Originals Basic Insulin, at Physiological Concentrations, Enhances the Polymorphonuclear Leukocyte Chemotactic Properties
Summary We investigated the influence of insulin on human polymorphonuclear leukocyte (PMN) chemotaxis induced by mediators in a microchamber assay. Insulin increased, with a dose-response relationship, chemotaxis induced by formyl-methionyl-leucyl-phenylalanine, calcium ionophore and phorbol-miristyl acetate (p = 0.0057, p = 0.0001 and p = 0.0215, respectively). The hormone effect was present also at the physiological concentration of 40 iiU/ml. Our data show that insulin affects P M N activity in normal subjects and therefore support the hypothesis that insulin deficiency may be responsible for the impaired PMN function observed in diabetic patients in poor metabolic control. Key words Insulin — Polymorphonuclear Leukocytes — fMLP - PMA - Calciumlonophore
Introduction Infection was a frequent cause of death in diabetic patients before insulin treatment was available. Even in the treated subjects, infection has a worse prognosis once established, especially in those who are not well controlled {Rayfield, Ault, Keusch, Brothers, Neclamias and Smith 1982). Furthermore, fatal pneumonia, staphylococcal and mycotic infections and tuberculosis are more frequent in these subjects {Rayfield et al. 1982). Among other factors, the increased morbidity and mortality were attributed to impairment of granulocyte function {Wilson 1986). Different aspects of polymorphonuclear leukocytes (PMN) in diabetes were studied: adherence {Bagdade, Stewart and Walters 1978), chemotaxis {Movat and Baum 1971; Molenaar, Palumbo, Wilson and Pitts 1976; Fikrig, Reddy, Orti, Herod and Suntharalingam 1977), phagocytosis {Bagdade, Root and Dulger 1974), killing {Dziatkowiak, Kowalski and Denys 1982) and, recently, chemiluminescence {Shah, Wallin and Eilen 1983), superoxide anion production {Shah, Wallin and Eilen 1983) and superoxide dismutase ac-
Horm. metab. Res. 24 (1992) 225 - 228 © Georg Thieme Verlag Stuttgart • New York
tivity {Nath, Chad and Rathi 1984). Although sometimes normal PMN function had been found {Fikrig et al. 1977), impaired activities were generally described and they induced different authors to study the mechanism of this impairment. Various factors would seem to play a role: increased blood glucose may induce an increase in aldose reductase activity {Wilson, Tomlinson and Reeves 1987) or lead to non-enzymatic glycosylation of proteins involved in PMN function {Nielson and Hindson 1989; Hostetter 1990); furthermore, the plasma may contain some P M N inhibitory factor {Pereira, Sasnnomiya and Leme l987). The role of insulin has been studied only marginally. Even though the entry of glucose into the P M N is widely considered to be insulin-independent, studies by Esmann and other authors {Esmann 1963; Esmann 1972; Martin, McKinney, Green and Becker 1953; Dumm 1957; Dumm 1959) demonstrated that diabetic leukocytes display a decreased rate of glycolysis, decreased lactate production and decreased glycogen synthesis; these abnormalities were corrected by in vivo insulin treatment. A depressed glycogen-transferase activity in diabetic subjects was shown to increase in response to insulin administration {Esmann, Hedeskov and Rosell Perez 1968). A correction of impaired PMN functions, including adherence {Bagdade, Stewart and Walters 1978), chemotaxis {Kjersem, Hilsted, Madsbad, Staer-Johansen and Wandall 1975), phagocytosis and bactericidal rates {Rayfield et al. 1982; Bagdade, Root and Dulger 1974), was described in diabetic subjects after improved metabolic control obtained by insulin treatment. In summary, many data obtained in vivo are in favor of insulin influencing PMN carbohydrate metabolism and ameliorating impaired PMN functions in diabetic subjects. The effects of insulin treatment in vivo, however, are not necessarily due to a direct hormone effect: actually, they could also be attributed to the improvement of metabolic control, with a reduction of blood glucose concentrations. Unfortunately, only few studies evaluated the influence of insulin per se, added in vitro to PMN preparations: these studies gave different results according to the parameter investigated. Insulin at 100 LiU/ml was shown to increase chemotactic activity of diabetic PMN in response to casein {Movat and Baum 1971). Studying chemiluminescence and superoxide anion production of normal and diabetic leukocytes after stimulation with PMA, Shah, Wallin and Eilen (1983) could not demonstrate an effect of insulin. On the other hand, insulin at 0.36 nM (50 nU/ml) was shown to increase in vitro glucose
Received: 26 Feb. 1991
Accepted: 8 Jan. 1992 after revision
Downloaded by: University of Illinois. Copyrighted material.
F. Cavalot, G. Anfossi, I. Russo, E. Mularoni, P. Massucco, S. Burzacca, L. Mattiello and M. Trovati Department of Clinical and Biological Sciences of the University of Torino, San Luigi Gonzaga Hospital, Orbassano, Torino, Italy
Horm. metab. Res. 24 (1992)
F. Cavalot, G. Anfossi, I. Russo, E. Mularoni, P. Massucco et al.
Fig. 1 Effect of increasing concentrations of insulin on PMN chemotaxis induced by 1 nM fMLP. The statistical significance of the test was evaluated by ANOVA for repeated measurements (p = 0.0057). The difference between insulin concentrations of 0 and 40 u,U/ml was statistically significant (p = 0.0068) by t-test for paired data.
Fig. 2 Effect of increasing insulin concentrations on PMN chemotaxis induced by 10 nM calcium ionophore. The statistical significance of the test was evaluated by ANOVA for repeated measurements (p = 0.0001). The difference between insulin concentrations of 0 and 40 u.U/ml was statistically significant (p = 0.0002) by t-test for paired data.
consumption of human non diabetic leukocytes by about 40 % without influencing the permeability of the plasma membrane to glucose (Lewsaux, Marchand, Hang Than Ha and Cartier 1975). Finally, insulin receptors were demonstrated on PMN plasma membrane (Fussganger, Kahn, Roth and De Meyts 1976).
methionyl-leucyl-phenylalanine (fMLP) (Sigma, St. Louis, MO) was used at 1 nM, phorbol-miristyl-acetate (PMA) (Sigma) and A23187 (Sigma) were used at 10 nM. Lyophylized insulin, obtained by Sigma and therefore without the preservatives of the preparations for clinical use, was dissolved in TTBSA with 10 mM calcium and magnesium, the same medium employed for the chemoattractants, and added to both chemoattractant solution and cell suspension at the following concentrations: 0 |xU/ml, 40 uU/ml, 80 u.U/ml, 160 uU/ml and 320 uU/ml. PMN were suspended at the concentration of 2.5 x 105/ml in TTBSA containing 10 mM calcium and magnesium. 25 ul of chemoattractant solution were put in the lower part of the well, a 5 nm pore polycarbonate filter (Neuroprobe) was overlaid and 50 ul of cell suspension were put in the upper part of each well. The assay was conducted at 37°C for 2hrs in5%C0 2 /95% O2.
The aim of our study was to further clarify the direct influence of insulin on PMN functions. In particular we investigated: a) whether insulin modifies the human PMN chemotactic properties in response to agents showing different mechanisms of action; b) whether this insulin effect is concentration-dependent; c) whether insulin influences PMN chemotaxis already at the physiological concentration of 40 uU/ml. Materials and Methods Preparation
of PMN
Blood, obtained from 15 normal volunteers, age 27.4 + 2.0 years, 9 males and 6 females, was collected in 20 U/ml heparin, mixed with an equal volume of RPMI 1640, stratified over Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and centrifuged at 1350 rpm for 20 min. Mononuclear cells and platelets were discarded, while the bottom layer containing PMN and red blood cells was suspended in 2.5% gelatin (Difco, Detroit, MI) and sedimented at 37 °C. The supernatant containing the PMN was centrifuged at 2500 rpm for 20 min. To eliminate residual red blood cells, the pellet was submitted to hypotonic lysis with distilled water containing 10 mM EDTA for 20 sec and then the solution was made physiological with an equivalent amount of 1.8% NaCl. The cells were then washed 3 times with Tris Tyrode's buffer without calcium and magnesium containing 0.25% BSA and 5.5 mM glucose (TTBSA). Cells were stained by Giemsa and showed to be more than 90% PMN, with the other cells being lymphocytes and monocytes.
Chemotaxis assay It was conducted in 48-well microchemotaxis chambers (Neuroprobe, Bethesda, MD). Chemoattractant solutions were prepared in TTBSA containing calcium and magnesium. Formyl-
At the end of the incubation the filter was removed, fixed in methanol, stained with Harris' hematoxilin and then with eosin. Cells in the upper part of the filter were removed, while the cells that had migrated in the lower part of the filter were counted. Four 400x fields per well were counted. Each insulin concentration was run in six wells and the mean value was considered. Each PMN preparation was studied using all the above mentioned different insulin concentrations (0, 40, 80, 160, 320 u.U/ml). fMLP, PMA and A23187 were employed in 5,5, and 7 sets of experiments, respectively.
Calculation of results Values are mean + standard error of the mean in the number of experiments indicated. Statistical significance was evaluated by ANOVA for repeated measurements and, when appropriate, by Student's t-test for paired data. Results In the absence of insulin the number of PMN per high power field was 40.3 + 5.9 with 1 nM fMLP, 60.2 ± 10.5 with 10 nM A23187 and 77.3+13.2 with 10 nM PMA. Insulin added to upper and lower wells in the presence of a chemotactic agent in the lower well was shown to stimulate in a dose-dependent manner the number of cells that migrated: the effect was shown with fMLP (Fig. 1), with A23127 (Fig. 2) and with PMA (Fig. 3) (p = 0.0057, p = 0.0001 and
Downloaded by: University of Illinois. Copyrighted material.
226
Insulin and Polymorphonuclear Leukocyte
Chemotaxis
Horm. metab. Res. 24 (1992)
227
to evaluate whether insulin affects P M N function in diabetic patients at the same concentrations observed in healthy subjects, our study suggests the importance of an appropriate insulin therapy to prevent and to treat infections in diabetic patients.
Fig. 3 Effect of increasing concentrations of insulin on PMN chemotaxis induced by 10 nM PMA. The statistical significance of the test was evaluated by ANOVA for repeated measures (p = 0.0215). The difference between the insulin concentrations of 0 and 40 jj,U/ml was significant (p = 0.050) by t-test for paired data.
p = 0.0215 respectively when ANOVA for repeated measures was applied. The phenomenon was already statistically significant at the physiological insulin concentration of 40 uU/ml (p = 0.0068, p = 0.0002 and p = 0.050 respectively, as evaluated by Student's t-test for paired data). Discussion Our data show that insulin, at physiological glucose concentrations, increases PMN migration towards a positive gradient of stimulatory substances (chemotaxis). Our observations could explain, at least in part, the previously quoted literature reports on the influence of insulin treatment on PMN in diabetic patients. The effect of insulin was present with all the mediators used. They are known to activate PMN through different intracellular pathways as Sandborg and Smolen (1988) clearly reviewed: actually, fMLP acts through a plasma membrane receptor and G-proteins are most probably involved in signal transduction; the calcium ionophore A23187 increases intracellular calcium concentrations; PMA directly stimulates protein kinase C, thus by-passing membrane mechanisms. Our observation would suggest that insulin in some way stimulates general PMN responsiveness to the mediators by mechanisms that need to be further investigated. The effect of insulin is already present at the concentration of 40 uU/ml that is frequently attained in the everyday life of both healthy subjects and insulin-treated diabetic patients: this fact is in favor of a physiological insulin role in the modulation of PMN function. Recently, we demonstrated that insulin physiologically modulates also platelet function {Trovati, Anfossi, Cavalot, Massucco, Mularoni and Emanuelli 1988). In conclusion, our data support the hypothesis that insulin treatment ameliorates PMN function not only by the attainment of a better metabolic control — as the studies in vivo could suggest - but also through an effect directly exerted by the hormone itself. The depressed PMN function frequently described in poorly controlled diabetic patients could therefore be attributed not only to the elevated blood glucose concentrations, but also to the insulin deficiency per se or to deficient insulin action. Even if further studies are needed
Bagdade, J. D., R. K. Root, R. J. Dulger: Impaired leukocytes function in patients with poorly controlled diabetes. Diabetes 23: 9—15 (1974) Bagdade, J. D., M. Stewart, E. Walters: Impaired granulocyte adherence. A reversible defect in host defense in patients with poorly controlled diabetes. Diabetes27:677-681 (1978) Dumm, M. E.: Glucose utilization and lactate production by leukocytes of patients with diabetes mellitus. Proc. Soc. Exp. Biol. Med. 95:571-574(1957) Dumm, M. E.: Effect of tolbutamide on glucose uptake by leukocytes of patients with diabetes mellitus. J. Appl. Physiol. 14: 1023 — 1025 (1959) Dziatkowiak, H., M. Kowahki, A. Denys: Phagocytic and bactericidal activity of granulocytes in diabetic children. Diabetes 31: 1041 — 1043 (1982) Esmann, V.: Effect of insulin on human leukocytes. Diabetes 12: 545— 549 (1963) Esmann, V., C. J. Hedeskov, N. Rosell Perez: UDP-glucose-glucan glycosyl-transferase activity of polymorphonuclear leukocytes from diabetic subjects. Diabetologia 4:181-187(1968) Esmann, V .The diabetic leukocyte. Enzyme 13: 32—55(1972) Fikrig, J. M., C. M. Reddy, E. Orti, L. Herod, K. Suntharalingam: Diabetes and neutrophil chemotaxis. Diabetes 26:446—449 (1977) Fussgdnger, R. D., C. R. Kahn, J. Roth, P. De Meyts: Binding and degradation of insulin by human peripheral granulocytes. J. Biol. Chem. 251: 2761-2769 (1976) Hostetter, M. K.: Handicaps to host defense. Effect of hyperglycemia on C3 and Candida Albicans. Diabetes 39:271-275 (1990) Kjersem, H., J. Hilsted, J. Madsbad, K. Staer-Johansen, H. Wandall: The function of polymorphonuclear granulocytes (PMN) during normo- and hyperglycemia in juvenile diabetics. Diabetologia 19: 290 (1980) Lewsaux, J. P., J. C. Marchand, R. Hang Than Ha, P. Cartier: The influence of insulin on glucose permeability and metabolism of human granulocytes. Eur. J. Biochem. 58: 387 (1975) Martin, S. P., G. R. McKinney, R. Green, C. Becker: The influence of glucose, fructose and insulin on the metabolism of leukocytes of healthy and diabetic subjects. J. Clin. Invest. 32: 1171 — 1174 (1953) Molenaar, D. M., P. J. Palumbo, W. R. Wilson, R. E. Pitts: Leukocyte chemotaxis in diabetic patients and their non-diabetic first-degree relatives. Diabetes 25 (Suppl. 2): 880-883 (1976) Movat, A. G., J. Baum: Chemotaxis of polymorphonuclear leukocytes from patients with diabetes mellitus. N. Eng. J. Med. 284:621 -627 (1971) Nath, N., S. M. Chari, A. B. Rathi: Superoxide dismutase in diabetic polymorphonuclear leukocytes. Diabetes 33: 586-589 (1984) Nielson, C. P., D. A. Hindson: Inhibition of polymorphonuclear leukocyte respiratory burst by elevated glucose concentrations in vitro. Diabetes 38: 1031-1035(1989) Pereira, M. A. A, P. Sasnnomiya, J. G. Leme: Inhibition of leukocyte chemotaxis by factor in alloxan-induced diabetic rat plasma. Diabetes 36: 1307-1314(1987) Rayfield, E. J., M. J. Ault, G. T. Keusch, M. J. Brothers, C. Neclamias, A. Smith: Infection and diabetes: the case for glucose control. Am. J. Med. 72:439-450(1982) Sandborg, R. R., J. E. Smolen: Biology of disease. Early biological events in leukocytes activation. Lab. Invest. 59:300—320 (1988) Shah, V. S., J. D. Wallin, J. D. Eilen: Chemiluminescence and superoxide anion production by leucocytes from diabetic patients. J. Clin. Endocrinol. Metab. 57:402-409 (1983) Trovati, M., G. Anfossi, F. Cavalot, P. Massucco, E. Mularoni, G. Emanuelli: Insulin directly reduces platelet sensitivity to aggregat-
Downloaded by: University of Illinois. Copyrighted material.
References
ing agents. Studies in vitro and in vivo. Diabetes 37: 780-786 (1988) Wilson, R. M.: Neutrophil function in diabetes. Diabetic Medicine 3: 509-512(1986) Wilson, R. M., D. R. Tomlinson, W. G. Reeves: Neutrophil sorbitol production impairs oxidative killing in diabetes. Diabetic Medicine 4: 37-40 (1987)
F. Cavalot, G. Anfossi, I. Russo, E. Mularoni, P. Massucco et al. Requests for reprints should be addressed to: Prof. Marietta Trovati Diabetes Unit Department of Clinical and Biological Sciences University of Turin Ospedale San Luigi Gonzaga I-10043 Orbassano (Torino) (Italy)
Downloaded by: University of Illinois. Copyrighted material.
228 Horm. metab. Res. 24 (1992)
Summary We investigated the influence of insulin on human polymorphonuclear leukocyte (PMN) chemotaxis induced by mediators in a microchamber assay. Insulin increased, with a dose-response relationship, chemotaxis induced by formyl-methionyl-leucyl-phenylalanine, calcium ionophore and phorbol-miristyl acetate (p = 0.0057, p = 0.0001 and p = 0.0215, respectively). The hormone effect was present also at the physiological concentration of 40 iiU/ml. Our data show that insulin affects P M N activity in normal subjects and therefore support the hypothesis that insulin deficiency may be responsible for the impaired PMN function observed in diabetic patients in poor metabolic control. Key words Insulin — Polymorphonuclear Leukocytes — fMLP - PMA - Calciumlonophore
Introduction Infection was a frequent cause of death in diabetic patients before insulin treatment was available. Even in the treated subjects, infection has a worse prognosis once established, especially in those who are not well controlled {Rayfield, Ault, Keusch, Brothers, Neclamias and Smith 1982). Furthermore, fatal pneumonia, staphylococcal and mycotic infections and tuberculosis are more frequent in these subjects {Rayfield et al. 1982). Among other factors, the increased morbidity and mortality were attributed to impairment of granulocyte function {Wilson 1986). Different aspects of polymorphonuclear leukocytes (PMN) in diabetes were studied: adherence {Bagdade, Stewart and Walters 1978), chemotaxis {Movat and Baum 1971; Molenaar, Palumbo, Wilson and Pitts 1976; Fikrig, Reddy, Orti, Herod and Suntharalingam 1977), phagocytosis {Bagdade, Root and Dulger 1974), killing {Dziatkowiak, Kowalski and Denys 1982) and, recently, chemiluminescence {Shah, Wallin and Eilen 1983), superoxide anion production {Shah, Wallin and Eilen 1983) and superoxide dismutase ac-
Horm. metab. Res. 24 (1992) 225 - 228 © Georg Thieme Verlag Stuttgart • New York
tivity {Nath, Chad and Rathi 1984). Although sometimes normal PMN function had been found {Fikrig et al. 1977), impaired activities were generally described and they induced different authors to study the mechanism of this impairment. Various factors would seem to play a role: increased blood glucose may induce an increase in aldose reductase activity {Wilson, Tomlinson and Reeves 1987) or lead to non-enzymatic glycosylation of proteins involved in PMN function {Nielson and Hindson 1989; Hostetter 1990); furthermore, the plasma may contain some P M N inhibitory factor {Pereira, Sasnnomiya and Leme l987). The role of insulin has been studied only marginally. Even though the entry of glucose into the P M N is widely considered to be insulin-independent, studies by Esmann and other authors {Esmann 1963; Esmann 1972; Martin, McKinney, Green and Becker 1953; Dumm 1957; Dumm 1959) demonstrated that diabetic leukocytes display a decreased rate of glycolysis, decreased lactate production and decreased glycogen synthesis; these abnormalities were corrected by in vivo insulin treatment. A depressed glycogen-transferase activity in diabetic subjects was shown to increase in response to insulin administration {Esmann, Hedeskov and Rosell Perez 1968). A correction of impaired PMN functions, including adherence {Bagdade, Stewart and Walters 1978), chemotaxis {Kjersem, Hilsted, Madsbad, Staer-Johansen and Wandall 1975), phagocytosis and bactericidal rates {Rayfield et al. 1982; Bagdade, Root and Dulger 1974), was described in diabetic subjects after improved metabolic control obtained by insulin treatment. In summary, many data obtained in vivo are in favor of insulin influencing PMN carbohydrate metabolism and ameliorating impaired PMN functions in diabetic subjects. The effects of insulin treatment in vivo, however, are not necessarily due to a direct hormone effect: actually, they could also be attributed to the improvement of metabolic control, with a reduction of blood glucose concentrations. Unfortunately, only few studies evaluated the influence of insulin per se, added in vitro to PMN preparations: these studies gave different results according to the parameter investigated. Insulin at 100 LiU/ml was shown to increase chemotactic activity of diabetic PMN in response to casein {Movat and Baum 1971). Studying chemiluminescence and superoxide anion production of normal and diabetic leukocytes after stimulation with PMA, Shah, Wallin and Eilen (1983) could not demonstrate an effect of insulin. On the other hand, insulin at 0.36 nM (50 nU/ml) was shown to increase in vitro glucose
Received: 26 Feb. 1991
Accepted: 8 Jan. 1992 after revision
Downloaded by: University of Illinois. Copyrighted material.
F. Cavalot, G. Anfossi, I. Russo, E. Mularoni, P. Massucco, S. Burzacca, L. Mattiello and M. Trovati Department of Clinical and Biological Sciences of the University of Torino, San Luigi Gonzaga Hospital, Orbassano, Torino, Italy
Horm. metab. Res. 24 (1992)
F. Cavalot, G. Anfossi, I. Russo, E. Mularoni, P. Massucco et al.
Fig. 1 Effect of increasing concentrations of insulin on PMN chemotaxis induced by 1 nM fMLP. The statistical significance of the test was evaluated by ANOVA for repeated measurements (p = 0.0057). The difference between insulin concentrations of 0 and 40 u,U/ml was statistically significant (p = 0.0068) by t-test for paired data.
Fig. 2 Effect of increasing insulin concentrations on PMN chemotaxis induced by 10 nM calcium ionophore. The statistical significance of the test was evaluated by ANOVA for repeated measurements (p = 0.0001). The difference between insulin concentrations of 0 and 40 u.U/ml was statistically significant (p = 0.0002) by t-test for paired data.
consumption of human non diabetic leukocytes by about 40 % without influencing the permeability of the plasma membrane to glucose (Lewsaux, Marchand, Hang Than Ha and Cartier 1975). Finally, insulin receptors were demonstrated on PMN plasma membrane (Fussganger, Kahn, Roth and De Meyts 1976).
methionyl-leucyl-phenylalanine (fMLP) (Sigma, St. Louis, MO) was used at 1 nM, phorbol-miristyl-acetate (PMA) (Sigma) and A23187 (Sigma) were used at 10 nM. Lyophylized insulin, obtained by Sigma and therefore without the preservatives of the preparations for clinical use, was dissolved in TTBSA with 10 mM calcium and magnesium, the same medium employed for the chemoattractants, and added to both chemoattractant solution and cell suspension at the following concentrations: 0 |xU/ml, 40 uU/ml, 80 u.U/ml, 160 uU/ml and 320 uU/ml. PMN were suspended at the concentration of 2.5 x 105/ml in TTBSA containing 10 mM calcium and magnesium. 25 ul of chemoattractant solution were put in the lower part of the well, a 5 nm pore polycarbonate filter (Neuroprobe) was overlaid and 50 ul of cell suspension were put in the upper part of each well. The assay was conducted at 37°C for 2hrs in5%C0 2 /95% O2.
The aim of our study was to further clarify the direct influence of insulin on PMN functions. In particular we investigated: a) whether insulin modifies the human PMN chemotactic properties in response to agents showing different mechanisms of action; b) whether this insulin effect is concentration-dependent; c) whether insulin influences PMN chemotaxis already at the physiological concentration of 40 uU/ml. Materials and Methods Preparation
of PMN
Blood, obtained from 15 normal volunteers, age 27.4 + 2.0 years, 9 males and 6 females, was collected in 20 U/ml heparin, mixed with an equal volume of RPMI 1640, stratified over Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and centrifuged at 1350 rpm for 20 min. Mononuclear cells and platelets were discarded, while the bottom layer containing PMN and red blood cells was suspended in 2.5% gelatin (Difco, Detroit, MI) and sedimented at 37 °C. The supernatant containing the PMN was centrifuged at 2500 rpm for 20 min. To eliminate residual red blood cells, the pellet was submitted to hypotonic lysis with distilled water containing 10 mM EDTA for 20 sec and then the solution was made physiological with an equivalent amount of 1.8% NaCl. The cells were then washed 3 times with Tris Tyrode's buffer without calcium and magnesium containing 0.25% BSA and 5.5 mM glucose (TTBSA). Cells were stained by Giemsa and showed to be more than 90% PMN, with the other cells being lymphocytes and monocytes.
Chemotaxis assay It was conducted in 48-well microchemotaxis chambers (Neuroprobe, Bethesda, MD). Chemoattractant solutions were prepared in TTBSA containing calcium and magnesium. Formyl-
At the end of the incubation the filter was removed, fixed in methanol, stained with Harris' hematoxilin and then with eosin. Cells in the upper part of the filter were removed, while the cells that had migrated in the lower part of the filter were counted. Four 400x fields per well were counted. Each insulin concentration was run in six wells and the mean value was considered. Each PMN preparation was studied using all the above mentioned different insulin concentrations (0, 40, 80, 160, 320 u.U/ml). fMLP, PMA and A23187 were employed in 5,5, and 7 sets of experiments, respectively.
Calculation of results Values are mean + standard error of the mean in the number of experiments indicated. Statistical significance was evaluated by ANOVA for repeated measurements and, when appropriate, by Student's t-test for paired data. Results In the absence of insulin the number of PMN per high power field was 40.3 + 5.9 with 1 nM fMLP, 60.2 ± 10.5 with 10 nM A23187 and 77.3+13.2 with 10 nM PMA. Insulin added to upper and lower wells in the presence of a chemotactic agent in the lower well was shown to stimulate in a dose-dependent manner the number of cells that migrated: the effect was shown with fMLP (Fig. 1), with A23127 (Fig. 2) and with PMA (Fig. 3) (p = 0.0057, p = 0.0001 and
Downloaded by: University of Illinois. Copyrighted material.
226
Insulin and Polymorphonuclear Leukocyte
Chemotaxis
Horm. metab. Res. 24 (1992)
227
to evaluate whether insulin affects P M N function in diabetic patients at the same concentrations observed in healthy subjects, our study suggests the importance of an appropriate insulin therapy to prevent and to treat infections in diabetic patients.
Fig. 3 Effect of increasing concentrations of insulin on PMN chemotaxis induced by 10 nM PMA. The statistical significance of the test was evaluated by ANOVA for repeated measures (p = 0.0215). The difference between the insulin concentrations of 0 and 40 jj,U/ml was significant (p = 0.050) by t-test for paired data.
p = 0.0215 respectively when ANOVA for repeated measures was applied. The phenomenon was already statistically significant at the physiological insulin concentration of 40 uU/ml (p = 0.0068, p = 0.0002 and p = 0.050 respectively, as evaluated by Student's t-test for paired data). Discussion Our data show that insulin, at physiological glucose concentrations, increases PMN migration towards a positive gradient of stimulatory substances (chemotaxis). Our observations could explain, at least in part, the previously quoted literature reports on the influence of insulin treatment on PMN in diabetic patients. The effect of insulin was present with all the mediators used. They are known to activate PMN through different intracellular pathways as Sandborg and Smolen (1988) clearly reviewed: actually, fMLP acts through a plasma membrane receptor and G-proteins are most probably involved in signal transduction; the calcium ionophore A23187 increases intracellular calcium concentrations; PMA directly stimulates protein kinase C, thus by-passing membrane mechanisms. Our observation would suggest that insulin in some way stimulates general PMN responsiveness to the mediators by mechanisms that need to be further investigated. The effect of insulin is already present at the concentration of 40 uU/ml that is frequently attained in the everyday life of both healthy subjects and insulin-treated diabetic patients: this fact is in favor of a physiological insulin role in the modulation of PMN function. Recently, we demonstrated that insulin physiologically modulates also platelet function {Trovati, Anfossi, Cavalot, Massucco, Mularoni and Emanuelli 1988). In conclusion, our data support the hypothesis that insulin treatment ameliorates PMN function not only by the attainment of a better metabolic control — as the studies in vivo could suggest - but also through an effect directly exerted by the hormone itself. The depressed PMN function frequently described in poorly controlled diabetic patients could therefore be attributed not only to the elevated blood glucose concentrations, but also to the insulin deficiency per se or to deficient insulin action. Even if further studies are needed
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References
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228 Horm. metab. Res. 24 (1992)
