Dm
ORIGINAL ARTICLES
Leucocyte Mobilization and Release of Neutrophil Elastase Following Acute Insulin-induced Hypoglycaemia in Normal Humans A. Collier, A.W. Patrick, D.A. Hepburn, D. Bell, M. Jacksona,J. Dawes”, B.M. Frier Diabetic Department, Royal Infirmary and aB/ood Components Assay Croup, Edinburgh, Scotland
Insulin-induced hypoglycaemia in humans is associated with the rapid mobilization of leucocytes in peripheral blood. The aim of the present study was to determine whether neutrophil activation, manifested in plasma by neutrophil elastase concentration, occurs in response to insulin-induced hypoglycaemia. Acute hypoglycaemia (mean blood glucose 1.3 k 0.2 mmol I-’; mean 2 SD) was induced with intravenous insulin in 15 normal human subjects, and provoked an increase in the neutrophil count from 3.4 (range 1.9-6.5) to 10.7 (9.4-16.3) x lo9 I-’ (p < 0.001), and in the total leucocyte counts from 5.7 (4.1-8.1) to 12.8 (11.3-18.6) x 10’ I-’ (p < 0.001), with associated elevations in plasma neutrophil elastase concentration from 21 (12-34) to 29 (14-70) Fg I-’ (p < 0.05), and in total neutrophil elastase concentration from 5.90 (3.1 3-8.20) to 25.20 (23.00-52.00) mg I-’ ( p < 0.001). As neutrophil elastase is implicated in the development of vascular disease, this rise in response to hypoglycaemia may be of pathological importance in insulin-treated diabetic patients. KEY WORDS
Hypoglycaemia Neutrophils Leucocytes Plasma neutrophil elastase Total neutrophil elastase
Introduction The brisk counter-regulatory hormonal response to acute insulin-induced hypoglycaemia in normal humans includes the secretion of catecholamines and cortisol.’ These hormones induce a biphasic elevation of leucocytes in peripheral blood, with an initial rise in the total lymphocyte count followed by a later rise in the granulocyte count.24 The secretion of catecholamines causes haemoconcentration with an acute rise in blood haematocrit,*r3 a reduction of plasma and an associated increase in blood viscosity,6 with concomitant platelet activation and aggregation and fibrinolysis.’-” It has been suggested that when these acute changes are superimposed upon established diabetic microangiopathy, they may encourage intravascular coagulation, reduce capillary blood flow, and precipitate capillary closure, thus aggravating the microvascular complications of diabetes. ‘ O f ’ Plasma and total neutrophil elastase concentrations are elevated in diabetic patients, and elastase has been implicated in the development of diabetic vascular d i ~ e a s e . ’ ~ ,The ’ ~ aim of the present study was to determine whether neutrophil activation, manifested by
Correspondence to: A. Collier, Level 7, Gartnavel General Hospital, 1053 Gt Western Road, Glasgow, G12 OYN, UK
506
0742-3071190/060506-04$05.00
0 1990 by John Wiley
& Sons, Ltd.
a rise of plasma neutrophil elastase, accompanies the leucocyte mobilization which occurs in response to insuIi n-i nduced hypogl ycaem ia in non-d iabetic subjects.
Subjects and Methods
.
Fifteen healthy male volunteers, aged 23-39 years, were studied in a supine position after an overnight fast. Smoking and the ingestion of caffeine were prohibited for the preceding 12 h. All subjects gave informed consent and the study had the approval of the local Medical Ethical Advisory Committee. An intravenous cannula was inserted into an antecubital vein, and basal blood samples were taken after resting for 30 min. At time zero, human insulin (Human Actrapid, Novo Laboratories, Basingstoke, UK) was given as an IV bolus injection in a dose of 0.125 U kg-body-weight-’ to induce acute hypoglycaemia. Blood glucose was monitored at the bedside using a meter (Reflolux, BCL, Lewes, UK), and heart rate and blood pressure were measured frequently throughout the study. Serial blood sampling for ‘the measurement of blood glucose was performed until 120 min after identification of the acute autonomic reaction, which coincided with the nadir of blood glucose. Sampling was timed from the reaction (= R ) to account for individual variability in the time of onset of acute hypoglycaemia after the administration of insulin. Accepted 19 February 1990
DIABETIC MEDICINE, 1990; 7: 506-509
DT17 Blood glucose was measured using a Yellow Springs Glucose Oxidase Analyser (Yellow Spring Instruments, Yellow Springs, OH, USA). Venous blood for plasma neutrophil (PNE) and total neutrophil elastase (TNE) measurements was collected at three time-points (baseline, R + 45 min, and R + 120 rnin). The blood was anticoagulated with 31.2 g IF1 trisodium citrate in 50 g I-’ Hepes buffer for measurement of plasma neutrophil elastase, and 10 ml I-’ Triton X-100 was added to measure total neutrophil elastase. The sample for PNE was centrifuged at 1500 Xg for 10 min at 4 “C and the plasma aspirated. Both plasma and lysed whole blood were stored at -20 “C and assayed within a week of sampling. A full blood count was performed using a Coulter S Plus IV (Coulter Electronics, Luton, UK) and a differential leucocyte count was performed manually on blood films stained with May-Grunwald/Giemsa. Human neutrophil elastase was measured by a specific radioimmunoassay using rabbit polyclonal antiserum. The antigen was purified from human neutrophils following leucapheresis. The antibody was absolutely specific for neutrophil elastase and did not cross-react with pancreatic or platelet elastase. Neutrophil elastase was measured either as the free enzyme or as a complex with its inhibitors, a,-antitrypsin and a2-macroglobulin.14 In brief, 50 pl of standardhample was added to 50 pI 1251-elastase(10 pg IF1) and 50 pl anti-elastase antibody (dilution 1:3000) and made up to 200 pl with buffer comprising 0.05 mol 1-l phosphate (pH 7.4), 0.6 mol IF1 NaCI, 2 mmol.I-’ EDTA, 130 g I-’ heparin, 20 ku IF1 aprotinin, and 20 ml I-] heat-inactivated horse serum. Samples were incubated overnight at room temperature and separated with donkey anti-rabbit immunoglobulin immobilized on Sepharose. After shaking for 45 min at room temperature the bound complex was separated from the free complex by sedimentation under gravity through a 100 g IF1 sucrose solution, then aspirated and counted on a NE 1600 gamma counter (Nuclear Enterprises, Edinburgh, UK). Plasma neutrophil elastase and total neutrophil elastase are expressed as pg IF1 and mg I-l, respectively. The inter- and intra-assay coefficients of variation were less than 5 %.
Sta tis tica I Analysis Results are expressed as mean (SD), or as median (range) where the data were not normally distributed. The data from the three time-points were compared using the Wilcoxon rank sum test with the Bonferroni correction.
ResuI t s
Glucose All subjects experienced an acute autonomic reaction (time = R ) typical of acute hypoglycaemia with sudden NEUTROPHIL ELASTASE AND HYPOCLYCAEMIA
O R I G I N A L ARTICLES onset of sweating and a tachycardia at approximately 20 min (range 18-28 min) after the administration of insulin. The blood glucose fell from a basal value of 4.6 (0.2) mmol IF’ to a nadir of 1.3 (0.2) mmol I-’ (p < O.Ol), which coincided with time R, with subsequent recovery to basal values. The changes in heart rate and blood pressure were similar to those described previously in normal humans in response to acute hypoglycaemia.lo,’’
Leucocytes and Elastase The neutrophil count rose from a basal value of 3.4 (range 1.9-6.5) to 10.7 (9.4-16.3) X109 I-’ at time R 120 min ( p < 0.001), while the total leucocyte count increased from 5.7 (4.1-8.1) to 12.8 (11.3-18.6) x 1 0 9 I-’ ( p < 0.001) (Figure 1). The plasma neutrophil elastase concentration rose from a basal value of 21 (12-34) to 29 (14-70) p g I-] at time R + 120 min (p < 0.05) with 11 out of 1 5 subjects having a higher concentration of PNE at time R + 120 min. Two subjects had a higher concentration of PNE at time R 45 min, which declined to a sub-basal level at time R + 120 min. There was a pronounced rise in total neutrophil elastase from 5.90 (3.1 3-8.20) to 25.20 (23.00-52.00) mg IF’ (p < 0.001) (Figure 2). A correlation was demonstrated between the total neutrophil elastase concentration and the neutrophil count before the induction of hypoglycaemia (r = 0.74; p < 0.001), but no correlation was observed between total neutrophil elastase and the neutrophil count at either time R 45 min or time R 120 min. No correlation was found between the plasma neutrophil elastase and the neutrophil count at any of the three times of measurement.
+
+
+
+
Discussion The mobilization of leucocytes which is provoked by acute hypoglycaemia in humans comprises an early rapid lymphocytosis which i s adrenergically-mediated, and a later rise in the neutrophil count which appears to be mediated primarily by c ~ r t i s o l .Leucocytes ~ have been implicated in the development of vascular endothelial damage in humans, through their release of acid and neutral proteases, including neutrophil elastase.12-15 Neutrophil elastase is a potent proteolytic enzyme which is capable of destroying host as well as foreign protein. It is present in high concentration in the azurophilic granules of neutrophils and in myeloid precursor^.^^,^^ In the present study a radioimmunoassay specific for neutrophil elastase was used which measures neutrophil elastase with equal facility in either its free form or when complexed to its inhibitors, a,-antitrypsin and a*macroglobulin. Although most of the plasma neutrophil
507
Dm
ORIGINAL ARTICLES P
ORIGINAL ARTICLES
Leucocyte Mobilization and Release of Neutrophil Elastase Following Acute Insulin-induced Hypoglycaemia in Normal Humans A. Collier, A.W. Patrick, D.A. Hepburn, D. Bell, M. Jacksona,J. Dawes”, B.M. Frier Diabetic Department, Royal Infirmary and aB/ood Components Assay Croup, Edinburgh, Scotland
Insulin-induced hypoglycaemia in humans is associated with the rapid mobilization of leucocytes in peripheral blood. The aim of the present study was to determine whether neutrophil activation, manifested in plasma by neutrophil elastase concentration, occurs in response to insulin-induced hypoglycaemia. Acute hypoglycaemia (mean blood glucose 1.3 k 0.2 mmol I-’; mean 2 SD) was induced with intravenous insulin in 15 normal human subjects, and provoked an increase in the neutrophil count from 3.4 (range 1.9-6.5) to 10.7 (9.4-16.3) x lo9 I-’ (p < 0.001), and in the total leucocyte counts from 5.7 (4.1-8.1) to 12.8 (11.3-18.6) x 10’ I-’ (p < 0.001), with associated elevations in plasma neutrophil elastase concentration from 21 (12-34) to 29 (14-70) Fg I-’ (p < 0.05), and in total neutrophil elastase concentration from 5.90 (3.1 3-8.20) to 25.20 (23.00-52.00) mg I-’ ( p < 0.001). As neutrophil elastase is implicated in the development of vascular disease, this rise in response to hypoglycaemia may be of pathological importance in insulin-treated diabetic patients. KEY WORDS
Hypoglycaemia Neutrophils Leucocytes Plasma neutrophil elastase Total neutrophil elastase
Introduction The brisk counter-regulatory hormonal response to acute insulin-induced hypoglycaemia in normal humans includes the secretion of catecholamines and cortisol.’ These hormones induce a biphasic elevation of leucocytes in peripheral blood, with an initial rise in the total lymphocyte count followed by a later rise in the granulocyte count.24 The secretion of catecholamines causes haemoconcentration with an acute rise in blood haematocrit,*r3 a reduction of plasma and an associated increase in blood viscosity,6 with concomitant platelet activation and aggregation and fibrinolysis.’-” It has been suggested that when these acute changes are superimposed upon established diabetic microangiopathy, they may encourage intravascular coagulation, reduce capillary blood flow, and precipitate capillary closure, thus aggravating the microvascular complications of diabetes. ‘ O f ’ Plasma and total neutrophil elastase concentrations are elevated in diabetic patients, and elastase has been implicated in the development of diabetic vascular d i ~ e a s e . ’ ~ ,The ’ ~ aim of the present study was to determine whether neutrophil activation, manifested by
Correspondence to: A. Collier, Level 7, Gartnavel General Hospital, 1053 Gt Western Road, Glasgow, G12 OYN, UK
506
0742-3071190/060506-04$05.00
0 1990 by John Wiley
& Sons, Ltd.
a rise of plasma neutrophil elastase, accompanies the leucocyte mobilization which occurs in response to insuIi n-i nduced hypogl ycaem ia in non-d iabetic subjects.
Subjects and Methods
.
Fifteen healthy male volunteers, aged 23-39 years, were studied in a supine position after an overnight fast. Smoking and the ingestion of caffeine were prohibited for the preceding 12 h. All subjects gave informed consent and the study had the approval of the local Medical Ethical Advisory Committee. An intravenous cannula was inserted into an antecubital vein, and basal blood samples were taken after resting for 30 min. At time zero, human insulin (Human Actrapid, Novo Laboratories, Basingstoke, UK) was given as an IV bolus injection in a dose of 0.125 U kg-body-weight-’ to induce acute hypoglycaemia. Blood glucose was monitored at the bedside using a meter (Reflolux, BCL, Lewes, UK), and heart rate and blood pressure were measured frequently throughout the study. Serial blood sampling for ‘the measurement of blood glucose was performed until 120 min after identification of the acute autonomic reaction, which coincided with the nadir of blood glucose. Sampling was timed from the reaction (= R ) to account for individual variability in the time of onset of acute hypoglycaemia after the administration of insulin. Accepted 19 February 1990
DIABETIC MEDICINE, 1990; 7: 506-509
DT17 Blood glucose was measured using a Yellow Springs Glucose Oxidase Analyser (Yellow Spring Instruments, Yellow Springs, OH, USA). Venous blood for plasma neutrophil (PNE) and total neutrophil elastase (TNE) measurements was collected at three time-points (baseline, R + 45 min, and R + 120 rnin). The blood was anticoagulated with 31.2 g IF1 trisodium citrate in 50 g I-’ Hepes buffer for measurement of plasma neutrophil elastase, and 10 ml I-’ Triton X-100 was added to measure total neutrophil elastase. The sample for PNE was centrifuged at 1500 Xg for 10 min at 4 “C and the plasma aspirated. Both plasma and lysed whole blood were stored at -20 “C and assayed within a week of sampling. A full blood count was performed using a Coulter S Plus IV (Coulter Electronics, Luton, UK) and a differential leucocyte count was performed manually on blood films stained with May-Grunwald/Giemsa. Human neutrophil elastase was measured by a specific radioimmunoassay using rabbit polyclonal antiserum. The antigen was purified from human neutrophils following leucapheresis. The antibody was absolutely specific for neutrophil elastase and did not cross-react with pancreatic or platelet elastase. Neutrophil elastase was measured either as the free enzyme or as a complex with its inhibitors, a,-antitrypsin and a2-macroglobulin.14 In brief, 50 pl of standardhample was added to 50 pI 1251-elastase(10 pg IF1) and 50 pl anti-elastase antibody (dilution 1:3000) and made up to 200 pl with buffer comprising 0.05 mol 1-l phosphate (pH 7.4), 0.6 mol IF1 NaCI, 2 mmol.I-’ EDTA, 130 g I-’ heparin, 20 ku IF1 aprotinin, and 20 ml I-] heat-inactivated horse serum. Samples were incubated overnight at room temperature and separated with donkey anti-rabbit immunoglobulin immobilized on Sepharose. After shaking for 45 min at room temperature the bound complex was separated from the free complex by sedimentation under gravity through a 100 g IF1 sucrose solution, then aspirated and counted on a NE 1600 gamma counter (Nuclear Enterprises, Edinburgh, UK). Plasma neutrophil elastase and total neutrophil elastase are expressed as pg IF1 and mg I-l, respectively. The inter- and intra-assay coefficients of variation were less than 5 %.
Sta tis tica I Analysis Results are expressed as mean (SD), or as median (range) where the data were not normally distributed. The data from the three time-points were compared using the Wilcoxon rank sum test with the Bonferroni correction.
ResuI t s
Glucose All subjects experienced an acute autonomic reaction (time = R ) typical of acute hypoglycaemia with sudden NEUTROPHIL ELASTASE AND HYPOCLYCAEMIA
O R I G I N A L ARTICLES onset of sweating and a tachycardia at approximately 20 min (range 18-28 min) after the administration of insulin. The blood glucose fell from a basal value of 4.6 (0.2) mmol IF’ to a nadir of 1.3 (0.2) mmol I-’ (p < O.Ol), which coincided with time R, with subsequent recovery to basal values. The changes in heart rate and blood pressure were similar to those described previously in normal humans in response to acute hypoglycaemia.lo,’’
Leucocytes and Elastase The neutrophil count rose from a basal value of 3.4 (range 1.9-6.5) to 10.7 (9.4-16.3) X109 I-’ at time R 120 min ( p < 0.001), while the total leucocyte count increased from 5.7 (4.1-8.1) to 12.8 (11.3-18.6) x 1 0 9 I-’ ( p < 0.001) (Figure 1). The plasma neutrophil elastase concentration rose from a basal value of 21 (12-34) to 29 (14-70) p g I-] at time R + 120 min (p < 0.05) with 11 out of 1 5 subjects having a higher concentration of PNE at time R + 120 min. Two subjects had a higher concentration of PNE at time R 45 min, which declined to a sub-basal level at time R + 120 min. There was a pronounced rise in total neutrophil elastase from 5.90 (3.1 3-8.20) to 25.20 (23.00-52.00) mg IF’ (p < 0.001) (Figure 2). A correlation was demonstrated between the total neutrophil elastase concentration and the neutrophil count before the induction of hypoglycaemia (r = 0.74; p < 0.001), but no correlation was observed between total neutrophil elastase and the neutrophil count at either time R 45 min or time R 120 min. No correlation was found between the plasma neutrophil elastase and the neutrophil count at any of the three times of measurement.
+
+
+
+
Discussion The mobilization of leucocytes which is provoked by acute hypoglycaemia in humans comprises an early rapid lymphocytosis which i s adrenergically-mediated, and a later rise in the neutrophil count which appears to be mediated primarily by c ~ r t i s o l .Leucocytes ~ have been implicated in the development of vascular endothelial damage in humans, through their release of acid and neutral proteases, including neutrophil elastase.12-15 Neutrophil elastase is a potent proteolytic enzyme which is capable of destroying host as well as foreign protein. It is present in high concentration in the azurophilic granules of neutrophils and in myeloid precursor^.^^,^^ In the present study a radioimmunoassay specific for neutrophil elastase was used which measures neutrophil elastase with equal facility in either its free form or when complexed to its inhibitors, a,-antitrypsin and a*macroglobulin. Although most of the plasma neutrophil
507
Dm
ORIGINAL ARTICLES P
