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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-1619
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 7 2738-2748
Copyright © 2006 by The Endocrine Society

Insulin Differentially Regulates Monocyte and Polymorphonuclear Neutrophil Functions in Healthy Young and Elderly Humans

Stéphane Walrand, Christelle Guillet, Yves Boirie and Marie-Paule Vasson

Unité de Nutrition Humaine (S.W., C.G., Y.B.), Unité Mixte de Recherche Université d’Auvergne/Institut National de la Recherche Agronomique, Centre de Recherche en Nutrition Humaine, Centre Hospitalier Universitaire de Clermont-Ferrand, 63009 Clermont-Ferrand, France; and Laboratoire de Biochimie (M.-P.V.), Biologie Moléculaire et Nutrition, Faculté de Pharmacie, Centre de Recherche en Nutrition Humaine, 63001 Clermont-Ferrand, France

Address all correspondence and requests for reprints to: Stéphane Walrand, Ph.D., 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
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Insulin can regulate immune cell function. Aging is associated with various degrees of insulin resistance together with reduced immune cell activity.

Objective: We investigated the hypothesis that blood monocytes and polymorphonuclear neutrophils (PMNs) are less responsive to the action of insulin in elderly subjects.

Design-Intervention: We evaluated the effect of hyperinsulinemia (0.7 mU/kg–1 fat-free mass per minute–1) on monocyte and PMN activity using a 4-h euglycemic clamp technique.

Participants: Eight young (24 ± 6 yr old) and nine elderly (69 ± 4 yr old) healthy volunteers participated in the study.

Main Outcome Measures: Monocyte and PMN receptor expression and density were measured using flow cytometric detection. PMN chemotaxis toward formyl-Met-Leu-Phe (fMLP) was evaluated using a two-compartment chamber. PMN and monocyte phagocytosis was determined by measuring the engulfment of opsonized particles. Microbicidal functions were determined based on the production of reactive oxygen species (ROS) and bactericidal protein by stimulated cells.

Results: The density of PMN and monocyte insulin receptors was not affected by age or insulin clamp treatment regardless of the age. Insulin was able to regulate the expression of receptors involved in PMN action in the young-adult group only. PMN chemotaxis was up-regulated by insulin in both groups. In contrast, although insulin stimulated phagocytosis and bactericidal activity in young-adult subjects, the ability of PMN to adapt to physiological hyperinsulinemia was blunted in the older group. The effect of insulin on monocyte bactericidal properties seemed to be limited, although a suppressive action on fMLP-induced ROS production was detected in young adults.

Conclusions: We confirmed the presence of the insulin receptor on monocyte and PMN membranes. We revealed that insulin has a limited action on monocyte function. Insulin has a priming effect on the main PMN functions. Immune cell function adapted poorly to insulin infusion in the elderly subjects.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
AS THE HUMAN body enters its senior years, its ability to fight off infection and other health problems, such as neoplastic diseases, diminishes significantly. Many factors can contribute to this phenomenon, such as the presence of comorbid conditions, environmental factors, and the senescence of normal defense mechanisms in elderly subjects, termed immunosenescence. The immune system, which is responsible for fighting most of the diseases, simply does not function as efficiently in older adults as in younger people.

Given that 60–70% of blood leukocytes are granulocytes and over 90% of granulocytes are neutrophils, polymorphonuclear neutrophils (PMNs) are the largest fraction of white blood cells. PMNs possess a variety of functions including chemotaxis, adhesion to the endothelium and foreign agents, phagocytosis, and microbicidal activity. PMNs are able to penetrate and migrate into infected tissues and destroy invading microorganisms after internalization by producing multiple toxic agents such as reactive oxygen species (ROS), proteases (elastase), and proteins interfering with bacterial development (lactoferrin). Studies on PMN function in aged people have yielded conflicting results, but it is generally agreed that, as with other immune system components, there is a general decline in PMN number and functional activities (1, 2, 3). PMNs from elderly subjects often exhibit a diminished in vitro migration activity against chemoattractant agents (4, 5). However, an in vivo evaluation of PMN migration using the skin window technique reported quantitatively normal-range results in elderly subjects, indicating that neither endogenous generation of chemotactic compounds nor PMN movement mechanisms are affected by the aging process (6). Stimulated PMN adhesion seems to remain unchanged with age (6, 7, 8), although there have been reports on inhibited PMN adherence to human venous endothelial cells in elderly people (7).

Normal (4, 9) or impaired (10, 11) phagocytotic activity has also been documented in aged subjects. PMN phagocytosing foreign matter undergo a series of biochemical events that lead to a variety of microbicidal defensive responses including ROS production and lytic enzyme release. ROS are generated during a complex process known as the respiratory burst (12), during which superoxide anion (O2.–) is formed immediately after the reduction of molecular oxygen by single electrons through the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system. O2.– is then rapidly transformed through enzyme activity [superoxide dismutase, catalase, myeloperoxidase (MPO)] into other ROS, which include hydrogen peroxide (H2O2), hydroxyl radicals, and hypochlorous acid (13). The importance of O2.– and H2O2 productions for PMN bactericidal activity is well known because patients with chronic granulomatous disease that do not produce enough O2.– and H2O2 become more susceptible to bacterial infection (14). Different groups using different techniques and types of stimulating agents have all found a significant reduction in ROS production by stimulated PMN in healthy elderly donors (for review, see Ref. 1).

Some authors (15, 16) found that there are no differences between blood monocyte functions from young and old humans in their ability to kill bacteria or generate cytokines, although others observed that the capacity of tissue macrophages from rodents to present antigen and to produce cytokines declined with age (17, 18). Other groups (17, 18) reported that in vitro release of H2O2 and nitric oxide by activated monocytes-macrophages was 50% lower in old than adult rodents. Because the ability of macrophages to secrete ROS, reactive nitrogen intermediate, and cytokines correlates closely with their ability to perform two critical effector functions, intracellular killing of microorganism and lysis of tumor cells, the diminished response of macrophages to activating signals also may be one aspect of the impaired immune response in advanced age.

It is generally difficult to detect solely age-dependent changes in the immune system, and consequently the literature presents conflicting data. Other factors, such as chronic disease, nutrition, and lifestyle, have a much more profound effect on immunity and thus further conceal more subtle age-related changes (19, 20). To overcome this problem, Ligthart et al. (21), working under the SENIEUR EURAGE protocol, set strict admission criteria for human immunogerontology that include clinical information, laboratory data, and immunopharmacological interferences. This protocol is not the only way to define healthy, but it is well established and clear in its criteria, thus enabling readers to understand what healthy means in studies following this protocol (22).

The combination of age-associated changes in immune function may cause an increased susceptibility to pathogens in elderly people. Additionally, the risk of infectious diseases is 2- to 4-fold higher in patients with diabetes, or even impaired glucose tolerance without hyperglycemia, than in healthy subjects (23, 24, 25). Elderly people, even nondiabetic subjects, are often prone to silent reduced-insulin sensitivity (26, 27, 28), which may worsen the age-associated immune dysfunction. Importantly, we recently demonstrated that insulin under strict euglycemia is able to prime PMN function in adult healthy humans (29). The conclusion of this study was that insulin modulates PMN activity not only by gaining a better metabolic control, as suggested by studies in diabetic patients, but also through a direct effect of the hormone. Insulin may act as an immunoregulatory agent to turn immune cells to a primed state, which prepares the cell for a greater immune response (29). The present study was designed to address the hypothesis that blunted monocyte and PMN activity in older people is associated with reduced insulin sensitivity. To address this hypothesis, monocyte and PMN functions were determined in healthy, i.e. selected according to the SENIEUR protocol, elderly human subjects under strict euglycemia and physiological insulin concentrations to understand the aged-related action of insulin on leukocytes without hypo- or hyperglycemic interference.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Characteristics of the subjects

Young (n = 8) and elderly (n = 9) male subjects participated in the study. The SENIEUR protocol (21) was used to select healthy adult and older volunteers (Table 1Go). Briefly, each volunteer presented normal results on physical examination and blood biochemistry analysis. In addition, all of the subjects presented no underlying acute or chronic disease liable to affect the immune system. None of the volunteers suffered from cancer or cardiac, liver, brain, or kidney disease. All volunteers were also checked for signs of infection and inflammation, and none were taking medication likely to affect the immune system, such as antiinflammatory drugs, hormones, or analgesics. None had been recently vaccinated. Furthermore, all of the subjects were fully ambulant and sedentary, with similar activity levels between groups as assessed by a physical activity questionnaire. All the participants were nondiabetic, nonobese, and nonsmokers.


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  		TABLE 1. Subject characteristics

 
The purpose and potential risks of the study were fully explained, and written informed consent was obtained from each participant. Our institution is authorized by the French Ministry of Health to perform experiments on healthy subjects. The study was carried out in accordance with the Declaration of Helsinki guidelines, after being approved by the Ethics Committee of the Auvergne region.

Experimental procedures

Given that nutritional intakes can affect immune status, notably in older individuals (19, 30), each subject received a standardized diet (1.6 x predicted resting energy expenditure, 16% proteins) for 7 d before the study to normalize energy and protein intakes. The volunteers came to the clinical research center 1 to 2 wk before the study day, and a dietitian performed a diet assessment to determine and explain food needs to the subjects. At the same time, body composition (fat mass, fat-free mass) was measured by whole body dual x-ray absorptiometry (DPX-IQ, Lunar, Madison, WI).

On the day of the experiment, sensitivity of monocytes and PMNs to the in vivo action of insulin was measured by using the hyperinsulinemic euglycemic clamp technique, as previously described (29). Briefly, after an overnight fasting (12 h), two catheters were inserted in the right antecubital vein for the infusion of insulin and glucose. 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 per minute for 4 h using a calibrated syringe pump. Glycemia was measured every 5 min using an automated glucose analyzer (Glucose Analyzer 2; Beckman, Fullerton, CA), and glucose infusion (20% dextrose) was performed to maintain glycemia at a constant fasting level (0.9 g/liter). An amino acid solution (10% Primene; Clintec Clinical Nutrition, Velizy-Villacoublay, France) was also infused at a rate of 0.020 ml·kg–1·min–1 to avoid any insulin-mediated decrease in plasma amino acid concentrations, which would have affected immune status (31). Blood samples were taken in EDTA Vacutainers before insulin infusion and at exactly 4 h after the beginning of insulin clamping to assay monocyte and PMN functions.

Characterization of insulin sensitivity

Insulin sensitivity was characterized using a continuous infusion of D-[6,6-2H2]glucose (Eurisotop, Gif-sur-Yvette, France) at a rate of 0.05 mg·kg–1·min–1. Glucose disposal rate (GDR) was calculated from the dilution of labeled glucose in plasma using the single compartmental model (32). Insulin sensitivity was characterized by the GDR to insulinemia ratio (33). The tracer used to measure insulin sensitivity was checked for chemical and isotopic impurities using gas chromatography-mass spectrometry (Hewlett-Packard 5971A, Palo Alto, CA). After preparation, the solutions of labeled glucose were tested for sterility and pyrogenicity and were membrane filtered through a 0.22-µm-pore-size filter throughout the experiment.

Insulin sensitivity was also evaluated by calculating M value according to the following equation (34): M value = rate of perfused glucose during the clamp/[glycemia during the clamp x (insulinemia during the clamp – basal insulinemia)].

Data on insulin sensitivity are presented in Table 1Go.

Biochemical and hematological characteristics

Plasma insulin and IGF-I concentrations were measured by RIA (Table 1Go; CIS, Gif-sur-Yvette, France). Plasma albumin and C-reactive protein levels were determined by immunonephelometry (Array protein system, Beckman-Coulter, Villepinte, France) and turbidimetry, respectively, using human antibodies (Dako, Trappes, France).

Blood cellularity (leukocyte number and differential count) was assessed using a Coulter counter (Coultronics, Margency, France).

Analysis of monocyte and PMN functions

Insulin receptor density on monocyte and PMN membranes. Insulin receptor density was measured by flow cytometry (Beckman-Coulter) after preparing the blood with the Q-Prep Epics immunology workstation (Beckman-Coulter). A sandwich reaction with an antiinsulin receptor detected with a fluorescein isothiocyanate (FITC)-conjugated anti-antibody was used (Beckman-Coulter). Insulin receptor density was evaluated using the Epics Immuno Brite kit (Beckman-Coulter). This kit comprises five vials containing particles of a specific fluorescence intensity (blank, medium-low, medium, medium-high, bright). Five hundred microliters from each of the five bottles were dispensed into a sample tube, and the fluorescence of each particle population was recorded by flow cytometry. The fluorescence intensity values measured were mapped 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 give receptor density.

Results are expressed as the number of membrane receptors per cell (receptor density). Intraassay and interassay coefficients of variation were recorded using a lyophilized preparation of human immune leukocytes that exhibited surface antigens (Cyto-Trol control cells; Beckman-Coulter) and a suspension of fluorospheres of similar size and fluorescence intensity (Flow-Count fluorosphere, Beckman-Coulter). Intraassay and interassay coefficients of variation were less than 2% for all flow cytometric measurements.

PMN migration

We previously set up a novel and accurate method combining the multiwell insert system (Becton Dickinson, Meylan, France) and flow cytometry to measure PMN chemotaxis (29). Briefly, 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 then centrifuged at 700 x g for 30 min at 20 C. PMNs were then collected on the corresponding layer and washed twice in PBS (Sigma). PMNs were then 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 PMNs (1.106) were added to a multiwell insert system (Becton Dickinson) with a 3-µm polyethylene terephthalate membrane placed in a 24-well plate. RPMI 1640 medium (Sigma) with or without 10–7 mol/liter of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) was added in the lower chamber to measure directed migration (DM) and spontaneous migration (SM), respectively. PMNs were then allowed to migrate through the membrane for 90 min at 37 C in a humidified atmosphere containing 5% CO2. After migration, PMNs in the lower chambers were removed and counted using a flow cytometer (Epics XL; Beckman-Coulter). The chemotaxis index was calculated as the ratio of the number of PMNs that migrated in the lower chamber in response to fMLP (DM) to the number of cells that migrated spontaneously (SM).

PMN receptor expression and density

PMN subsets were quantified by immunoreaction with fluorochrome-conjugated monoclonal antibodies (Beckman-Coulter). The panel of monoclonal antibodies used to measure PMN receptor expression and density was FITC anti-CD11b, FITC anti-CD15, and FITC anti-CD62L. CD11b is an integrin involved in a variety of cell-matrix and cell-cell adhesion functions, in particular at the endothelial level (35). A quantitative increase in CD11b surface expression is linked to a transient activation of PMN-endothelium interaction and an enhanced transmigration of PMN across the endothelium. CD15, i.e. the Lewis carbohydrate antigen, is the ligand of the endothelium cell-leukocyte adhesion molecule-1, exclusively expressed on the cell surface of activated human endothelial cells (36). CD62L is also used by PMN for diapedesis across the endothelial surface and for binding to opsonized microorganisms (37).

PMN receptor expression and density were measured by flow cytometry (Beckman-Coulter) after preparing the blood with the Q-Prep Epics immunology workstation (Beckman-Coulter) as described for the insulin receptor. Results are expressed as the percentages and absolute numbers [giga (G)/liter] of PMNs that expressed receptors and as the number of membrane receptors per PMN (receptor density).

Monocyte and PMN phagocytosis

Phagocytosis, i.e. the ability of cells to internalize microorganisms, is a membrane-dependent mechanism. Therefore, whole blood instead of isolated monocytes and PMNs was used to avoid any potential alteration or modification of the membrane composition during the cell isolation procedure. Basically, monocyte and PMN phagocytosis was evaluated by measuring ROS production induced by the engulfment of zymosan particles opsonized (OZ) with AB serum (Sigma). Five hundred microliters of whole blood were rapidly treated with a gentle hemolytic solution (0.15 mol/liter NH4Cl, 12 mmol/liter NaHCO3, 0.1 mmol/liter EDTA) at room temperature to avoid cell membrane disruption. Leukocytes were then washed twice in PBS and adjusted to 106 cells/ml in RPMI 1640 medium. The cells were preincubated for 15 min with 1 µmol/liter of 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 cell oxidative burst during which nonfluorescent intracellular DHR-123 is oxidized to highly fluorescent rhodamine 123 (Rh-123) by H2O2 (38). Individual monocytes and PMNs were identified and counted, and Rh-123 fluorescence emission was measured by flow cytometry (Beckman-Coulter). Results were expressed as the percentage of monocytes and PMNs able to engulf OZ particles and as the fluorescence intensity of positive cells, i.e. the phagocytic index.

Monocyte and PMN bactericidal activity

Monocytes and PMNs exhibit various bactericidal systems. One of the most powerful is undoubtedly the production of highly toxic ROS. Therefore, monocyte and PMN bactericidal function was determined by measuring ROS production after cell activation and evaluating MPO expression. PMNs also produce lactoferrin in phagosomes to trap iron and limit bacterial division and development. ROS production after phorbol myristate acetate (PMA; 10–6 mol/liter) or fMLP (10–5 mol/liter) stimulation was measured using the DHR-123 probe method, as previously described (38). MPO and lactoferrin expression and content were measured after permeabilization and fixation of cells by treating 50 µl whole blood with saponin and formaldehyde (IntraPrep permeabilization reagent; Immunotech, Marseille, France), respectively. After permeabilization, FITC anti-MPO and phycoerythrin antilactoferrin antibodies were added, and the blood preparation was incubated for 15 min at room temperature in the dark and then rinsed with PBS. Intracellular MPO and lactoferrin expression (in percents and giga per liter) and density (number of MPO and lactoferrin molecules per cell) were then analyzed by flow cytometry, as described above for the membrane receptors.

Statistical analysis

Data are presented as means ± SEM, and statistical analysis was performed by using StatView program 4.02 software (Abacus Concepts, Berkley, CA). The experimental design comprised two fixed crossed factors with the factor age as two classes (young-adult and aged) and the factor clamp as two classes (before and after clamp). A two-way ANOVA was used to discriminate among the effects of age, clamp, and their interactions after adjustment for body mass index (BMI). The level of significance was set at P < 0.05 for this test. When the ANOVA indicated significant differences, an post hoc Newman-Keuls test was performed to identify differences between individual means. A Student’s t test was also used when appropriate (subject characteristics). Pearson’s product moment was used to calculate correlations. P < 0.05 was considered as significant for these tests.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Markers related to insulin sensitivity

As shown in Table 1Go, fasting plasma glucose and insulin levels were not different between the two groups of different ages. As expected, blood insulin concentration rose significantly during the hyperinsulinemic clamp (P < 0.001 vs. baseline insulin level) and reached physiological postprandial values in both groups. We used two different ways to investigate insulin sensitivity, and both clearly showed that despite there being no modification in basal blood glucose and insulin values, elderly people were characterized by a significant reduction in insulin sensitivity, regardless of the marker used (P < 0.05 vs. young-adult; see Table 1Go). Interestingly, the prevalence of insulin resistance in elderly population has been related to age-induced modifications in body composition, in particular an increase in fat mass (39). We observed a higher BMI in the aged volunteers (P < 0.05 vs. young adult) together with a tendency toward increased fat mass (P = 0.07 vs. young adult).

White blood cell counts

Although there were no changes in absolute PMN numbers between young-adult and aged volunteers (Table 1Go), we observed a significant decrease in lymphocyte and monocyte counts in elderly people (P < 0.05 vs. young adult), as commonly described in immunogerontological studies, even in SENIEUR protocol-selected subjects.

Insulin receptor density

Insulin receptor density of PMNs and monocytes was not modified by the insulin infusion regardless of the age of the subjects (Fig. 1Go).


Figure 1
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  		FIG. 1. Monocyte and PMN membrane insulin receptor density in healthy young-adult and elderly subjects before and after hyperinsulinemic euglycemic clamp. Data are presented as means ± SEM; n = 8 or 9 subjects per group. Two-way ANOVA was performed to discriminate among the effects of age, clamp, and their interaction (NS) + Newman-Keuls test (NS). Insulin receptor density is expressed as the number of membrane receptors per cell.

 
Monocyte functions

Monocyte phagocytosis. Although no modification of the percentage of phagocytic monocytes was observed, the monocyte phagocytic index was decreased in the elderly subjects in comparison with the younger group regardless of the period (age effect, P < 0.05, Table 2Go).


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  		TABLE 2. Monocyte and PMN phagocytosis indexes in healthy young-adult and elderly subjects before and after the hyperinsulinemic euglycemic clamp

 
Monocyte bactericidal activity. A decrease in the number of monocytes able to produce ROS after PMA stimulation and in the amount of ROS generated by this cell was observed in the elderly group regardless of the period (age effect, P < 0.05, Table 3Go). There was a significant increase in the proportion of monocytes able to produce ROS after fMLP stimulation only in the young adults after the insulin clamping (P < 0.05). The insulin treatment down-regulated the fMLP-induced ROS production in monocytes of the young-adult subjects. This index was significantly decreased in the older group, compared with the young adults regardless of the period (age and clamp effects and their interaction, P < 0.05). Although MPO density in monocytes was unchanged, the number of cell-expressing MPO was decreased in the elderly subjects before the clamping period (P < 0.05).


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  		TABLE 3. Monocyte and PMN bactericidal indexes in healthy young-adult and elderly subjects before and after the hyperinsulinemic euglycemic clamp

 
PMN functions

PMN chemotaxis. When an infectious agent enters the body, PMN first uses its migration activity to reach the infected tissue. Clearly, PMN chemotaxis was up-regulated by hyperinsulinemia both in the young-adult and elderly populations (clamp effect, P < 0.05, Fig. 2Go). Changes in PMN migration were related to the increase in migration directed toward fMLP (clamp effect, P < 0.05), whereas the spontaneous ability of PMN to migrate remained unchanged (results not shown).


Figure 2
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  		FIG. 2. PMN chemotactic response in healthy young-adult and elderly subjects before and after hyperinsulinemic euglycemic clamp. Data are presented as means ± SEM; n = 8 or 9 subjects per group. Two-way ANOVA was performed to discriminate among the effects of age, clamp, and their interaction (clamp effect, P < 0.05) + Newman-Keuls test (a != b with P < 0.05). PMN chemotaxis is expressed as the ratio of directed migration toward fMLP to spontaneous migration (DM/SM). Insulin increased PMN chemotaxis in both groups (P < 0.05).

 
PMN receptor expression. PMN is able to bind to the endothelium and penetrate the target tissue. Furthermore, the cells use these adhesive properties to trap the invading microorganisms. Overall, the number of PMNs expressing CD11b, CD15, and CD62L receptors (Table 4Go) as well as their densities, i.e. the number of membrane receptors per cell (Figs. 3–5GoGoGo), presented age- and insulin infusion-dependent changes. The insulin clamp significantly increased the absolute count, i.e. in giga per liter, of CD11b-, CD15-, and CD62L-positive cells in young-adult volunteers, whereas there was no change in the older group (age and clamp effects, P < 0.05). The percentage of PMNs expressing CD62L receptor was also reduced in the elderly group, regardless of the period considered, i.e. before or after the clamp (P < 0.05). Interestingly, insulin down-regulated CD15 and CD62L receptor densities in both groups (P < 0.05 clamp effect, Figs. 4Go and 5Go). However, there was an increase in CD11b density (age effect, P < 0.05, Fig. 3Go) and a decrease in CD62L density (age and clamp effect, P < 0.05, Fig. 5Go) in the elderly subjects.


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  		TABLE 4. PMN receptor expression in healthy young-adult and elderly subjects before and after the hyperinsulinemic euglycemic clamp

 

Figure 3
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  		FIG. 3. PMN membrane CD11b density in healthy young-adult and elderly subjects before and after hyperinsulinemic euglycemic clamp. Data are presented as means ± SEM; n = 8 or 9 subjects per group. Two-way ANOVA was performed to discriminate among the effects of age, clamp, and their interaction (age effect, P < 0.05) + Newman-Keuls test (a != b with P < 0.05). CD11b density is expressed as the number of membrane receptors per PMN. PMN CD11b density was increased in elderly subjects, regardless of the period (P < 0.05).

 

Figure 4
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  		FIG. 4. PMN membrane CD15 density in healthy young-adult and elderly subjects before and after hyperinsulinemic euglycemic clamp. Data are presented as means ± SEM; n = 8 or 9 subjects per group. Two-way ANOVA was performed to discriminate among the effects of age, clamp, and their interaction (clamp effect, P < 0.05) + Newman-Keuls test (a != b with P < 0.05). CD15 density is expressed as the number of membrane receptors per PMN. Insulin decreased CD15 density on PMN surface in both groups (P < 0.05).

 

Figure 5
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  		FIG. 5. PMN membrane CD62L density in healthy young-adult and elderly subjects before and after hyperinsulinemic euglycemic clamp. Data are presented as means ± SEM; n = 8 or 9 subjects per group. Two-way ANOVA was performed to discriminate among the effects of age, clamp, and their interaction (age and clamp effects, P < 0.05) + Newman-Keuls test (a != b != c != d with P < 0.05). CD62L density is expressed as the number of membrane receptors per PMN. CD62L density was basically decreased in elderly (P < 0.05). Insulin reduced PMN CD62L density in both groups (P < 0.05).

 
PMN phagocytosis. After binding, PMNs internalize the microorganism to be destroyed. We investigated the percentage of blood PMNs able to internalize a particle, i.e. OZ, and determined the phagocytic index based on the ROS production induced by this internalization. The results indicated that insulin infusion had a stimulating effect on the percentage of phagocytic PMNs (age and clamp effects, P < 0.05) and their ability to internalize particles (age and clamp effects, and their interaction, P < 0.05) in the young-adult group (Table 2Go). However, PMN phagocytosis was affected by age, with a lower number of phagocytic PMNs in the elderly group, regardless of period (age and clamp effects, and their interaction, P < 0.05). Furthermore, the PMN phagocytic index was not stimulated by insulin in the elderly group (P < 0.05, Table 2Go).

PMN bactericidal activity. Once internalized in PMN phagosomes, bacterial elements have to be eliminated. For this purpose, PMNs produce various highly toxic compounds including ROS. There was an increase in the number of PMNs able to produce ROS after PMA or fMLP activation and in the amount of ROS produced after insulin clamping in the young-adult group (age and clamp effects, P < 0.05, Table 3Go). However, there was a decrease in the number of bactericidal PMNs in the elderly group (P < 0.05). In addition, the priming effect of insulin on ROS production was blunted in the elderly group after fMLP activation (age effect, P < 0.05). These observations were confirmed by measuring the number of PMNs expressing MPO, a key enzyme in ROS metabolism, which increased after the insulin treatment in the young-adult subjects but not the elderly group (age and clamp effects, P < 0.05, Table 3Go). Surprisingly, MPO density in the cell was reduced after the insulin infusion, regardless of group age (clamp effect, P < 0.05).

We also measured the expression of lactoferrin, which participates in the bactericidal function of PMNs by trapping the iron present into the phagosome. A stimulating effect of insulin on the absolute number of PMNs expressing lactoferrin was observed only in the younger group (age and clamp effects, P < 0.05, Table 3Go), whereas lactoferrin density inside the cell decreased after the clamp period in both two groups (clamp effect, P < 0.05).

Correlations. Table 5Go lists the receptors that correlated significantly with PMN functions. All the significant associations were positively correlated. CD11b density was closely related to PMA-induced ROS production both before (r = 0.5534, P < 0.05) and after (r = 0.3291, P < 0.05) the insulin treatment in the elderly group. The same pattern was observed with fMLP-induced ROS generation in the younger group (r = 0.6589, P < 0.05 before insulin and r = 0.6034, P < 0.05 after insulin). Although the relationship between CD15 density and PMA-induced ROS production was statistically significant in both groups (r = 0.7790 and r = 0.5562 in the young and elderly populations, respectively), correlations were found between CD15 density and fMLP-induced ROS production (r = 0.4184, P < 0.05), CD15 density and phagocytosis (r = 0.9160, P < 0.05) and CD15 density and chemotactic index (r = 0.6909, P < 0.05) only in young people before clamp. CD62L density was positively correlated to fMLP-induced ROS production (r = 0.7461, P < 0.05 before insulin and r = 0.7515, P < 0.05 after insulin) in elderly people, but this link was nonsignificant in the younger group. CD62L density was significantly related to phagocytosis in both groups before (r = 0.7395 and r = 0.4974 in young and elderly, respectively) and after (r = 0.7863 and r = 0.8314 in young and elderly, respectively) the insulin clamp. Surprisingly, insulin receptor density was correlated only with phagocytosis in the elderly group, regardless of the period (r = 0.8060 and r = 0.5237 before and after insulin infusion, respectively). Insulin receptor density was also related to the migration capacity of PMNs in both the young (r = 0.6498, P < 0.05) and elderly (r = 0.3900, P < 0.05) groups but only after insulin infusion (Table 5Go).


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  		TABLE 5. Significant correlations between PMN receptor densities and PMN functions in healthy young-adult and elderly subjects before and after the hyperinsulinemic euglycemic clamp

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This study was designed to investigate the effect of insulin on monocyte and PMN functions in elderly humans because it has previously been shown that insulin hormone has a strong regulating effect on the functional activities of immune cells (29, 40, 41, 42, 43, 44). Generally speaking, the priming action of insulin on PMN activity may be seen as the body providing a global defense to support primary immune response against exposure to antigens, which is enhanced by food intake (19, 45, 46). Our H1 hypothesis was that the aging-induced reduction in insulin sensitivity plays a role in the age-related weakening of the immune system (for review, see Ref. 1), particularly after food intake (19). Hence, alterations in immune cell function may partly explain the higher prevalence of infective episodes in the older population. To focus specifically on our target subject and render our data interpretable, we selected a healthy population to avoid confounding factors such as chronic disease or medication liability to modify immune status and consequently mask the aging effect, and we developed a batch of markers able to give an overview of monocyte and PMN activities because alterations in one function may affect the overall ability of the cell to destroy microorganisms (14).

Taken together, our results showed a profound age-associated alteration of monocyte functions, i.e. phagocytosis and fMLP- and PMA-induced ROS production. In addition, insulin had a limited regulating effect on this cell in young people, and this action was suppressed in the older group. Aging also affects the ability of PMNs to be primed by insulin. The insulin-mediated regulation of PMN receptor expression, phagocytosis, and bactericidal activity was blunted in the older group. Therefore, the evidence is strong enough to reject a null hypothesis in favor of the H1 hypothesis. These observations could be related to the relative insulin resistance detected in our nondiabetic elderly subjects, although there was no correlation between the measurements for whole-body insulin sensitivity and monocyte and PMN functions. In addition, no change in insulin receptor expression on monocyte and PMN surfaces has been detected. However, the intracellular signaling pathway involved in the action of insulin on monocyte and PMN functions is not yet fully understood (47, 48) and may contribute to the altered response in elderly subjects. Furthermore, we did not measure insulin receptor affinity, and therefore, we cannot use quantification of the receptor density alone as evidence that insulin receptor function was not defective. Previous studies in mononuclear cells (49, 50) have clearly shown that insulin receptor affinity is modified in diabetic patients as well as healthy subjects receiving insulin.

Diabetic patients exhibit clearly altered PMN functions, and it has often been suggested that PMN activity could be restored by controlling hyperglycemia with insulin (51, 52). However, the present study design, i.e. euglycemia, suggests that insulin may act directly on PMN function independently of its effect on glucose uptake, in accordance with many in vitro assays (41, 42, 43, 44, 47, 48, 53). In addition, this is the first report of the priming action of physiological hyperinsulinemia on PMN function in healthy elderly people. Chronologically, activity displayed by PMNs is migration through blood and tissues toward a gradient of chemical compounds produced by the infected bacteria bed. Insulin is able to stimulate PMN migration and deformability when the cell is chemically attracted by fMLP. PMN migration is dependent on fMLP receptor expression, membrane composition, and deformability and intracellular biochemical processes such as calcium fluxes or cytoskeleton movement (54). Hence, chemotaxis is a high ATP-demanding function and, incidentally, it is dependent on glucose catabolism (55, 56).

Interestingly, although PMNs do not require insulin to uptake glucose, glucose use and glycogen metabolism inside PMNs are both insulin dependent (57). Therefore, the optimized glucose use in PMNs during insulin infusion may contribute to their stimulatory action on cell migration. Previous in vitro data (58) have indicated that the chemotactic effect of fMLP is regulated by physiological glucose concentrations in the medium by inducing locomotor activity in otherwise nonlocomotive cells. More interestingly, these authors revealed that insulin is needed to sustain glucose-activated PMN migration (47, 53). Taken together, these results support the hypothesis that insulin resistance may play a direct role in impaired PMN function, e.g. chemotaxis, observed in diabetic patients with poor metabolic control (59). Furthermore, insulin receptor expression was correlated with PMN chemotaxis in both young and elderly subjects after the insulin treatment.

This observation is further evidence of the potential action of insulin on PMN function. Importantly, the action of insulin on PMN chemotaxis was not modified by age in our study. Similarly, aging did not influence the down-regulation by insulin of receptors used by PMN to interact with the endothelial cells, such as CD15. Adhesion molecules are especially important in PMN rolling on the vessel wall, a process that can precede firm attachment and extravasation during inflammation or infection. Therefore, both PMN trafficking and recruitment to sites of injury are mediated by these molecules. As previously shown (60), increased plasma insulin concentrations inhibit PMN adherence to the endothelium and consequently increase the number of circulating PMNs able to migrate into the target tissue. This observation was confirmed in our study by the reduced density of PMN receptors involved in the interaction with the endothelial barrier. A decreased expression of the PMN receptors used to conjugate with endothelium may therefore represent a potential mechanism by which insulin increases PMN counts in the blood.

It was also reported that insulin reduces the expression of endothelial molecules, such as intracellular adhesion molecule-1 and monocyte chemoattractant protein-1, in vivo (61) and in vitro (62, 63). This observation suggests that insulin inhibits leukocyte adherence to the endothelium by reducing the expression of chemoattractant and adhesion molecules in both leukocyte and endothelium cells. Although insulin led to a down-regulation of PMN receptor density in both groups, the increased numbers of circulating PMNs expressing these membrane antigens in the younger population after insulin infusion was not observed in the older group. We observed that the number of CD11b-positive cells was basically lower and failed to increase with insulin in the elderly group. However, the receptor density of this adhesion molecule was higher in elderly subjects both before and after insulin infusion. This phenomenon may be interpreted as a compensatory mechanism in aged PMNs, i.e. an increase in receptor density to compensate for the reduced number of PMNs expressing the receptor. Also, considering that CD11b density is basically different in young and elderly people, it is possible that the effect of insulin on the inhibition of PMN adhesion to the endothelium through this particular receptor may also differ with age.

In contrast with both CD11b and CD15, the density of CD62L, which is a protein that promotes the interaction of PMNs with endothelial cells and specific opsonins deposited on bacterial membranes, was severely depressed in elderly subjects. This phenomenon may reduce the ability of elderly PMNs to internalize and therefore destroy foreign invaders. Accordingly, phagocytosis and bacterial poisoning by ROS or lactoferrin were also affected in the elderly group before and/or after the insulin clamp. Interestingly, the same result, i.e. a decrease in functional activity, was observed in monocytes of elderly subjects. The monocyte phagocytic index and ROS production in the presence of PMA or fMLP was basically reduced in the older group. Alteration of monocyte functions with age has already been described (17, 18) and is likely to contribute to the decline of immune system in elderly. Our baseline data revealed that PMNs from elderly subjects displayed an fMLP-triggered ROS responsiveness, which overlapped that seen in the younger population, whereas a significant decrease in respiratory burst was observed with the presence of PMA in the same population. The differences observed in PMN oxidative burst response in older people according to the stimulant used may be related to the different biochemical pathways activated by these two commonly used soluble stimulating agents. However, although these two compounds induce different intracellular pathways, the ultimate action is the activation of protein kinase C, which in turn phosphorylates and therefore activates the components of NADPH oxidase, which is the main ROS-producing enzyme (13, 64). Surprisingly, the action of insulin on PMN oxidative burst was maintained using PMA but depressed after fMLP activation in elderly subjects, whereas insulin was able to stimulate PMN oxidative burst in young-adult subjects regardless of the stimulant used.

It has already been reported that one of the main in vitro actions of insulin on PMNs is to increase its ROS production (29, 48). The activation of the biochemical insulin cascade in PMNs is able to phosphorylate NADPH oxidase, which probably triggers the activation of the ROS pathway. In terms of ROS production, this pathway could underlie the cross-interaction between insulin and PMN stimulants, and it appears to be altered in elderly subjects with a reduced insulin sensitivity. Interestingly, using PMA, PMNs from type 2 diabetic patients produced markedly less ROS, compared with control subjects (51). Furthermore, an in vitro addition of insulin or an increased glucose concentration in the PMN medium failed to restore PMN-generated ROS production in these diabetic patients (51). Therefore, the insulin-mediated regulation of ROS production seems to be weaker in subjects with type 2 diabetes mellitus as well as elderly people with reduced insulin sensitivity. By contrast with PMNs, fMLP-induced ROS production in monocytes was reduced by the insulin infusion in young people. This is the only change that was observed in monocyte functional activity after the insulin-clamping period. This observation suggests that insulin action on ROS production by stimulated leukocytes is cell and stimulant specific.

An important point is that previous data (61, 65) have revealed that insulin infusion in obese nondiabetic subjects or against a background of myocardial infarction resulted in decreased ROS production by monocytes. These authors have proposed that insulin exerts an antiinflammatory effect on monocyte-macrophage cells that in the long term prove to be antiatherogenic.

Decreased MPO content in PMNs after the insulin infusion may also participate in the promotion of oxidative burst by insulin because MPO is involved in the elimination of ROS, particularly H2O2. Intravenous insulin infusion in healthy humans was also associated with a decrease in PMN elastase (66), another enzyme participating in the bactericidal process. Taken together, these data demonstrated that insulin affects the concentrations of proteins involved in the bactericidal function of PMNs, probably through an increase in PMN granule secretion. In the study, MPO and lactoferrin contents in PMNs were modified to the same extent by insulin treatment in young-adult and elderly subjects. Surprisingly, no modification in MPO expression was noticed in monocytes after the insulin-clamping period. Here again, insulin action was different according to the cell considered. Additional studies are needed to investigate the specific role of insulin in the functional regulation of each of these two leukocyte populations.

Of note, the analysis of whether PMN measurements correlated with receptor measurements revealed strong differences between young and elderly subjects. Overall, the expression of PMN receptors was more closely related to PMN activity in young subjects than elderly subjects. In addition, some of the PMN functions correlated with receptor expressions in young and elderly people both before and after the insulin infusion, showing that these particular functions are closely related to PMN receptor expression. However, the receptor expression-related adaptation of PMN activity to insulin was different between the two groups, revealing a significant effect of age on the regulation of PMN activity by insulin.

In conclusion, our data demonstrate a suppressive action of insulin on fMLP-induced ROS production in monocytes. Although this action was the only one detected in monocytes, it is consistent with an antiinflammatory effect of insulin on this cell. The stimulatory effect of insulin on PMN function was also confirmed in the present study. As already proposed for IGF-I (67), insulin should be classified as a PMN primer, i.e. an agent able to prepare PMNs for a faster and stronger response. It is important to underline that the very similar structure of IGF-I and insulin suggests that insulin may also have acted through the IGF-I receptor in our study. Furthermore, the decline in IGF-I concentration observed in the elderly subjects may have contributed to the weaker PMN response. Our data demonstrated that reduced insulin sensitivity may act in combination with intrinsic age-associated disturbances to affect immune status in older subjects. The intracellular insulin pathway in immune cells needs to be clearly determined, although it has long been thought that leukocytes can live and act without this hormone.


   Footnotes
 
First Published Online April 18, 2006

Abbreviations: BMI, Body mass index; DHR-123, dihydrorhodamine 123; DM, directed migration; FITC, fluorescein isothiocyanate; fMLP, formyl-methionyl-leucyl-phenylalanine; G, giga; GDR, glucose disposal rate; MPO, myeloperoxidase; NADPH, nicotinamide adenine dinucleotide phosphate; O2.–, superoxide anion; OZ, opsonized zymosan; PMA, phorbol myristate acetate; PMN, polymorphonuclear neutrophil; Rh-123, rhodamine 123; ROS, reactive oxygen species; SM, spontaneous migration.

Received July 20, 2005.

Accepted April 11, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Walrand S, Vasson MP, Lesourd B 2003 The role of nutrition in immunity of the aged. In: Perdigon G, Fuller R, eds. Gut flora, nutrition and immunity. Oxford, UK: Blackwells; 237–269
  2. Lord JM, Butcher S, Killampali V, Lascelles D, Salmon M 2001 Neutrophil ageing and immunosenescence. Mech Ageing Dev 122:1521–1535[CrossRef][Medline]
  3. Ginaldi L, De Martinis M, D’Ostilio A, Marini L, Loreto MF, Quaglino D 1999 The immune system in the elderly: III. Innate immunity. Immunol Res 20:117–126[Medline]
  4. Niwa Y, Kasama T, Miyachi Y, Kanoh T 1989 Neutrophil chemotaxis, phagocytosis and parameters of reactive oxygen species in human aging: cross-sectional and longitudinal studies. Life Sci 44:1655–1664[CrossRef][Medline]
  5. Polignano A, Tortorella C, Venezia A, Jirillo E, Antonaci S 1994 Age-associated changes of neutrophil responsiveness in a human healthy elderly population. Cytobios 80:145–153[Medline]
  6. Biasi D, Carletto A, Dell’Agnola C, Caramaschi P, Montesanti F, Zavateri G, Zeminian S, Bellavite P, Bambara LM 1996 Neutrophil migration, oxidative metabolism, and adhesion in elderly and young subjects. Inflammation 20:673–681[CrossRef][Medline]
  7. Damtew B, Spagnuolo PJ, Goldsmith GG, Marino JA 1990 Neutrophil adhesion in the elderly: inhibitory effects of plasma from elderly patients. Clin Immunol Immunopathol 54:247–255[CrossRef][Medline]
  8. Tortorella C, Ottolenghi A, Pugliese P, Jirillo E, Antonaci S 1993 Relationship between respiratory burst and adhesiveness capacity in elderly polymorphonuclear cells. Mech Ageing Dev 69:53–63[CrossRef][Medline]
  9. Chan SS, Monteiro HP, Deucher GP, Abud RL, Abuchalla D, Junqueira VB 1998 Functional activity of blood polymorphonuclear leukocytes as an oxidative stress biomarker in human subjects. Free Radic Biol Med 24:1411–1418[CrossRef][Medline]
  10. Emanuelli G, Lanzio M, Anfossi T, Romano S, Anfossi G, Calcamuggi G 1986 Influence of age on polymorphonuclear leukocytes in vitro: phagocytic activity in healthy human subjects. Gerontology 32:308–316[Medline]
  11. Nagel JE, Han K, Coon PJ, Adler WH, Bender BS 1986 Age differences in phagocytosis by polymorphonuclear leukocytes measured by flow cytometry. J Leukoc Biol 39:399–407[Abstract]
  12. Dahlgren C, Karlsson A 1999 Respiratory burst in human neutrophils. J Immunol Methods 232:3–14[CrossRef][Medline]
  13. Casimir CM, Teahan CG 1994 The respiratory burst of neutrophils and its deficiency. In: Hellewell PG, Williams TJ, eds. Immunopharmacology of neutrophil. London: Academic Press; 27–54
  14. Smith RM, Curnutte JT 1991 Molecular basis of chronic granulomatous disease. Blood 77:673–686[Free Full Text]
  15. Gardner ID, Lim ST, Lawton JW 1981 Monocyte function in ageing humans. Mech Ageing Dev 16:233–239[Medline]
  16. Nielsen H, Blom J, Larsen SO 1984 Human blood monocyte function in relation to age. Acta Pathol Microbiol Immunol Scand [C] 92:5–10[Medline]
  17. Davila DR, Edwards 3rd CK, Arkins S, Simon J, Kelley KW 1990 Interferon-{gamma}-induced priming for secretion of superoxide anion and tumor necrosis factor-{alpha} declines in macrophages from aged rats. FASEB J 4:2906–2911[Abstract]
  18. Ding A, Hwang S, Schwab R 1994 Effect of aging on murine macrophages. Diminished response to IFN-{gamma} for enhanced oxidative metabolism. J Immunol 153:2146–2152[Abstract]
  19. Walrand S, Moreau K, Caldefie F, Tridon A, Chassagne J, Portefaix G, Cynober L, Beaufrere B, Vasson MP, Boirie Y 2001 Specific and nonspecific immune responses to fasting and refeeding differ in healthy young adult and elderly persons. Am J Clin Nutr 74:670–678[Abstract/Free Full Text]
  20. Schroder AK, Rink L 2003 Neutrophil immunity of the elderly. Mech Ageing Dev 124:419–425[CrossRef][Medline]
  21. Ligthart GJ, Corberand JX, Fournier C, Galanaud P, Hijmans W, Kennes B, Muller-Hermelink HK, Steinmann GG 1984 Admission criteria for immunogerontological studies in man: the SENIEUR protocol. Mech Ageing Dev 28:47–55[CrossRef][Medline]
  22. Stephensen CB 2001 Examining the effect of a nutrition intervention on immune function in healthy humans: what do we mean by immune function and who is really healthy anyway? Am J Clin Nutr 74:565–566[Free Full Text]
  23. Bessman AN, Sapico FL 1992 Infections in the diabetic patient: the role of immune dysfunction and pathogen virulence factors. J Diabetes Complications 6:258–262[CrossRef][Medline]
  24. Boyko EJ, Fihn SD, Scholes D, Abraham L, Monsey B 2005 Risk of urinary tract infection and asymptomatic bacteriuria among diabetic and nondiabetic postmenopausal women. Am J Epidemiol 161:557–564[Abstract/Free Full Text]
  25. Moutschen MP, Scheen AJ, Lefebvre PJ 1992 Impaired immune responses in diabetes mellitus: analysis of the factors and mechanisms involved. Relevance to the increased susceptibility of diabetic patients to specific infections. Diabetes Metab 18:187–201
  26. Kesavadev JD, Short KR, Nair KS 2003 Diabetes in old age: an emerging epidemic. J Assoc Physicians India 51:1083–1094[Medline]
  27. Sowers JR 2004 Diabetes in the elderly and in women: cardiovascular risks. Cardiol Clin 22:541–551[CrossRef][Medline]
  28. Grant RW, Meigs JB 2004 Should the insulin resistance syndrome be treated in the elderly? Drugs Aging 21:141–151[CrossRef][Medline]
  29. Walrand S, Guillet C, Boirie Y, Vasson MP 2004 In vivo evidences that insulin regulates human polymorphonuclear neutrophil functions. J Leukoc Biol 76:1104–1110[Abstract/Free Full Text]
  30. Walrand S, Chambon-Savanovitch C, Felgines C, Chassagne J, Raul F, Normand B, Farges MC, Beaufrere B, Vasson MP, Cynober L 2000 Aging: a barrier to renutrition? Nutritional and immunologic evidence in rats. Am J Clin Nutr 72:816–824[Abstract/Free Full Text]
  31. Cynober LA 2002 Plasma amino acid levels with a note on membrane transport: characteristics, regulation, and metabolic significance. Nutrition 18:761–766[CrossRef][Medline]
  32. Matsuda M, DeFronzo RA 1999 Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care 22:1462–1470[Abstract/Free Full Text]
  33. Jacquez JA 1992 Theory of production rate calculations in steady and non-steady states and its application to glucose metabolism. Am J Physiol 262:E779–E790
  34. Katz A, Nambi SS, Mather K, Baron AD, Follmann DA, Sullivan G, Quon MJ 2000 Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. J Clin Endocrinol Metab 85:2402–2410[Abstract/Free Full Text]
  35. Mazzone A, Ricevuti G 1995 Leukocyte CD11/CD18 integrins: biological and clinical relevance. Haematologica 80:161–175[Abstract/Free Full Text]
  36. Capurro M, Ballare C, Bover L, Portela P, Mordoh J 1999 Differential lytic and agglutinating activity of the anti-Lewis(x) monoclonal antibody FC-2.15 on human polymorphonuclear neutrophils and MCF-7 breast tumor cells. In vitro and ex vivo studies. Cancer Immunol Immunother 48:100–108[CrossRef][Medline]
  37. Bevilacqua MP, Nelson RM 1993 Selectins. J Clin Invest 91:379–387[Medline]
  38. Walrand S, Valeix S, Rodriguez C, Ligot P, Chassagne J, Vasson MP 2003 Flow cytometry study of polymorphonuclear neutrophil oxidative burst: a comparison of three fluorescent probes. Clin Chim Acta 331:103–110[CrossRef][Medline]
  39. Coon PJ, Rogus EM, Drinkwater D, Muller DC, Goldberg AP 1992 Role of body fat distribution in the decline in insulin sensitivity and glucose tolerance with age. J Clin Endocrinol Metab 75:1125–1132[Abstract]
  40. Oldenborg PA 1999 Effects of insulin on N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated production of reactive oxygen metabolites from normal human neutrophils. Inflamm Res 48:404–411[CrossRef][Medline]
  41. Okouchi M, Okayama N, Shimizu M, Omi H, Fukutomi T, Itoh M 2002 High insulin exacerbates neutrophil-endothelial cell adhesion through endothelial surface expression of intercellular adhesion molecule-1 via activation of protein kinase C and mitogen-activated protein kinase. Diabetologia 45:556–559[CrossRef][Medline]
  42. Okouchi M, Okayama N, Imai S, Omi H, Shimizu M, Fukutomi T, Itoh M 2002 High insulin enhances neutrophil transendothelial migration through increasing surface expression of platelet endothelial cell adhesion molecule-1 via activation of mitogen activated protein kinase. Diabetologia 45:1449–1456[CrossRef][Medline]
  43. Cavalot F, Anfossi G, Russo I, Mularoni E, Massucco P, Burzacca S, Mattiello L, Trovati M 1992 Insulin, at physiological concentrations, enhances the polymorphonuclear leukocyte chemotactic properties. Horm Metab Res 24:225–228[Medline]
  44. Cavalot F, Anfossi G, Russo I, Mularoni E, Massucco P, Burzacca S, Mattiello L, Trovati M 1993 Insulin stimulates the polymorphonuclear leukocyte chemokinesis. Horm Metab Res 25:321–322[Medline]
  45. Komaki G, Kanazawa F, Sogawa H, Mine K, Tamai H, Okamura S, Kubo C 1997 Alterations in lymphocyte subsets and pituitary-adrenal gland-related hormones during fasting. Am J Clin Nutr 66:147–152[Abstract/Free Full Text]
  46. Chandra RK 1997 Food hypersensitivity and allergic disease: a selective review. Am J Clin Nutr 66:526S–529S
  47. Oldenborg PA, Sehlin J 1998 Insulin-stimulated chemokinesis in normal human neutrophils is dependent on D-glucose concentration and sensitive to inhibitors of tyrosine kinase and phosphatidylinositol 3-kinase. J Leukoc Biol 63:203–208[Abstract]
  48. Safronova VG, Gabdoulkhakova AG, Miller AV, Kosarev IV, Vasilenko RN 2001 Variations of the effect of insulin on neutrophil respiratory burst. The role of tyrosine kinases and phosphatases. Biochemistry (Mosc) 66:840–849[CrossRef][Medline]
  49. DeFronzo R, Deibert D, Hendler R, Felig P, Soman V 1979 Insulin sensitivity and insulin binding to monocytes in maturity-onset diabetes. J Clin Invest 63:939–946[Medline]
  50. Helderman JH 1984 Acute regulation of human lymphocyte insulin receptors. Analysis by the glucose clamp. J Clin Invest 74:1428–1435[Medline]
  51. Shah SV, Wallin JD, Eilen SD 1983 Chemiluminescence and superoxide anion production by leukocytes from diabetic patients. J Clin Endocrinol Metab 57:402–409[Abstract]
  52. Thomson GA, Fisher BM, Gemmell CG, MacCuish AC, Gallacher SJ 1997 Attenuated neutrophil respiratory burst following acute hypoglycaemia in diabetic patients and normal subjects. Acta Diabetol 34:253–256[CrossRef][Medline]
  53. Oldenborg PA, Sehlin J 1999 Hyperglycemia in vitro attenuates insulin-stimulated chemokinesis in normal human neutrophils. Role of protein kinase C activation. J Leukoc Biol 65:635–640[Abstract]
  54. Van Haastert PJ, Devreotes PN 2004 Chemotaxis: signalling the way forward. Nat Rev Mol Cell Biol 5:626–634[CrossRef][Medline]
  55. McMurray RW, Bradsher RW, Steele RW, Pilkington NS 1990 Effect of prolonged modified fasting in obese persons on in vitro markers of immunity: lymphocyte function and serum effects on normal neutrophils. Am J Med Sci 299:379–385[Medline]
  56. Mowat A, Baum J 1971 Chemotaxis of polymorphonuclear leukocytes from patients with diabetes mellitus. N Engl J Med 284:621–627[Medline]
  57. Esmann V 1983 The polymorphonuclear leukocyte in diabetes mellitus. J Clin Chem Clin Biochem 21:561–567[Medline]
  58. Oldenborg PA, Sehlin J 1999 The glucose concentration modulates N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated chemokinesis in normal human neutrophils. Biosci Rep 19:511–523[Medline]
  59. Calvet HM, Yoshikawa TT 2001 Infections in diabetes. Infect Dis Clin North Am 15:407–421, viii[CrossRef][Medline]
  60. Baumgartner-Parzer SM, Tschoepe D 1997 Role of adhesion molecules in diabetes mellitus. Horm Metab Res 29:613
  61. Dandona P, Aljada A, Mohanty P, Ghanim H, Hamouda W, Assian E, Ahmad S 2001 Insulin inhibits intranuclear nuclear factor {kappa}B and stimulates I{kappa}B in mononuclear cells in obese subjects: evidence for an anti-inflammatory effect? J Clin Endocrinol Metab 86:3257–3265[Abstract/Free Full Text]
  62. Aljada A, Ghanim H, Saadeh R, Dandona P 2001 Insulin inhibits NF{kappa}B and MCP-1 expression in human aortic endothelial cells. J Clin Endocrinol Metab 86:450–453[Abstract/Free Full Text]
  63. Aljada A, Saadeh R, Assian E, Ghanim H, Dandona P 2000 Insulin inhibits the expression of intercellular adhesion molecule-1 by human aortic endothelial cells through stimulation of nitric oxide. J Clin Endocrinol Metab 85:2572–2575[Abstract/Free Full Text]
  64. Braga PC, Sala MT, Dal Sasso M, Pecile A, Annoni G, Vergani C 1998 Age-associated differences in neutrophil oxidative burst (chemiluminescence). Exp Gerontol 33:477–484[Medline]
  65. Chaudhuri A, Janicke D, Wilson MF, Tripathy D, Garg R, Bandyopadhyay A, Calieri J, Hoffmeyer D, Syed T, Ghanim H, Aljada A, Dandona P 2004 Anti-inflammatory and profibrinolytic effect of insulin in acute ST-segment-elevation myocardial infarction. Circulation 109:849–854[Abstract/Free Full Text]
  66. Collier A, Patrick AW, Hepburn DA, Bell D, Jackson M, Dawes J, Frier BM 1990 Leucocyte mobilization and release of neutrophil elastase following acute insulin-induced hypoglycaemia in normal humans. Diabet Med 7:506–509[Medline]
  67. Bjerknes R, Aarskog D 1995 Priming of human polymorphonuclear neutrophilic leukocytes by insulin-like growth factor I: increased phagocytic capacity, complement receptor expression, degranulation, and oxidative burst. J Clin Endocrinol Metab 80:1948–1955[Abstract]




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