254-260
Clinicalrheumatology, 1992, ll, N ~ 2
Inhibition of Superoxide Anion Release from Human Polymorphonuclear Leukocytes by N-AcetyI-Galactosamine and N-AcetyI-Glucosamine M. K A M E L ,
M. A L N A H D I *
Summary This study shows that N-acetyl-galactosamine and N-acetyl-glucosamine can diminish the production of superoxide anion from cytochalasin-B treated PMNs stimulated with FMLP. Inhibition ranged from 80.9% to 1.8%. N-acetyl-galactosamine was superior to N-acetyl-glucosamine, but both showed their action in a dose - related fashion. The mannosamine may diminish the superoxide production, but without a statistical significance. Other sugars such as L-fucose, D-fucose and D-glucose failed to induce inhibition of superoxide generation. Previous reports showed that sugars interfere with carbohydrates lectins interaction. This study shows that aminosugars can do more than interfere with carbohydrates-lectin interaction. The mechanism is not completely known yet. The question whether aminosugars affect cell-cell interaction, regulation of respiratory burst, inflammatory mediators functions, or the glucose uptake and utilization needs further study. Key words : Galactosamine, Glucosamine, Polymorphonuclear Leukocyte, Enzyme Secretion, Superoxide Anion, Rheumatoid Arthritis.
INTRODUCTION Oxygen-free radicals are an important group of inflammatory mediators that cause tissue destruction in rheumatoid arthritis (1-3). During an inflammation, PMNs and macrophages are the most important sources of superoxide anion generation (4). Exposure of neutrophils to suitable stimuli results in the activation of an oxygen-consuming metabolic pathway. The cellular manifestations of this pathway include increase in oxygen uptake, superoxide production and glucose consumption. This group of events, taken together, are designated the "respiratory burst" (5-7). Current studies show that N-acetyl-galactosamine and glucosamine can inhibit binding of gonococci to human neutrophils (8). The activity of human leukocyte inhibiting factor (LIF) could be eluted with N-acetyl-glucosamine, fucose and galactose (9). N-acetyl-galactosamine and N-acetyl-glucosamine enhance the recognition and phagocytosis of particles by human neutrophils (10).
Department of Rheumatology, Azhar University, Cairo, Egypt. Dr. Fakhry Hospital, Dhahran, Saudi Arabia; *Department of Internal Medicine, King Faisal University, Dammam, Saudi Arabia.
The chemotactic response of PMN to FMLP normally provokes the release of enzymes and superoxide anion (11). This was shown to be inhibited with the Wheat Germ Agglutinin (WGA). The inhibition mechanism could possibly by explained either by attaching to the N-acetyl-glucosamine residue on the PMN plasma membrane, or by blocking the recycling of a preparation of FMLP receptors required for continuous migration (12). In this study, we tested the effects of aminosugars on the production of superoxide from the cytochalasin-B treated polymorphonuclear leukocytes stimulated with the chemotactic peptide FMLP. MATERIALS AND METHODS Cell preparation Neutrophils were prepared from the peripheral venous blood of normal healthy adult donors by centrifugation on Hypaque Ficoll (13) followed by a standard technique of dextran sedimentation, and hypotonic lysis of erythrocyte as described (14). The suspension of isolated cells of 95 - 98% of the polymorphonuclear leukocytes was made in HEPES buffer, pH 7.45, which was used for all subsequent incubations of the cells.
Aminosugars inhibit superoxide production
Table I :
255
Percent release of superoxide anion from human PMNs
Sugars
N A galactosamine NA galactosamine NA galactosamine NA glucosamine N A glucosamine N A glucosamine Mannosamine Mannosamine D-fucose D-fucose D-glucose
Final conc
No Exp
Mean %
SEM %
p Value
Signif
10mg/ml 5mg/ml lmg/ml 10mg/ml 5mg/ml lmg/ml 5mg/ml lmg/ml 10mg/ml 5tng/ml 10mg/ml
4 4 4 5 4 4 3 2 2 4 3
19.1 59.7 77.9 48.3 78.4 98.2 98.4 98.6 95.5 98.9 100
1.2 7.4 4.9 12.7 9.3 1.7 2.1 1.4 3.6 0.8 00
0.007 0.001 0.006 0.01 0.1 0.4 0.3 0.1 0.5 0.1 00
S+§ + S § ++ S. ++ S+ + NS NS NS NS NS NS NS
Data are presented as the m e a n percentage, SEM and p value of the superoxide anion release of identical series of experiments, in the presence of aminosugars and without, versus the control FMLP. T h e m e a n of the population (FMLP) was considered 100%,
Cell viability
Cell viability was determined by monitoring release of the cytoplasmic enzyme, lactic dehydrogenase as described (15). Enzyme release was expressed as percentage of total activity, measured in aliquot of cells lysed with triton • (final concentration 0.2%). No significant difference was observed between sugar-treated and untreated cells. Generation of superoxide anion
The O~ release of cytochalasin-B treated neutrophils was measured as superoxide-dismutase-inhibitable reduction of cytochrome C. Duplicate reaction mixtures containing 1.25 x 106 neutrophils, 75 nM of cytochrome C, and the tested sugar in subsequent concentrations, making a final volume of 1 ml/cuvette, were incubated at 37~ Absorption at 551 nm was determined by Beckman spectrophotometer Model 25 (16,17). The molar quantity of superoxide anion generation was calculated from. the difference in absorption at 551 nm between ferricyanide-oxidized and potassium thiosulphite reduced cytochrome C. The results are the mean of duplicate determinations, and were expressed in nanomole cytochrome C/3min/1 x 106 PMN as described in laboratory manual of neutrophil functions (18).
peroxide-dismutase-inhibitable reduction of cytochrome C assay for superoxide anion generation (19). The absorption was determined using Beckman spectrophotometer at 551.2 nm at room temperature. We added 100 gl of cytochrome C 9 mg / ml, xanthine oxidase 100 ~tl of 10 ~tl/ml stock solution diluted 1:10 to make a final concentration of 1:1000. Hypoxanthine oxidase 100 ~tl of stock solution 2 mg/ml and diluted 1:10 to make a final concentration of 2 mg/100ml. The superoxide dismutase 10 ~tl was added to the sample cuvette only, and then to both cuvettes, to make sure that the system was working. Finally, 100 gl of the tested sugar was added. These reagents make a solution of 1 ml, which was incubated for 5 minutes at room temperature. Results were calculated by measuring the curve slope of each sample.
Materials
D-glucose, L(-) fucose, D( + ) fucose, N-acetyl-galactosamine N-acetyl-glucosamine, L(-) mannose and D-mannosamine hydrochloride, were obtained from Sigma Chemical Company (Milwaukee, WI). FMLP (N.Formyl -methionyl-leucyl-phenylalanine) came from Peninsula Laboratories (San Carlos, CA). Other materials were reagent grade.
Statistics Xantine and hypoxanthine oxidase assay
This assay was done to assess and exclude a possible inhibitory effect of the tested sugar material on the su-
Statistical tests were run on an Apple Computer using the Microplan program and population t-test. The mean population of the control was considered 100%. The re-
256
M. Kamel, M. Alnahdi 2O
Z
NA-gala
NA-gala
13_
cD O
|
16
F1,
CO O 12
O
E
I
cO O
I
o
4
o
FMLP
I )
O
E o
cid
0
I
I
time min
3
CO
Z
FMLP
13_ 2o c.o O
NA-gluco
cO O
O
16
[]
NA-gluco []
/
12
E
s
cO O
8
O O
E
4
a
O
t'--
C
0
1
2
3
time min Fig. 1 : (A-B) Typical experiment records show that both the NA glactosamine and N A glucosamine can diminish the release of the superoxide anion generation when added to a suspension contained 106 cell/ml of P M N s treated by cytochalasin-B equilibrated at 37~ and stimulated with FMLP. The given n u m b e r s refer to the concentrations of the added sugars in mg/ml. NA-Gala (N-A-galactosamine), N-A-Gluco. (N-Acetyl-glucosamine) and F M L P (N Formyl-Methionyl-Leucyl-Phenylalanine). N A - G a l a is a more potent inhibitor than N-AGluco and both showed their action in a dose-related fashion.
Aminosugars inhibit superoxide production
257
percent release of 0 - 2 generation by aminosugars and control 120 1
5
1
5
lC
I
ao
~ 6o o
4O
| 1
2O
0
m
FMLP N,A.Gluco. Msnn08. D.Fucose D,Olucose N.A.C~atsc. Fig. 2: This diagram shows the mean percent release of the superoxide by PMNs with or without arninosugars. The reference control is FMLP which is considered 100%. (Mannos : is Mannosamine).
suits are given in Mean +_ SEM, p
Clinicalrheumatology, 1992, ll, N ~ 2
Inhibition of Superoxide Anion Release from Human Polymorphonuclear Leukocytes by N-AcetyI-Galactosamine and N-AcetyI-Glucosamine M. K A M E L ,
M. A L N A H D I *
Summary This study shows that N-acetyl-galactosamine and N-acetyl-glucosamine can diminish the production of superoxide anion from cytochalasin-B treated PMNs stimulated with FMLP. Inhibition ranged from 80.9% to 1.8%. N-acetyl-galactosamine was superior to N-acetyl-glucosamine, but both showed their action in a dose - related fashion. The mannosamine may diminish the superoxide production, but without a statistical significance. Other sugars such as L-fucose, D-fucose and D-glucose failed to induce inhibition of superoxide generation. Previous reports showed that sugars interfere with carbohydrates lectins interaction. This study shows that aminosugars can do more than interfere with carbohydrates-lectin interaction. The mechanism is not completely known yet. The question whether aminosugars affect cell-cell interaction, regulation of respiratory burst, inflammatory mediators functions, or the glucose uptake and utilization needs further study. Key words : Galactosamine, Glucosamine, Polymorphonuclear Leukocyte, Enzyme Secretion, Superoxide Anion, Rheumatoid Arthritis.
INTRODUCTION Oxygen-free radicals are an important group of inflammatory mediators that cause tissue destruction in rheumatoid arthritis (1-3). During an inflammation, PMNs and macrophages are the most important sources of superoxide anion generation (4). Exposure of neutrophils to suitable stimuli results in the activation of an oxygen-consuming metabolic pathway. The cellular manifestations of this pathway include increase in oxygen uptake, superoxide production and glucose consumption. This group of events, taken together, are designated the "respiratory burst" (5-7). Current studies show that N-acetyl-galactosamine and glucosamine can inhibit binding of gonococci to human neutrophils (8). The activity of human leukocyte inhibiting factor (LIF) could be eluted with N-acetyl-glucosamine, fucose and galactose (9). N-acetyl-galactosamine and N-acetyl-glucosamine enhance the recognition and phagocytosis of particles by human neutrophils (10).
Department of Rheumatology, Azhar University, Cairo, Egypt. Dr. Fakhry Hospital, Dhahran, Saudi Arabia; *Department of Internal Medicine, King Faisal University, Dammam, Saudi Arabia.
The chemotactic response of PMN to FMLP normally provokes the release of enzymes and superoxide anion (11). This was shown to be inhibited with the Wheat Germ Agglutinin (WGA). The inhibition mechanism could possibly by explained either by attaching to the N-acetyl-glucosamine residue on the PMN plasma membrane, or by blocking the recycling of a preparation of FMLP receptors required for continuous migration (12). In this study, we tested the effects of aminosugars on the production of superoxide from the cytochalasin-B treated polymorphonuclear leukocytes stimulated with the chemotactic peptide FMLP. MATERIALS AND METHODS Cell preparation Neutrophils were prepared from the peripheral venous blood of normal healthy adult donors by centrifugation on Hypaque Ficoll (13) followed by a standard technique of dextran sedimentation, and hypotonic lysis of erythrocyte as described (14). The suspension of isolated cells of 95 - 98% of the polymorphonuclear leukocytes was made in HEPES buffer, pH 7.45, which was used for all subsequent incubations of the cells.
Aminosugars inhibit superoxide production
Table I :
255
Percent release of superoxide anion from human PMNs
Sugars
N A galactosamine NA galactosamine NA galactosamine NA glucosamine N A glucosamine N A glucosamine Mannosamine Mannosamine D-fucose D-fucose D-glucose
Final conc
No Exp
Mean %
SEM %
p Value
Signif
10mg/ml 5mg/ml lmg/ml 10mg/ml 5mg/ml lmg/ml 5mg/ml lmg/ml 10mg/ml 5tng/ml 10mg/ml
4 4 4 5 4 4 3 2 2 4 3
19.1 59.7 77.9 48.3 78.4 98.2 98.4 98.6 95.5 98.9 100
1.2 7.4 4.9 12.7 9.3 1.7 2.1 1.4 3.6 0.8 00
0.007 0.001 0.006 0.01 0.1 0.4 0.3 0.1 0.5 0.1 00
S+§ + S § ++ S. ++ S+ + NS NS NS NS NS NS NS
Data are presented as the m e a n percentage, SEM and p value of the superoxide anion release of identical series of experiments, in the presence of aminosugars and without, versus the control FMLP. T h e m e a n of the population (FMLP) was considered 100%,
Cell viability
Cell viability was determined by monitoring release of the cytoplasmic enzyme, lactic dehydrogenase as described (15). Enzyme release was expressed as percentage of total activity, measured in aliquot of cells lysed with triton • (final concentration 0.2%). No significant difference was observed between sugar-treated and untreated cells. Generation of superoxide anion
The O~ release of cytochalasin-B treated neutrophils was measured as superoxide-dismutase-inhibitable reduction of cytochrome C. Duplicate reaction mixtures containing 1.25 x 106 neutrophils, 75 nM of cytochrome C, and the tested sugar in subsequent concentrations, making a final volume of 1 ml/cuvette, were incubated at 37~ Absorption at 551 nm was determined by Beckman spectrophotometer Model 25 (16,17). The molar quantity of superoxide anion generation was calculated from. the difference in absorption at 551 nm between ferricyanide-oxidized and potassium thiosulphite reduced cytochrome C. The results are the mean of duplicate determinations, and were expressed in nanomole cytochrome C/3min/1 x 106 PMN as described in laboratory manual of neutrophil functions (18).
peroxide-dismutase-inhibitable reduction of cytochrome C assay for superoxide anion generation (19). The absorption was determined using Beckman spectrophotometer at 551.2 nm at room temperature. We added 100 gl of cytochrome C 9 mg / ml, xanthine oxidase 100 ~tl of 10 ~tl/ml stock solution diluted 1:10 to make a final concentration of 1:1000. Hypoxanthine oxidase 100 ~tl of stock solution 2 mg/ml and diluted 1:10 to make a final concentration of 2 mg/100ml. The superoxide dismutase 10 ~tl was added to the sample cuvette only, and then to both cuvettes, to make sure that the system was working. Finally, 100 gl of the tested sugar was added. These reagents make a solution of 1 ml, which was incubated for 5 minutes at room temperature. Results were calculated by measuring the curve slope of each sample.
Materials
D-glucose, L(-) fucose, D( + ) fucose, N-acetyl-galactosamine N-acetyl-glucosamine, L(-) mannose and D-mannosamine hydrochloride, were obtained from Sigma Chemical Company (Milwaukee, WI). FMLP (N.Formyl -methionyl-leucyl-phenylalanine) came from Peninsula Laboratories (San Carlos, CA). Other materials were reagent grade.
Statistics Xantine and hypoxanthine oxidase assay
This assay was done to assess and exclude a possible inhibitory effect of the tested sugar material on the su-
Statistical tests were run on an Apple Computer using the Microplan program and population t-test. The mean population of the control was considered 100%. The re-
256
M. Kamel, M. Alnahdi 2O
Z
NA-gala
NA-gala
13_
cD O
|
16
F1,
CO O 12
O
E
I
cO O
I
o
4
o
FMLP
I )
O
E o
cid
0
I
I
time min
3
CO
Z
FMLP
13_ 2o c.o O
NA-gluco
cO O
O
16
[]
NA-gluco []
/
12
E
s
cO O
8
O O
E
4
a
O
t'--
C
0
1
2
3
time min Fig. 1 : (A-B) Typical experiment records show that both the NA glactosamine and N A glucosamine can diminish the release of the superoxide anion generation when added to a suspension contained 106 cell/ml of P M N s treated by cytochalasin-B equilibrated at 37~ and stimulated with FMLP. The given n u m b e r s refer to the concentrations of the added sugars in mg/ml. NA-Gala (N-A-galactosamine), N-A-Gluco. (N-Acetyl-glucosamine) and F M L P (N Formyl-Methionyl-Leucyl-Phenylalanine). N A - G a l a is a more potent inhibitor than N-AGluco and both showed their action in a dose-related fashion.
Aminosugars inhibit superoxide production
257
percent release of 0 - 2 generation by aminosugars and control 120 1
5
1
5
lC
I
ao
~ 6o o
4O
| 1
2O
0
m
FMLP N,A.Gluco. Msnn08. D.Fucose D,Olucose N.A.C~atsc. Fig. 2: This diagram shows the mean percent release of the superoxide by PMNs with or without arninosugars. The reference control is FMLP which is considered 100%. (Mannos : is Mannosamine).
suits are given in Mean +_ SEM, p
