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Originally published online as doi:10.1189/jlb.0104050 on September 2, 2004

Published online before print September 2, 2004
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(Journal of Leukocyte Biology. 2004;76:1104-1110.)
© 2004 by Society for Leukocyte Biology

In vivo evidences that insulin regulates human polymorphonuclear neutrophil functions

S. Walrand*,{dagger},1, C. Guillet*, Y. Boirie* and M-P. Vasson{dagger}

* Unité du Métabolisme Protéino-Energétique, UMR Université d’Auvergne/INRA CHU de Clermont-Ferrand, and
{dagger} Laboratoire de Biochimie, Biologie Moléculaire et Nutrition, EA 2416, Faculté de Pharmacie, Centre de Recherche en Nutrition Humaine, Clermont-Ferrand, France

1 Correspondence: Unité du Métabolisme Protéino-Energétique, Laboratoire de Nutrition Humaine, BP 321, 58 rue Montalembert, 63009 Clermont-Ferrand Cedex 1, France. E-mail: swalrand{at}clermont.inra.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polymorphonuclear neutrophils (PMN) are able to destroy invasive mircoorganisms by a wide variety of functions. Whereas insulin does not stimulate hexose transport in PMN, previous reports have clearly shown that this hormone regulates glucose metabolism inside this cell, raising the question of insulin action on PMN functions in humans. It is interesting that in vitro studies established a strong relationship between specific binding of insulin to its PMN membrane receptor and the activation of the main PMN functions. Therefore, investigation in healthy subjects under strict euglycemia and physiological insulinemia was performed to understand the in vivo-specific action of insulin on PMN functions without hyperglycemia interferences. We determined numerous PMN functions before and after hyperinsulinemia (0.5 mU/kg/min) and euglycemia (0.9 g/l) clamp for 4 h in eight adult healthy volunteers (24±6 years). The total number of PMN and the number of PMN expressing CD11b, CD15, CD62L, and CD89 were significantly increased over baseline (P<0.001), whereas the density of these receptors was down-regulated (P<0.01) by insulin. PMN chemotaxis (+117%, P<0.05), phagocytosis (+29%, P<0.001), and bactericidal (+17–25%, P<0.001) capacities were increased during the insulin clamp (P<0.05). Therefore, insulin treatment may modulate PMN functions not only by attainment of a better metabolic control, as suggested by in vivo studies in diabetic patients, but also through a direct effect of insulin.

Key Words: PMN • chemotaxis • phagocytosis • CD expression • reactive oxygen species production


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
After a microorganism has gained entry in the body, the first line of host defense is the nonspecific immune effector system, namely the phagocytic cells such as polymorphonuclear neutrophils (PMN), which possess a great variety of functions that consist in a sequence of events including chemotaxis, adhesion to endothelium and to foreign agents, phagocytosis, and microbicidy [1 ]. With respect to the destruction mechanisms, activated PMN destroy microorganisms by producing toxic reative oxygen species (ROS), proteases such as elastase, and other compounds interfering with bacterial growth and metabolism, such as lactoferrin.

Cellular functions of human PMN including motility, phagocytosis, and bactericidal activity require energy derived from glucose [2 ]. Whereas insulin does not stimulate hexose transport in this immune cell, previous reports have clearly shown that this hormone is able to regulate glucose metabolism in PMN [3 ]. A 50% reduced PMN glucose use and glycolysis were found in a group of patients suffering from diabetes mellitus [4 ]. As the energy production of leukocytes mainly depends on aerobic glycolysis, it is to be expected that the ATP production in diabetic cells is depressed. In addition, insulin-requiring diabetic patients, receiving half of their maintenance dose of insulin, showed normalization of glucose use and glycolysis in PMN. Glycogen synthesis was also severely depressed by 40–60% in leukocytes of diabetic patients [5 ]. Other works confirmed the in vitro effect of insulin on glucose use in PMN and also demonstrated the stimulating role of this hormone on glycogen synthesis [5 ]. When the same problem was investigated under in vivo conditions, a significant increase in glucose use and lactate and glycogen formation was observed in leukocytes after treatment of diabetic patients with insulin [6 ]. Taken as a whole, these observations partly explained that poorly controlled diabetic patients are prone to infection, which may be an important risk factor of increased morbidity and mortality.

Severe infection in diabetes millitus often arises from an altered function of blood components [7 ]. It has been recognized for many years that patients with diabetes demonstrate an impaired PMN function for which a number of different and contradictory mechanisms have been described (for review, see ref. [8 ]). For instance, data showed that bactericidal ROS production by activated PMN is reduced [9 ], normal [10 ], or increased [11 ] in patients with type 1 diabetes mellitus. The discrepancies among these investigations might be partly explained by the various metabolic controls of the selected population and by the procedure used to evaluate ROS generation in PMN. In addition, there is controversy about correlation between intrinsic defects in PMN function and the metabolic control of the disease. The effects of insulin-induced hypoglycemia on the PMN respiratory burst were investigated in diabetic or nondiabetic subjects [10 ]. In this study, ROS production by PMN was decreased in response to hypoglycemia in healthy and diabetic subjects, this phenomenon being more pronounced in nondiabetic volunteers. These data demonstrated that metabolic control of glucose homeostasis by insulin treatment might correct impaired PMN function in patients with diabetes mellitus. It is interesting that in vitro studies [12 , 13 ] also established the existence of a correlation between specific binding of insulin to its receptor and its effects on PMN functions. 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 a deficient insulin action [7 ]. However, the in vivo-specific effect of insulin on the main PMN functions has not been well defined. Therefore, investigations in healthy subjects under strict euglycemia and physiological plasma insulin concentrations were performed to understand the action of insulin on PMN function without hyperglycemia interferences.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects’ characteristics
Young, healthy subjects were included in the study (Table 1 ). As subject selection is of crucial importance in immunological studies, all volunteers were selected according to the same admission criteria. All volunteers were checked for acute and chronic diseases and for signs of infection and inflammation, and none was taking medications that would affect the immune system (anti-inflammatory drugs, hormones, and analgesics). None was recently vaccinated. In addition, all of the subjects were fully ambulant and sedentary, with a similar activity level, as assessed by a physical activity questionnaire. All participants were nondiabetic, nonimpaired glucose-tolerant, and nonobese subjects (see Table 1 ).


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  		Table 1. Subjects’ Characteristics (n=8)

 
Before the study, all the subjects received a standardized diet (1.6x the predicted resting energy expenditure and containing 15% proteins) for 4 days (stabilization period) to normalize energy and protein intakes, which can affect the immune status.

Our institution is authorized by the French Ministry of Health to perform experiments on healthy volunteers. The study was carried out after approval by the Ethics Committee from the Auvergne area and was performed in accordance with the ethical standards of the Declaration of Helsinki. All the subjects provided written informed consent in accordance with the Ethics Committee guidelines for the protection of human subjects.

Biochemical and hematological characteristics
Plasma insulin concentrations were measured by radioimmunoassay (CIS, Gif-sur-Yvette, France). Plasma albumin and C-reactive protein levels were determined by immunonephelemetry and turbidimetry (Array protein system, Beckman-Coulter, Villepinte, France), respectively, with the use of human antibodies (Dako, Trappes, France).

The assessment of blood cellularity (leukocyte number and differential count) was determined with the use of a Coulter counter (Coultronics, Margency, France) distinguishing the five principal white blood cell types: lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

Hyperinsulinemic, euglycemic clamp protocol
Sensitivity of PMN to the in vivo action of insulin was measured using the hyperinsulinemic, euglycemic clamp technique. To avoid hyperinsulinemia-related hypoaminoacidemia, which may affect immune cell functions [18 ], amino acid solution was also infused during the clamp. After an overnight fasting (12 h), two catheters were inserted in the right antecubital vein for the infusion of insulin, amino acid mixture, and glucose, respectively. Human insulin (Actrapid Hmge, Novo Nordisk Pharmaceutique S.A., Boulogne-Billancourt, France) was given at a constant infusion rate of 0.7 mU/kg–1 fat-free mass/min–1 for 4 h by using a calibrated syringue pump (Orchestra DPS, Fresenius Vial, France). Plasma insulin level was determined in basal-fasted conditions and after the 4 h infusion (see Table 1 ). Blood glucose concentration was "clamped" at a predetermined level (e.g., fasting level) by titrating a variable-rate glucose infusion. Practically, glycemia was measured at the bedside every 5 min during the 4 h of the infusion by an automate glucose analyzer (Glucose Analyzer 2, Beckman, Fullerton, CA). On the basis of the plasma glucose concentration, glucose infusion (20% dextrose, Braun Medical AG, Emmenbrücke, Switzerland) was performed to keep glycemia constant [0.3 g/l]. Amino acid mixture solution (10%, Primene®, Clintec Clinical Nutrition, Vélizy-Villacoublay, France) was also infused during the insulin clamp at a rate of 0.020 ml/kg/min to avoid any insulin-mediated decrease in plasma amino acid concentration.

Insulin sensitivity was evaluated by calculating the M value using the following equation [14 ]: M value = rate of perfused glucose during the clamp/[glycemia during the clampx(insulinemia during the clamp–basal insulinemia)].

Characterization of insulin sensitivity was also realized by using a continuous infusion of D-[6,6-2H2]-glucose (0.05 mg.kg–1.min–1, Euriso-Top, Gif-sur Yvette, France) throughout the clamp. Isotopic and chemical purities of the tracer were checked by gas chromatography mass spectrometry (Hewlett-Packard 5971A, Palo Alto, CA). Solution of labeled glucose was tested for sterility and pyrogenicity and was membrane-filtered through a 0.22-µm pore-size filter throughout the experiment. GDR was calculated from the dilution of labeled glucose in plasma using a monocompartment model and Steele’s equations [16 ]. Insulin sensitivity was characterized by the ratio GDR/insulinema [15 ] (Table 1) .

Analysis of PMN functions
Blood samples were taken in EDTA vacutainers before insulin infusion and 4 h after the beginning of insulin clamp to determine PMN functions.

PMN receptor expressions and densities
All antibodies and products used for the determination of PMN receptor expressions and densities were purchased from Beckman-Coulter. PMN receptor expressions were measured by flow cytometry (Epics XL, Beckman-Coulter) after preparing the blood with the Q-Prep Epics immunology work station (Beckman-Coulter).

PMN subsets were quantified by immunoreaction with fluorochrome-conjugated monoclonal antibodies (mAb). PMN receptor densities were determined by using the Epics Immuno Brite® kit. The kit is composed of five vials containing particles of a specific fluorescent intensity (blank, medium-low, medium, medium-high, bright). From each of the five bottles, 500 µL was dispensed into a sample tube, and the fluorescence of each particle population was recorded by flow cytometry. The fluorescence intensity values measured were graphed against the corresponding fluorochrome density of each of the five particle populations to obtain a straight line. The fluorescence intensity of each sample was reported in the graph to deduce the receptor density.

Insulin receptor density was measured by using a sandwich reaction with an anti-insulin receptor detected with a fluorescein isothiocyanate (FITC)-conjugated antiantibody. The panel of mAb used to measure the other PMN receptor expressions and densities was as follows: FITC anti-CD11b, FITC anti-CD15, FITC anti-CD62L, phycoerythrin (PE) anti-CD89. Results are expressed as the percentages and absolute numbers [Giga/liter (G/L)] of PMN that expressed receptors and as the number of membrane receptors per PMN (receptor density).

Intra-assay and interassay coefficients of variations (CVs) were recorded by using a lyophilized preparation of human immune leukocytes, which exhibited surface antigens (Cyto-Trol Control Cells®, Beckman-Coulter), and a suspension of fluorospheres, which were uniform in size and fluorescence intensity (Flow-Count Fluorosphere®, Beckman-Coulter). Intra-assay and interassay CVs were <2% for all measurements made with the flow cytometer (PMN receptor expressions and functions).

PMN chemotaxis assay
Whole blood samples were carefully layered onto a discontinuous Ficoll-Hypaque density gradient (1.077 and 1.119 g/cm3, Sigma, Saint-Quentin-Fallavier, France) and spun (700 g, 30 min, 20°C). PMN were then collected on the corresponding layer and washed twice in phosphate-buffered saline (PBS; Sigma). PMN were tested for purity (>95%) and viability (>95%) using May-Grunwald-Giemsa staining and the Trypan blue dye exclusion test, respectively. The final cell suspension was then counted in a Malassez chamber (MC2, Clermont-Ferrand, France). Freshly isolated PMN (1x106) were added to a Multiwell insert system (Becton Dickinson, Meylan, France) with a 3-µm polyethylene terephthalate membrane placed in a 24-well plate. RPMI-1640 medium (Sigma), with or without 10–7 mole/L of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP), was added in the lower chamber to measure directed and spontaneous migrations (DM and SM), respectively. PMN were then allowed to migrate for 90 min at 37°C in a humidified atmosphere containing 5% CO2. After migration, PMN of the lower chambers were removed and counted using the flow cytometer. The chemotaxis index corresponding to the ratio of the number of PMN that migrated in the lower chamber in response to fMLP (DM) to the number of cells that migrated spontaneously (SM) was calculated. Results were presented as SM-stimulated migration toward fMLP and chemotactic index, i.e., DM/SM.

PMN phagocytosis index
PMN phagocytosis was evaluated indirectly by measuring the ROS production induced by the engulfment of AB serum opsonized zymosan (OZ; Sigma). Whole blood (500 µl) was treated rapidly with a hemolytic solution (0.15 mol/L NH4Cl, 12 mmole/L NaHCO3, 0.1 mmole/L EDTA). Leukocytes were then washed twice using PBS and adjusted to 106 cells/ml with RPMI-1640 medium. Cells were preincubated for 15 min with 1 µmol/L dihydrorhodamine 123 (DHR 123; Sigma) in a water bath with permanent horizontal agitation at 37°C. OZ solution was then added to the medium. This results in a PMN oxidative burst during which nonfluorescent, intracellular DHR 123 is oxidized to highly fluorescent rhodamine 123 (Rh 123) by hydrogen peroxide [17 ]. Individual PMN were discerned and counted, and Rh 123 fluorescence emission was measured by using flow cytometry analysis. Results were expressed as the percentage of PMN able to engulfe OZ and as the fluorescence intensity of positive PMN, i.e., the phagocytic index.

PMN bactericidal indexes
PMN microbicidy was determined by measuring the ROS generation after PMN activation and by evaluating PMN myeloperoxidase (MPO) and lactoferrin expressions. ROS production after phorbol myristate acetate (PMA; 10–6 mole/L) or fMLP (10–5 mole/L) stimulation was measured by using the DHR 123 probe method, as described previously [17 ]. PMN MPO and lactoferrin expressions and contents were measured after permeabilization and fixation of the cells by treating 50 µL whole blood with saponin and formaldehyde (IntraPrep Permeabilization® reagent, Immunotech, Marseille, France), respectively. After permeabilization, FITC anti-MPO and PE antilactoferrin antibodies were added, and the blood preparation was incubated during 15 min at room temperature in the dark and washed once with PBS. The intracellular MPO and lactoferrin expressions (in percents and G/L) and densities (number of MPO and lactoferrin molecules per PMN) were thereafter analyzed by flow cytometry as described above for membrane receptor expressions.

Statistical analysis
Data are presented as means ± SEM, and statistical analysis was performed using PCEM software (Deltasoft, Grenoble, France). A repeated measure ANOVA was used. The level of significance was set at P < 0.05 for this test. When the ANOVA indicated significant differences, the Newman-Keüls test was performed to identify differences between individual means. A P < 0.05 was considered significant for this test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Plasma insulin concentration
During insulin clamp, plasma insulin concentration rose significantly and reached physiological, postprandial values (P<0.001 vs. basal; Table 1 ).

White blood cell counts
The total leukocyte count was increased during the clamp (P<0.01 vs. basal; Fig. 1 ). This variation was partly a result in a rise in PMN number (P<0.01 vs. basal), whereas lymphocyte count decreased (P<0.05 vs. basal) during the insulin infusion (Fig. 1) .



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  		Figure 1. White blood cell counts before and after insulin clamp in adult healthy subjects. Data are presented as means ± SEM. White blood cell counts are expressed in G/L. Repeated-measure ANOVA + Newman-Keüls test. *, P < 0.05, and **, P < 0.01, versus before insulin clamp.

 
Indexes related to PMN function
PMN receptor expressions and densities
Insulin receptor density did not vary according to the clamping period, i.e., before and after insulin treatment (Fig. 2 ).



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  		Figure 2. PMN receptor densities before and after insulin clamp in adult healthy subjects. Data are presented as means ± SEM. Receptor densities are expressed in number of membrane receptors per PMN. Repeated-measure ANOVA + Newman-Keüls test. **, P < 0.01, versus before insulin clamp.

 
The percentage of PMN expressing CD11b, CD15, CD62L, and CD89 was not altered by the insulin clamp (Table 2 ). The absolute number of PMN expressing CD11b+, CD15+, CD62L+, or CD89+ was significantly increased by insulin infusion (P<0.01 vs. basal). Insulin clamp also significantly down-regulated PMN receptor densities, whatever the receptor considered (P<0.01 vs. basal; Fig. 2 ).


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  		Table 2. PMN Receptor Expressions before and after Insulin Clamp in Adult Healthy Subjects

 
PMN chemotaxis
PMN chemotaxis ability (DM/SM ratio) was higher during insulin clamp than during basal conditions (P<0.05 vs. basal; Fig. 3 ). The increase in PMN migration was related to a rise in DM capacity toward fMLP (P<0.05 vs. basal; Fig. 3 ).



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  		Figure 3. PMN chemotaxis ability before and after insulin clamp in adult healthy subjects. Data are presented as means ± SEM. PMN chemotaxis is expressed as the spontaneous migration (SM), the directed migration (DM) toward fMLP, and the ratio DM/SM. Repeated-measure ANOVA + Newman-Keüls test. *, P < 0.05, versus before insulin clamp.

 
PMN phagocytosis indexes
The percentage of PMN able to ingest OZ particles was significantly increased after insulin infusion (P<0.05 vs. basal; Table 3 ). The fluorescence intensity corresponding to the phagocytic activity of PMN also rose significantly by insulin clamp (P<0.001 vs. basal).


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  		Table 3. PMN Phagocytosis Indexes before and after Insulin Clamp in Adult Healthy Subjects

 
PMN bactericidal indexes
The proportion of PMN able to produce ROS after PMA stimulation did not change (Table 4 ). However, the percentage of cells activated by fMLP and the absolute number of PMA- and fMLP-positive PMN increased after insulin clamp. In addition, ROS production by PMA- and fMLP-stimulated PMN was significantly increased by insulin (P<0.001 vs. basal; Table 4 ).


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  		Table 4. PMN Bactericidal Indexes before and after Insulin Clamp in Adult Healthy Subjects

 
The percentage of cell expressing MPO and lactoferrin was not modified by insulin infusion (Table 4) . However, the absolute number of PMN containing MPO and lactoferrin was significantly increased by insulin clamp (P<0.001 vs. basal). This result was partly a result of the variation in PMN count induced by insulin infusion (Fig. 1) . Conversely, MPO and lactoferrin densities inside PMN were significantly decreased by insulin (P<0.001 vs. basal; Table 4 ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrated that insulin is a strong regulator of the main PMN functions in nondiabetic, healthy subjects. The hallmark of diabetes, i.e., hyperglycemia, rather than a reduced insulin action, was supposed to be the only triggering factor for the impaired PMN activity in patients with diabetes mellitus [19 20 21 ]. However, a recent study [22 ] revealed that a large dose of insulin infusion to counteract insulin resistance during cardiac surgery had a beneficial effect on PMN function and incidence of infection in nondiabetic patients. In these investigations, high glucose levels were not improved by insulin infusion, suggesting that this hormone may act as an immune activator independently of glucose regulation. We also observed that insulin increased peripheral blood PMN counts, which thereby improved phagocytic potentiality. The mechanism by which insulin may increase PMN counts is unknown. However, there is considerable evidence to suggest that an increased plasma insulin level may increase PMN count by reducing leukocyte endothelium adherence through its effect on glycemia [23 ]. Hyperglycemia in nondiabetic or diabetic subjects resulted in an increase in leukocyte adhesion molecule expression, such as CD11b or CD18, and consequently, in leukocyte adherence. However, plasma glucose concentration was maintained at a fasting level, i.e., 0.9 g/L, throughout the present study. Consequently, the effect of insulin on PMN count and functions cannot be explained by the modulation of glycemia by this hormone. Our observation revealed that insulin is able to down-regulate the adhesion molecule density, i.e., CD11b, CD15, CD62L, and CD89, at the PMN membrane level. CD11b, CD15, CD62L, and CD89 are adhesion receptors that promote interaction of PMN with each other, with endothelial cells, and with specific opsonins, such as complement fractions, deposited on invading microorganisms [24 ]. This finding suggests that insulin might diminish the adhesion potentiality of circulating PMN by decreasing the expression of the receptor found on their surface, leading to an increased PMN count. Okouchi et al. [25 , 26 ] have recently studied the in vitro effect of a high insulin level on PMN endothelial adhesion and transmigration across human umbilical vein endothelial cell-culture inserts. In spite of no effect of the hormonal treatment on PMN adhesion to the endothelium, these authors found that PMN transmigration was increased in the presence of insulin. These observations can be explained, as shown by our results, by the stimulating effect of insulin on PMN migration ability. The biochemical mechanism of insulin effect on PMN chemotaxis is still unknown. However, chemotaxis is a highly glucose-dependent, biochemical pathway [27 28 29 ], and high insulin levels may improve glucose metabolism within PMN [5 ]. In addition, a series of studies by Oldenborg and Sehlin [30 , 31 ] and by Cavalot et al. [13 , 32 ] revealed that insulin is able to in vitro, dose-dependently increase PMN migration capacity in healthy conditions. Although we showed that PMN were equipped with the insulin receptor, the signaling pathway in PMN involved in mediating the effects of this hormone on PMN chemotaxis from its receptor and downstream has been poorly described. As revealed by in vitro assays [30 ], the classical tyrosine phosphorylation pathway of this receptor seems to be a crucial, initial event in the mediation of the insulin effect on PMN migration. Indeed, the tyrosine kinase inhibitor genistein and the phosphatidylinositol 3-kinase inhibitor wortmanin did abolish the chemotactic-activating effect of insulin [30 ], suggesting that the PMN insulin receptor follows that general pattern.

One possible explanation for insulin-stimulated action on phagocytosis, i.e., the ability of PMN to engulf foreign agents, described in the present study might be its action on the cytoskeletal element, as it was postulated for PMN granule secretion [33 ]. However, the method used was rather indirect and dependent on the assumption that insulin did not affect the complement receptor-mediated respiratory burst. In addition, our observation has to be confirmed by using zymosan opsonized by homologous serum, i.e., prepared from the blood of each subject.

After engulfment of the microbial organism, PMN destroy it by producing toxic compounds such as ROS, lactoferrin, or proteases [34 , 35 ]. It is well known that the response of PMN to fMLP can be divided into two categories: chemotaxis, originating at low concentrations of the peptide, and respiratory burst, i.e., production of ROS, developing at a higher fMLP level. Binding of fMLP to its PMN receptor activates a G-protein-dependent biochemical cascade leading to the protein kinase C (PKC) activation [34 ]. PMA can freely cross the cell membrane and thanks to its analogy with diacylglycerol, can also stimulate PKC directly. PKC activation induces the assembly and the activation of the multicomponent system reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a transmembrane electron transport chain that reduces oxygen to ROS [17 ]. We documented, as already described by others [12 , 36 ], but only by using in vitro assays, a stimulating effect of insulin infusion on respiratory burst in PMN. Safronova et al. [12 ] demonstrated that the insulin action on bactericidal ROS generation in PMN was inhibited by the tyrosine kinase inhibitor tyrphostin. These authors postulated that binding of insulin to its PMN receptor activated the receptor-tyrosine kinase and subsequent phosphorylation of its substrates. The activation of this biochemical pathway may lead to the initiation of the mitogen-activated protein kinase (MAPK) cascade and consequently, possible additional phosphorylation of components of the NADPH oxidase. This probably caused increased activation of the ROS pathway [12 ]. Therefore, MAPK and PKC, causing phosphorylation of components of the NADPH oxidase, through insulin or fMLP and PMA pathways, respectively, could form the basis for the cross-interaction leading to the increase in ROS production described in the present study after insulin infusion. Insulin seemed to also possess a down-regulating action on enzymes involved in ROS elimination, as shown in this study for MPO. It was also found that MPO activity was reduced by in vitro insulin treatment in the cell-free supernatant of healthy PMN [37 ] and was altered in noncontrolled diabetic patients [38 ]. In addition, intravenous insulin infusion to induce hypoglycemia in healthy humans provoked an increase in the PMN count with associated elevation in plasma PMN elastase concentration [39 ]. Consistent with these data, MPO and lactoferrin might be spontaneously released from circulating PMN during the insulin clamp, reducing their concentrations in the cell. In addition, the decrease in MPO and lactoferrin content was associated with the rise in PMN count during insulin treatment. This observation might also suggest that the PMN, mobilized in response to the hormone, contained less MPO and lactoferrin in comparison with the PMN present in blood before the clamp.

In summary, the increased sensitivity of infections in diabetes has long been associated with changes in PMN functions [8 ]. Most studies have focused on the involvement of hyperglycemia in the defective PMN. Correction of impaired PMN functions, including chemotaxis, adherence, phagocytosis, and bactericidy, was indeed reported in diabetic subjects after improved metabolic control by insulin treatment. However, the effects of insulin treatment described in vivo in poor metabolic-controlled diabetic patients were not necessarily a result of an indirect effect of this hormone on glycemia regulation. Athough the entry of glucose into PMN is widely considered to be insulin-independent, former studies (for review, see ref. [5 ]) demonstrated that leukocytes of diabetic patients display a decreased rate of glycolysis, decreased lactate production, and decreased glycogen synthesis—these abnormalities being corrected by in vivo insulin treatment. Insulin also increased in vitro glucose consumption of PMN from nondiabetic human individuals without influencing the permeability of the plasma membrane to glucose. Finally, our data support the hypothesis that insulin treatment may normalize PMN functions, not only by attainment of a better metabolic control, as suggested by in vivo studies in diabetic patients, but also through a direct effect of insulin. It is interesting that the use of intensive insulin therapy substantially reduced morbidity and mortality as a result of multiple organ failure with a proven septic focus among nondiabetic, critically ill patients [40 ]. Taken as a whole, these observations emphasize the potential role of this hormone as an immunoregulator agent. Therefore, insulin action could be attributed to the improvement of glucose metabolism within PMN and to a direct immunostimulating effect on the main PMN functions. Before activation, PMN are usually turned to a primed state induced by agents, which prepare the cells for a faster and stronger response [12 ]. Amplification of PMN activity under priming may be a result of additive phosphorylation, which could represent the interaction between the signaling systems of the primer and the activator. Insulin is not classified as a primer in contrast with insulin-like growth factor-I [41 ], which has a similar structure, although there is a reason to believe that insulin may prime PMN.

Received January 29, 2004; revised July 15, 2004; accepted July 28, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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