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The functions of circulatory polymorphonuclear leukocytes in diabetic patients with and without diabetic triopathy
Life Sciences, Vol. 66, No. 19, pp. 1861-1870,2ooO Copyright 0 2000 Elsevicr Science Inc. Printed in the USA. All ri&s mservcd 0024-3205/001s_see front matter
PIISOO24-3205(00)00509-9
THE FUNCTIONS OF CIRCULATORY POLYMORPHONUCLEAR LEUKOCYTES IN DIABETIC PATIENTS WITH AND WITHOUT DIABETIC TRIOPATHY Masami Ohmori, Kazuhiro Harada, Yasuhiko Kitoh, Syou-ichiroh Nagasaka, Toshikazu Saito and Akio Fujimura Departments of Clinical Pharmacology and Medicine (Division of Endocrinology), Jichi Medical School, Tochigi 329-0498, Japan. (Receivedin finalformDecember9, 1999) Summary We determined circulatory polymorphonuclear leukocytes (PMN) functions of superoxide anion production, adhesion and aggregation in 38 type 2 diabetic patients with and without diabetic triopathy. Tumor necrosis factor (TNF)-a-stimulated superoxide production and N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated aggregation in diabetic patients with triopathy were significantly greater than those in diabetics without triopathy. The more diabetic complications existed, the more TNF-a-stimulated superoxide was produced by PMN. These results suggest that the activated PMN contributes to a progression of diabetic triopathy in type 2 diabetic patients. leukocyte., non-insulin-dependent diabetes mellitus, triopathy, superoxide, KeyWords: polymorphonuclear adhesion, aggregation
Diabetic triopathy (retinopathy, nephropathy and neuropathy) is the serious The pathogenesis is not fully complication in patients with diabetes mellitus. understood, but several mechanisms have been proposed. For example, dysfunction of platelet and vascular wall induced by glucose toxicity, glycation, advanced glycation end products (AGES), excessive reactive free radicals and decreased antioxidant properties are reported to cause diabetes-specific complications in these patients (1-G). Especially, reactive free radicals [superoxide anion (OZ.), hydrogen peroxide, hydroxyl radical] produced by polymorphonuclear leukocytes (PMN) might damage not only DNA, but membrane lipid bilayer and other essential cell components (7), which in turn cause Address for correspondence: Akio Fujimura, M.D., Department of Clinical Pharmacology, Jichi Medical School, 33 1 l- 1 Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan, Telephone: +81 285 58 7388, Telefax: +81 285 44 7562: E-mail: [email protected]
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In type 1 diabetes mellitus, 0, vascular complications in the diabetic patients. production by PMN in the patients with diabetic retinopathy is greater than that in the patients without the complication (8). This finding indicates that the enhanced 0,. production by circulating PMN contributes to a pathogenesis of diabetic retinopathy in the type 1 diabetic patients. However it is still unclear whether PMN is involved in the mechanism of diabetic organ damage in the type 2 diabetic patients. To address this issue, this study was undertaken to determine PMN capacities of 02production, adhesion and aggregation in type .2 diabetic patients with and without diabetic triopathy.
Materials
and Methods
Patients Thirty-eight type 2 diabetic patients with and without triopathy and seventeen nondiabetic control subjects were studied (Table 1.) There were no significant differences in sex ratio, age and body mass index between the type 2 diabetics and control groups. The Hb,,, was significantly higher, and the fasting blood sugar (FBS) tended to be higher in the type 2 diabetics than in the control group (Table 1.). While insulin, glibenclamide, gliclazide (a sulphonylurea) and epalrestat (an aldose reductase inhibitor) were given to the type 2 diabetic patients, other medications were similar between the two groups (data not shown). Patients with nephrotic syndrome and infection, and taking medications such as anti-inflammatory and anti-platelet were excluded. Diabetic nephropathy was defined as albuminuriaZ 20 mglday [n=172 (84 male, 88 female), SRL, Japan], which is similar to the previous criterion (albuminuria 2 21.6 mglday) (9). Diabetic neuropathy was defined as diminished nerve conduction Hbnlc was velocity, reduced deep tendon reflex and reduced feeling of vibration. measured by latex agglutination (LA) method, FBS by hexokinaseiglucose-6-phosphate dehydrogenase (Glu-HD) method, serum lipid peroxides by thiobarbituric acid (TBA) method and granulocyte elastase by enzyme immunoassay (EIA). Triopathy-score was defined as follows; 0 (no triopathy, n=lO), 1 (one and two triopathy, n=19): 2 (all triopathy, n=9>. Four out of 19 patients in the score-l group and 4 out of 9 patients in the score-2 group received epalrestat every day (the difference between the groups: ~~0.45 by x 2 test). All patients gave their informed consent to the aim and methods of this study. PMN prepara tion Blood sample was taken from forearm vein in the morning after an overnight fasting and heparinized using heparin 100 U/ml. Mixed leukocyte suspension was obtained after sedimentation in the presence of 1 % dextran in saline and gradient centrifugation with Lymphoprep (Nycomed Pharma AS, Oslo, Norway). Purified PMN suspension (cell viability> 95 %) was obtained by hypotonic lysis at 4 “C and washing twice in phosphate buffer. PMN was resuspended in Hanks’ balanced salt solution (Nissui Pharmaceutical Co., Tokyo, Japan) with 0.1 % human serum albumin (HSA) at the concentration of 6x 106cells/ml. Assay for superoxide anion production Oz. production by PMN was determined by the superoxide dismutase (SOD)-inhibitable
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cytochrome-C reduction method with a minor modification (10: 11). The following materials were placed on FBS-coated wells at a total volume of 200 ml/well; 1) PMN suspension contained 3x105 PMN, 2) tumor necrosis factor-u (TNF-tx: 100 U/ml, Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) or its control buffer, 3) SOD (200 U/ml: Sigma Chemical Co., St. Louis, USA) or its vehicle 4) cytochrome-C (12 mgiml. Sigma Chemical Co.: St. Louis, USA). After incubation for GO minutes at 37 ‘Y’. 0: production was determined by measurin, v the absorbance at 540 and 550 nm in a spectrophotometer (Beckman DU-640, Fullerton: CA). The assay was performed in duplicate. Intra- and inter-variability are 3.2 and 6.6 %: respectively.
Table
1. Characteristics and type
in control
2 diabetic
subjects
patients Mean rfrSE
Parameter
Sex (Men/Women)
Age (year)
Control
Type 2 diabetes
(n=17)
mellitus
16122
819 49.5-cl8.0
Body mass index
23.4 k3.2
HbAlc (%)
5.4kO.5
(n=38)
57.3k14.8 24.2k4.1 9.Ok2.0 p
FE%3(mg/dl)
93.5k11.2
137.8 k39.7
Assayfor PMN adherence Adhered PMN to FBS-coated wells was determined as protein content by Lowry method (12). Distilled Hz0 (0.1 ml) was added to the well. Three reagents (2 % sodium tartrate, 1 % &SO4 and 2 % NaGO, in 0.1 N NaOH) were mixed, and phenolreagent (Wako Pure Chemical Industries Ltd., Osaka, Japan) was diluted by same volume of HZ0 just before use. Fifteen minutes after, 1 ml of their mixture was placed to each well, and 0.1 ml of diluted phenol was added to each well. Thirty minutes later, protein content was determined by measuring the absorbance at 700 nm in a spectrophotometer. The assay was also performed in duplicate. Intra- and intervariability are 2.4 and 13.3 %, respectively. Assay for PMN aggregation
PMN aggregation was determined by an aggregometer (multichannel platelet aggregation analyzer, Mebanix Model PAT-2A: Tokyo, Japan) using PMN suspension which contained 1.2~10~ cells, and N-formyl-methionyl-leucyl-phenylalanine (FMLP, 10.; M, Sigma Chemical Co.: St. Louis, USA) or its control buffer at a total volume of 200 ml. Intra- and inter-variability are 2.5 and 3.1 %: respectively.
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Statistical analysis Results were expressed as the mean+SE. Data were analyzed by one-way ANOVA using Statview-J 4.02 (Abacus Concepts Inc., California, USA). A value of pcO.05 was regarded as significant.
Table 2. Biochemical subjects
profiles
and PMN functions
and type 2 diabetic
patients
Type 2 Parameter
Control
diabetes
(n=17)
mellitus (n=38)
Serum lipid peroxide (nmol/ml) Granulocyte
elastase
3.3f0.9
3.1 zkO.6
in control Mean + SE
Healthy* subjects (n=8) ( 3.5kO.5
) )
54.8f15.2
61 .l f22.3
( 63.3k9.3
UnsGmulated
0.28kO.05
0.25+0.03
(0.31 f0.07)
TNF-a-stimulated
1.26f0.20
1 .I
Unstimulated
3.66kO.40
4.8OkO.46
(3.35kO.47)
TNF-a-stimulated
10.53+0.82
12.04kO.66
(9.15fO.76)
0.10f0.03
(0.14+0.07)
5.og+o.i9
(4.36+0.50)
(kg/l) Superoxide
anion production
(nmol/3xlO~
Adhesion
cells/hr)
0+0.08
(1.21
f0.37)
(pg/well)
Aggregation
(%)
Unstimulated
0.09
kO.03
FMLP-stimulated
4.72kO.42
* The values in healthy subjects are shown for a comparison
purpose.
Results
No significant differences were observed in serum concentrations of lipid peroxides and granulocyte elastase, nor 02- production, adhesion and aggregation by PMN between the type 2 diabetic patients and control groups (Table 2.). Biochemical profiles of the type 2 diabetic patients with and without triopathy did not differ significantly (Table 3.). The FBS was significantly higher and tended to be higher in the patients with and without triopathy than in the control group. The Hb,,, was significantly higher in the type 2 diabetic patients with and without triopathy than in the control group. Serum lipid peroxide and granulocyte elastase of the patients with and without triopathy and control subjects did not differ.
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Unstimulated 02. production and aggregation of PMN of the patients with triopathy were similar to those in the group without triopathy (Table 4.) However, TNF-astimulated 02- production and FMLP-stimulated aggregation were significantly or tended to be greater in the patients with triopathy. Adhesion was similar among three groups [control and type 2 diabetics with and without triopathy (data not shown)]. TNF-a-stimulated Oz.production in the triopathy-score 0 group tended to be lower than that in the control group (p=O.O8) (Fig. 1). Such the difference decreased in the score 1 (p=O.36) and score 2 (p=O.41) groups. The 02. production in the triopathy-score 2 group was greater than that in the triopathy-score 0 and 1 groups. This parameter was There was a enhanced in a triopathy-score-dependent manner in the diabetic patients. significant correlation between the Oz- production and albuminuria (i-=0.39, n=38; ~~0.05). FMLP-stimulated aggregation was significantly higher in the patients with triopathy-score 2 than in other groups (control and type 2 diabetics with triopathy-score 0 and 1) (Fig. 2.). Adhesion was similar between the control and diabetic groups (Fig. 3.).
Table 3. Biochemical profiles in type 2 diabetic patients with and without triopathy tibm
FBS
Triopathy
(%)
(mddl)
Mean+=
Serum lipid
Granulocyte
peroxide
elastase
(nmol/mI)
(I.&l)
t (n=19)
140.4f37.5
9.OL1.7
3.2f0.8
66.7k25.0
- (n=19)
135.3f42.6
9.1i2.3
3.OkO.2
55.1k17.9
t (n=15)
148.2f48.4
9.0f2.3
3.2kO.8
60.7k26.1
- (n=23)
130.6k31.8
9.0f1.8
3.1f0.3
61.3f20.0
t (n=26)
142.7f41.8
8.9f2.0
3.1kO.7
63.9k24.4
- (n=12)
126.0f33.3
9.4k2.1
3.0f0.2
55.2k16.8
Retinopathy
Nephropathy
Neuropathy
Discussion
PMN is one of the major sources of oxygen free radicals, which are supposed to cause diabetic complication, together with other factors such as glucose itself and AGES (1, 2). Previous studies showed that PMN Oz. production in diabetic patients increased, decreased or did not change compared to that in the control subjects (8, 13, 14). In the
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present study, O,- production by PMN of the diabetic and control groups did not differ significantly although its production was enhanced in subje& with greater diabetic complications. Therefore, these diverse results might reside, in part; in the selection of diabetic patients. AEXJ EL-ASRAR et al. reported that PMN O?- production stimulated by phorbol myristate acetate (PMA) increased in the type 1 diabetic* patients with diabetic retinopathy (8). Protein kinase C (PKC) activity is also reported to increase in diabetic animals and is potentially involved in the development of diabetic triopathy (15). Undoubtedly, PMA direL%lystimulates PKC which induces a respiratory burst, and releases oxygen free radicals, elastase. cytokines and other metabolites (1G). However PMA is not a physiological stimulant. TNF-tr is a polypeptide cytokine and plays a role in inflammatory process (17). It exists as a membrane-bound precursor, which can be processed by a TNF-(r-converting enzyme.
As TNF-c~ is an endogenous factor, PMN functions stimulated
by this agent have
greater meaning in clinical situations. In this study, TNF-(x-stimulated Oy production by PMN increased in the type 2 diabetic patients with diabetic triopathy. Unidentified serum factor (s) in diabetic patients with retinopathy is also shown to enhance 0,. productions by normal PMN (18). In addition, it is recently proved that diabetic patients have significant defects of physiological antioxidant properties (urate. vitamin C; E and A, bilirubin) and decreased serum SOD activity (19. 20). Based on these data, we think that diabetic patients with triopathy are always under conditions of the excessive oxidative stress by hyperproduction and hypoconsumption of oxygen free radicals, which causes further organ damage. Some diabetic patients are at a high risk of d&eloping primary liver cancer: probably due to the excessive free radicals (2 1). On the contrary, other diabetics have more postoperative infections than non-diabetics. which might be induced by the reduced free radicals (22). As 0, production by PMN
Table 4. PMN functions
in type 2 diabetic patients with and without triopathy Mean+%
Superoxide anion production (nmoV3xl
Aggregation
OQells/hr)
(%)
Triopathy
t
(n=19)
UnsGmulated
TNF-a-stimulated
Unstimulated
0.27+0.05
1.23+0.11~
O.lli-0.04
FMLPstimulated
5.51f0.261
p
pco.05
Retinopathy
-I
- (n=19)
0.23f0.04
0.96kO.09
t
0.26+0.05
1.37+0.131
(n=15)
0.10f0.05
4.61k0.25
0.07+0.03
5.25kO.36
p405
Nephropathy - (n=23)
0.25kO.04
0.92+0.07 J
0.12+0.05
4.96kO.19
t (n=26)
0.26+0.04
1.24+0.091
0.12kO.04
5.17kO.24
0.07+0.05
4.92f0.30
pco.05
Neuropathy - (n=12)
0.24f0.05
0.76f0.06 -I
J
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increased in the type 2 diabetic patients with triopathy-score 2 and decreased in the patients with triopathy-score 0 and 1 in this study, we think that patients with diabetes mellitus are vulnerable to,bacterial infection or development of cancer which is depend on the status of free radical. Previous reports showed that adherence and aggregation of PMN in diabetic patients decreased or did not change compared to that in the control subjects (23-26). In this study, these functions of PMN did not differ significantly between the type 2 diabetic patients and control groups. However, PMN aggregation increased in the type 2 diabetic subgroup with triopathy-score 2. Therefore, we think that the elevated PMN aggregation also contributes to the deterioration of diabetic organ damage. Epalrestat inhibits neutrophil 0,. production stimulated by PMA in vitro (27). The drug might also inhibit ex vivo TNF-a-stimulated 0,. production, which, in turn, contributes to the difference between the control and diabetic groups. and between the score-0 and score-l (or 2) groups.
p=o.41 p=O.36
_:I*I-
-
0
A
B
C
D
unst imulat ed
A
B
C
-
D
TNF-a-st imulat ed
Fig. 1 Superoxide anion production by PMN with and without TNF-a stimulation in the control subjects and type 2 diabetic patients. A=control subjects (n=17); B: C and D=type 2 diabetic patients with triopathy-score 0 (n=lO), 1 (n=19) and 2 (n=S): respectively. mean? SE, * p
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7-l
3
6-
*
l
*
5’
In
4-
3-
2-
1 -
0ABCD
ABCD
FMLP-stimulated
unstimulated Fig. 2
PMN aggregation with and without FMLP-stimulation in the control subjects and type 2 diabetic patients. A=contol subjects (n=17); B, C and D=type 2 diabetic patients with triopathy-score 0 (n=lO), 1 (n=19) and 2 (n=9), respectively. meant SE: *pcO.O5 compared to D
+
w r
L+ t
A
B
CD
unstimulated
-
-
ABu
D
-
TNF-a-stimulated Fig. 3
PMN adherence with and without TNF-a-stimulation in the control subjects and type 2 diabetic patients. A=contol subjects (n=17); B, C and D=type 2 diabetic patients with triopathy-score 0 (n=lO), 1 (n=19) and 2 (n=9), respectively. mean*SE.
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The present findings that hyperfunction of PMN might be involved in the development of diabetic triopathy, offer the following potential drugs for the treatment of diabetic patients. First, a recombinant human anti-TNF-a antibody; which may be effective for diabetic triopathy, and does not change insulin sensitivity in the type 2 diabetic patients (28). Second, an inhibitor of TNF-a production. Third, a drug with a free radical scavenging effect. For example, in hypoglycemic agents, gliclazide has a free radical scavenging effect while glibenclamide does not (29). Many anti-hypertensive drugs such as calcium antagonist (nifedipine, nisoldipine) and angiotensin-converting enzyme inhibitor (captopril) have scavenging effec%s in vitro: but these effects are not always sufficient ex vivo (30-32). Fourth, a drug with a suppression of 0, production by PMN. Celiprolol (a @-blocker) has such an effe& in hypertensive patients ex vivo (33). In conclusion, the PMN functions increased in the type 2 diabetic patients with diabetic triopathy. The activated 02- production of PMN might contribute to the development of diabetic complications. Further studies are needed to evaluate the merit of modulating PMN functions for the treatment of diabetic patients.
Acknowledgment
We are grateful to Professor Shuichi Kitagawa of the Osaka City University Medical School for technical suggestions. We thank MS Chie Fukushima and Takami Kawaguchi for preparing the manuscript.
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PIISOO24-3205(00)00509-9
THE FUNCTIONS OF CIRCULATORY POLYMORPHONUCLEAR LEUKOCYTES IN DIABETIC PATIENTS WITH AND WITHOUT DIABETIC TRIOPATHY Masami Ohmori, Kazuhiro Harada, Yasuhiko Kitoh, Syou-ichiroh Nagasaka, Toshikazu Saito and Akio Fujimura Departments of Clinical Pharmacology and Medicine (Division of Endocrinology), Jichi Medical School, Tochigi 329-0498, Japan. (Receivedin finalformDecember9, 1999) Summary We determined circulatory polymorphonuclear leukocytes (PMN) functions of superoxide anion production, adhesion and aggregation in 38 type 2 diabetic patients with and without diabetic triopathy. Tumor necrosis factor (TNF)-a-stimulated superoxide production and N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated aggregation in diabetic patients with triopathy were significantly greater than those in diabetics without triopathy. The more diabetic complications existed, the more TNF-a-stimulated superoxide was produced by PMN. These results suggest that the activated PMN contributes to a progression of diabetic triopathy in type 2 diabetic patients. leukocyte., non-insulin-dependent diabetes mellitus, triopathy, superoxide, KeyWords: polymorphonuclear adhesion, aggregation
Diabetic triopathy (retinopathy, nephropathy and neuropathy) is the serious The pathogenesis is not fully complication in patients with diabetes mellitus. understood, but several mechanisms have been proposed. For example, dysfunction of platelet and vascular wall induced by glucose toxicity, glycation, advanced glycation end products (AGES), excessive reactive free radicals and decreased antioxidant properties are reported to cause diabetes-specific complications in these patients (1-G). Especially, reactive free radicals [superoxide anion (OZ.), hydrogen peroxide, hydroxyl radical] produced by polymorphonuclear leukocytes (PMN) might damage not only DNA, but membrane lipid bilayer and other essential cell components (7), which in turn cause Address for correspondence: Akio Fujimura, M.D., Department of Clinical Pharmacology, Jichi Medical School, 33 1 l- 1 Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan, Telephone: +81 285 58 7388, Telefax: +81 285 44 7562: E-mail: [email protected]
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In type 1 diabetes mellitus, 0, vascular complications in the diabetic patients. production by PMN in the patients with diabetic retinopathy is greater than that in the patients without the complication (8). This finding indicates that the enhanced 0,. production by circulating PMN contributes to a pathogenesis of diabetic retinopathy in the type 1 diabetic patients. However it is still unclear whether PMN is involved in the mechanism of diabetic organ damage in the type 2 diabetic patients. To address this issue, this study was undertaken to determine PMN capacities of 02production, adhesion and aggregation in type .2 diabetic patients with and without diabetic triopathy.
Materials
and Methods
Patients Thirty-eight type 2 diabetic patients with and without triopathy and seventeen nondiabetic control subjects were studied (Table 1.) There were no significant differences in sex ratio, age and body mass index between the type 2 diabetics and control groups. The Hb,,, was significantly higher, and the fasting blood sugar (FBS) tended to be higher in the type 2 diabetics than in the control group (Table 1.). While insulin, glibenclamide, gliclazide (a sulphonylurea) and epalrestat (an aldose reductase inhibitor) were given to the type 2 diabetic patients, other medications were similar between the two groups (data not shown). Patients with nephrotic syndrome and infection, and taking medications such as anti-inflammatory and anti-platelet were excluded. Diabetic nephropathy was defined as albuminuriaZ 20 mglday [n=172 (84 male, 88 female), SRL, Japan], which is similar to the previous criterion (albuminuria 2 21.6 mglday) (9). Diabetic neuropathy was defined as diminished nerve conduction Hbnlc was velocity, reduced deep tendon reflex and reduced feeling of vibration. measured by latex agglutination (LA) method, FBS by hexokinaseiglucose-6-phosphate dehydrogenase (Glu-HD) method, serum lipid peroxides by thiobarbituric acid (TBA) method and granulocyte elastase by enzyme immunoassay (EIA). Triopathy-score was defined as follows; 0 (no triopathy, n=lO), 1 (one and two triopathy, n=19): 2 (all triopathy, n=9>. Four out of 19 patients in the score-l group and 4 out of 9 patients in the score-2 group received epalrestat every day (the difference between the groups: ~~0.45 by x 2 test). All patients gave their informed consent to the aim and methods of this study. PMN prepara tion Blood sample was taken from forearm vein in the morning after an overnight fasting and heparinized using heparin 100 U/ml. Mixed leukocyte suspension was obtained after sedimentation in the presence of 1 % dextran in saline and gradient centrifugation with Lymphoprep (Nycomed Pharma AS, Oslo, Norway). Purified PMN suspension (cell viability> 95 %) was obtained by hypotonic lysis at 4 “C and washing twice in phosphate buffer. PMN was resuspended in Hanks’ balanced salt solution (Nissui Pharmaceutical Co., Tokyo, Japan) with 0.1 % human serum albumin (HSA) at the concentration of 6x 106cells/ml. Assay for superoxide anion production Oz. production by PMN was determined by the superoxide dismutase (SOD)-inhibitable
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cytochrome-C reduction method with a minor modification (10: 11). The following materials were placed on FBS-coated wells at a total volume of 200 ml/well; 1) PMN suspension contained 3x105 PMN, 2) tumor necrosis factor-u (TNF-tx: 100 U/ml, Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) or its control buffer, 3) SOD (200 U/ml: Sigma Chemical Co., St. Louis, USA) or its vehicle 4) cytochrome-C (12 mgiml. Sigma Chemical Co.: St. Louis, USA). After incubation for GO minutes at 37 ‘Y’. 0: production was determined by measurin, v the absorbance at 540 and 550 nm in a spectrophotometer (Beckman DU-640, Fullerton: CA). The assay was performed in duplicate. Intra- and inter-variability are 3.2 and 6.6 %: respectively.
Table
1. Characteristics and type
in control
2 diabetic
subjects
patients Mean rfrSE
Parameter
Sex (Men/Women)
Age (year)
Control
Type 2 diabetes
(n=17)
mellitus
16122
819 49.5-cl8.0
Body mass index
23.4 k3.2
HbAlc (%)
5.4kO.5
(n=38)
57.3k14.8 24.2k4.1 9.Ok2.0 p
FE%3(mg/dl)
93.5k11.2
137.8 k39.7
Assayfor PMN adherence Adhered PMN to FBS-coated wells was determined as protein content by Lowry method (12). Distilled Hz0 (0.1 ml) was added to the well. Three reagents (2 % sodium tartrate, 1 % &SO4 and 2 % NaGO, in 0.1 N NaOH) were mixed, and phenolreagent (Wako Pure Chemical Industries Ltd., Osaka, Japan) was diluted by same volume of HZ0 just before use. Fifteen minutes after, 1 ml of their mixture was placed to each well, and 0.1 ml of diluted phenol was added to each well. Thirty minutes later, protein content was determined by measuring the absorbance at 700 nm in a spectrophotometer. The assay was also performed in duplicate. Intra- and intervariability are 2.4 and 13.3 %, respectively. Assay for PMN aggregation
PMN aggregation was determined by an aggregometer (multichannel platelet aggregation analyzer, Mebanix Model PAT-2A: Tokyo, Japan) using PMN suspension which contained 1.2~10~ cells, and N-formyl-methionyl-leucyl-phenylalanine (FMLP, 10.; M, Sigma Chemical Co.: St. Louis, USA) or its control buffer at a total volume of 200 ml. Intra- and inter-variability are 2.5 and 3.1 %: respectively.
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Statistical analysis Results were expressed as the mean+SE. Data were analyzed by one-way ANOVA using Statview-J 4.02 (Abacus Concepts Inc., California, USA). A value of pcO.05 was regarded as significant.
Table 2. Biochemical subjects
profiles
and PMN functions
and type 2 diabetic
patients
Type 2 Parameter
Control
diabetes
(n=17)
mellitus (n=38)
Serum lipid peroxide (nmol/ml) Granulocyte
elastase
3.3f0.9
3.1 zkO.6
in control Mean + SE
Healthy* subjects (n=8) ( 3.5kO.5
) )
54.8f15.2
61 .l f22.3
( 63.3k9.3
UnsGmulated
0.28kO.05
0.25+0.03
(0.31 f0.07)
TNF-a-stimulated
1.26f0.20
1 .I
Unstimulated
3.66kO.40
4.8OkO.46
(3.35kO.47)
TNF-a-stimulated
10.53+0.82
12.04kO.66
(9.15fO.76)
0.10f0.03
(0.14+0.07)
5.og+o.i9
(4.36+0.50)
(kg/l) Superoxide
anion production
(nmol/3xlO~
Adhesion
cells/hr)
0+0.08
(1.21
f0.37)
(pg/well)
Aggregation
(%)
Unstimulated
0.09
kO.03
FMLP-stimulated
4.72kO.42
* The values in healthy subjects are shown for a comparison
purpose.
Results
No significant differences were observed in serum concentrations of lipid peroxides and granulocyte elastase, nor 02- production, adhesion and aggregation by PMN between the type 2 diabetic patients and control groups (Table 2.). Biochemical profiles of the type 2 diabetic patients with and without triopathy did not differ significantly (Table 3.). The FBS was significantly higher and tended to be higher in the patients with and without triopathy than in the control group. The Hb,,, was significantly higher in the type 2 diabetic patients with and without triopathy than in the control group. Serum lipid peroxide and granulocyte elastase of the patients with and without triopathy and control subjects did not differ.
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Unstimulated 02. production and aggregation of PMN of the patients with triopathy were similar to those in the group without triopathy (Table 4.) However, TNF-astimulated 02- production and FMLP-stimulated aggregation were significantly or tended to be greater in the patients with triopathy. Adhesion was similar among three groups [control and type 2 diabetics with and without triopathy (data not shown)]. TNF-a-stimulated Oz.production in the triopathy-score 0 group tended to be lower than that in the control group (p=O.O8) (Fig. 1). Such the difference decreased in the score 1 (p=O.36) and score 2 (p=O.41) groups. The 02. production in the triopathy-score 2 group was greater than that in the triopathy-score 0 and 1 groups. This parameter was There was a enhanced in a triopathy-score-dependent manner in the diabetic patients. significant correlation between the Oz- production and albuminuria (i-=0.39, n=38; ~~0.05). FMLP-stimulated aggregation was significantly higher in the patients with triopathy-score 2 than in other groups (control and type 2 diabetics with triopathy-score 0 and 1) (Fig. 2.). Adhesion was similar between the control and diabetic groups (Fig. 3.).
Table 3. Biochemical profiles in type 2 diabetic patients with and without triopathy tibm
FBS
Triopathy
(%)
(mddl)
Mean+=
Serum lipid
Granulocyte
peroxide
elastase
(nmol/mI)
(I.&l)
t (n=19)
140.4f37.5
9.OL1.7
3.2f0.8
66.7k25.0
- (n=19)
135.3f42.6
9.1i2.3
3.OkO.2
55.1k17.9
t (n=15)
148.2f48.4
9.0f2.3
3.2kO.8
60.7k26.1
- (n=23)
130.6k31.8
9.0f1.8
3.1f0.3
61.3f20.0
t (n=26)
142.7f41.8
8.9f2.0
3.1kO.7
63.9k24.4
- (n=12)
126.0f33.3
9.4k2.1
3.0f0.2
55.2k16.8
Retinopathy
Nephropathy
Neuropathy
Discussion
PMN is one of the major sources of oxygen free radicals, which are supposed to cause diabetic complication, together with other factors such as glucose itself and AGES (1, 2). Previous studies showed that PMN Oz. production in diabetic patients increased, decreased or did not change compared to that in the control subjects (8, 13, 14). In the
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present study, O,- production by PMN of the diabetic and control groups did not differ significantly although its production was enhanced in subje& with greater diabetic complications. Therefore, these diverse results might reside, in part; in the selection of diabetic patients. AEXJ EL-ASRAR et al. reported that PMN O?- production stimulated by phorbol myristate acetate (PMA) increased in the type 1 diabetic* patients with diabetic retinopathy (8). Protein kinase C (PKC) activity is also reported to increase in diabetic animals and is potentially involved in the development of diabetic triopathy (15). Undoubtedly, PMA direL%lystimulates PKC which induces a respiratory burst, and releases oxygen free radicals, elastase. cytokines and other metabolites (1G). However PMA is not a physiological stimulant. TNF-tr is a polypeptide cytokine and plays a role in inflammatory process (17). It exists as a membrane-bound precursor, which can be processed by a TNF-(r-converting enzyme.
As TNF-c~ is an endogenous factor, PMN functions stimulated
by this agent have
greater meaning in clinical situations. In this study, TNF-(x-stimulated Oy production by PMN increased in the type 2 diabetic patients with diabetic triopathy. Unidentified serum factor (s) in diabetic patients with retinopathy is also shown to enhance 0,. productions by normal PMN (18). In addition, it is recently proved that diabetic patients have significant defects of physiological antioxidant properties (urate. vitamin C; E and A, bilirubin) and decreased serum SOD activity (19. 20). Based on these data, we think that diabetic patients with triopathy are always under conditions of the excessive oxidative stress by hyperproduction and hypoconsumption of oxygen free radicals, which causes further organ damage. Some diabetic patients are at a high risk of d&eloping primary liver cancer: probably due to the excessive free radicals (2 1). On the contrary, other diabetics have more postoperative infections than non-diabetics. which might be induced by the reduced free radicals (22). As 0, production by PMN
Table 4. PMN functions
in type 2 diabetic patients with and without triopathy Mean+%
Superoxide anion production (nmoV3xl
Aggregation
OQells/hr)
(%)
Triopathy
t
(n=19)
UnsGmulated
TNF-a-stimulated
Unstimulated
0.27+0.05
1.23+0.11~
O.lli-0.04
FMLPstimulated
5.51f0.261
p
pco.05
Retinopathy
-I
- (n=19)
0.23f0.04
0.96kO.09
t
0.26+0.05
1.37+0.131
(n=15)
0.10f0.05
4.61k0.25
0.07+0.03
5.25kO.36
p405
Nephropathy - (n=23)
0.25kO.04
0.92+0.07 J
0.12+0.05
4.96kO.19
t (n=26)
0.26+0.04
1.24+0.091
0.12kO.04
5.17kO.24
0.07+0.05
4.92f0.30
pco.05
Neuropathy - (n=12)
0.24f0.05
0.76f0.06 -I
J
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increased in the type 2 diabetic patients with triopathy-score 2 and decreased in the patients with triopathy-score 0 and 1 in this study, we think that patients with diabetes mellitus are vulnerable to,bacterial infection or development of cancer which is depend on the status of free radical. Previous reports showed that adherence and aggregation of PMN in diabetic patients decreased or did not change compared to that in the control subjects (23-26). In this study, these functions of PMN did not differ significantly between the type 2 diabetic patients and control groups. However, PMN aggregation increased in the type 2 diabetic subgroup with triopathy-score 2. Therefore, we think that the elevated PMN aggregation also contributes to the deterioration of diabetic organ damage. Epalrestat inhibits neutrophil 0,. production stimulated by PMA in vitro (27). The drug might also inhibit ex vivo TNF-a-stimulated 0,. production, which, in turn, contributes to the difference between the control and diabetic groups. and between the score-0 and score-l (or 2) groups.
p=o.41 p=O.36
_:I*I-
-
0
A
B
C
D
unst imulat ed
A
B
C
-
D
TNF-a-st imulat ed
Fig. 1 Superoxide anion production by PMN with and without TNF-a stimulation in the control subjects and type 2 diabetic patients. A=control subjects (n=17); B: C and D=type 2 diabetic patients with triopathy-score 0 (n=lO), 1 (n=19) and 2 (n=S): respectively. mean? SE, * p
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7-l
3
6-
*
l
*
5’
In
4-
3-
2-
1 -
0ABCD
ABCD
FMLP-stimulated
unstimulated Fig. 2
PMN aggregation with and without FMLP-stimulation in the control subjects and type 2 diabetic patients. A=contol subjects (n=17); B, C and D=type 2 diabetic patients with triopathy-score 0 (n=lO), 1 (n=19) and 2 (n=9), respectively. meant SE: *pcO.O5 compared to D
+
w r
L+ t
A
B
CD
unstimulated
-
-
ABu
D
-
TNF-a-stimulated Fig. 3
PMN adherence with and without TNF-a-stimulation in the control subjects and type 2 diabetic patients. A=contol subjects (n=17); B, C and D=type 2 diabetic patients with triopathy-score 0 (n=lO), 1 (n=19) and 2 (n=9), respectively. mean*SE.
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The present findings that hyperfunction of PMN might be involved in the development of diabetic triopathy, offer the following potential drugs for the treatment of diabetic patients. First, a recombinant human anti-TNF-a antibody; which may be effective for diabetic triopathy, and does not change insulin sensitivity in the type 2 diabetic patients (28). Second, an inhibitor of TNF-a production. Third, a drug with a free radical scavenging effect. For example, in hypoglycemic agents, gliclazide has a free radical scavenging effect while glibenclamide does not (29). Many anti-hypertensive drugs such as calcium antagonist (nifedipine, nisoldipine) and angiotensin-converting enzyme inhibitor (captopril) have scavenging effec%s in vitro: but these effects are not always sufficient ex vivo (30-32). Fourth, a drug with a suppression of 0, production by PMN. Celiprolol (a @-blocker) has such an effe& in hypertensive patients ex vivo (33). In conclusion, the PMN functions increased in the type 2 diabetic patients with diabetic triopathy. The activated 02- production of PMN might contribute to the development of diabetic complications. Further studies are needed to evaluate the merit of modulating PMN functions for the treatment of diabetic patients.
Acknowledgment
We are grateful to Professor Shuichi Kitagawa of the Osaka City University Medical School for technical suggestions. We thank MS Chie Fukushima and Takami Kawaguchi for preparing the manuscript.
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