takahashi Y, Ibeke H, Wyman M, Kinoshita JH: Prevention of retinal vessel changes associated with diabetic retinopathy in galactose-fed dogs by aldose reductase inhibitors. Arch Ophthalmol 108: 1301–1309, 1990. 26. Takahashi Y, Wyman M, Frederick F III, Kador PF: Diabeteslike proliferative retinal changes in galactose-fed dogs. Arch Ophthalmol 110:1295–1302, 1992. 27. Kador PF, Takahashi Y, Myman M, Ferris F III: Diabeteslike proliferative retinal changes in galactose-fed dogs. Arch Ophthalmol 113:352–354, 1995. 28. Fukase S, Sato S, Secchi EF, Gery I, Kador PF: Decreased lymphocyte blastformation in long term galactose-fed dogs. Int J Diabetes 3:91–97, 1995.
12. Wierusz-Wysocka B, Wykretowicz A, Byks H, Sadurska K, Wysocki H: Polymorphonuclear neutrophils adherence, superoxide anion (O2-) production and HBA1 level in diabetic patients. Diabetes Res Clin Pract 21:109– 114, 1993.
29. Fukase S, Sato S, Mori K, Secchi EF, Kador PF: Polyol pathway and NADPH-dependent reductases in dog leukocytes. J Diabet Complications 10:304–313, 1996.
13. Olsen JA, Whitelaw CM, McHardy KC, Pearson DWM, Forrester JV: Soluble leukocyte adhesion molecules in diabetic retinopathy stimulate retinal capillary endothelial cell migration. Diabetologia 40:1166–1171, 1997.
30. Voyta JC, Via DP, Butterfield CE, Zetter BR: Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 99:2034–2040, 1984.
14. Shah SV, Wallin JD, Eilen SD: Chemiluminescence and superoxide anion production by leukocytes from diabetic patients. J Clin Endocrinol Metab 57:402–409, 1983.
31. Savill J, Haslett C: Granulocyte clearance by apoptosis in the resolution of inflammation. Semin Cell Biol 6:385– 393, 1995.
15. Freedman SF, Hatchell DL: Enhanced superoxide radical production by stimulated polymorphonuclear leukocytes in a cat model of diabetes. Exp Eye Res 55:767– 773, 1992.
32. Savill J: Apoptosis in resolution of inflammation. J Leukoc Biol 61:375–380, 1997.
16. Bullard SR, Hatchell DL, Cohen HJ, Rao KMK: Increased adhesion of neutrophils to retinal vascular endothelial
33. Darby IA, Bisucci T, Hewitson TD, MacLellan DG: Apoptosis is increased in a model of diabetes-impaired wound healing in genetically diabetic mice. Int J Biochem Cell Biol 29:191–200, 1997.
158
OHTA ET AL.
J Diab Comp 1999; 13:151–158
34. Takahashi Y, Kador PF: Lymphocytic thyroiditis in galactose-fed dogs. US-Japan Aldose Reductase Workshop, Feb. 12–16 Kona, Hawaii, p. 78.
39.
35. Schmid-Schonbein GW: The damaging potential of leukocyte activation in the microcirculation. Angiology 44:45–46, 1993.
40.
36. Darnay BG, Aggarwal BB: Early events in TNF signalling: A story of associations and dissociations. J Leukoc Biol 61:559–566, 1997. 37. Xia P, Inoguchi T, Kern T, Engerman RL, Oates PJ, King GL: Characterization of the mechanism for the chronic activation of diacylglycerol-protein kinase C pathway in diabetes and hyperglycemia. Diabetes 43:1122–1129, 1994. 38. Keogh RJ, Dunlop ME, Larkins RG: Effect of inhibition of aldose reductase on glucose flux, diacylglycerol for-
41.
42.
mation, protein kinase C, and phospholipase A2 activation. Metabolism 46:41–47, 1997. Kador PF, Takahashi Y, Sato S, Wyman M: Amelioration of diabetes-like retinal changes in galactose-fed dogs. Prevent Med 23:1925–1991, 1994. Neuenschwander H, Takahashi Y, Kador PF: Dosedependent reduction of retinal vessel changes associated with diabetic retinopathy in galactose-fed dogs by the aldose reductase inhibitor M79175. J Ocul Pharmacol Ther 13:517–528, 1997. Sato S, Takahashi Y, Wyman M, Kador PF: Progression of sugar cataract in the dog. Invest Ophthalmol Vis Sci 32:1925–1931, 1991. Sato S, Mori K, Wyman M, Kador PF: Dose-dependent prevention of sugar cataracts in galactose-fed dogs by the aldose reductase inhibitor M79175. Exp Eye Res 66:217–222, 1998.