Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2001;70:395-404.)
© 2001 by Society for Leukocyte Biology

Agonist-dependent failure of neutrophil function in diabetes correlates with extent of hyperglycemia

Linda M. McManus*,{dagger}, Rebecca C. Bloodworth*, Thomas J. Prihoda*,{ddagger}, Janet L. Blodgett§ and R. Neal Pinckard*,§

Departments of
* Pathology,
{dagger} Periodontics,
{ddagger} Psychiatry, and
§ Medicine, The University of Texas Health Science Center and
|| South Texas Veterans Administration Hospital, San Antonio, Texas

Correspondence: Linda M. McManus, Department of Pathology—MSC 7750, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. E-mail: mcmanus{at}uthscsa.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inexplicable controversies with regard to possible functional defects of neutrophilic polymorphonuclear leukocytes (PMNs) in diabetes persist. The purpose of the present study was to elucidate the relative effectiveness of several PMN agonists in stimulating lysosomal-enzyme secretion and leukotriene (LT) B4 production by PMNs isolated from diabetic subjects. Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) induced significantly less lysosomal-enzyme secretion and LTB4 production in diabetic-subject PMNs than in normal-subject PMNs. It is surprising that PMNs from these same diabetic subjects responded normally after stimulation with A23187, serum-opsonized zymosan, or phorbol myristate acetate. The in vitro responsiveness of PMNs stimulated with fMLP or PAF was inversely correlated with indices of in vivo glycemic control (fasting plasma glucose and glycated-hemoglobin levels). In combination, these results indicate that hyperglycemia is associated with sustained decreases in PMN function but only in response to agonists that initiate stimulus-response coupling via G-protein-coupled receptors. This agonist-selective reduction in PMN responsiveness may contribute to the compromised host defense associated with sustained hyperglycemia in diabetes.

Key Words: fMLP • GPCR • inflammation • LTB4 • PAF


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Infections represent a frequent and severe systemic complication of diabetes mellitus and are associated with sustained hyperglycemia [1 , 2 ]. However, the cellular, biochemical, or molecular basis for this decline in host defense leading to increased infections in diabetics remains to be conclusively established. Given the complex network of interacting cells and mediators that collectively contribute to innate and acquired immunity, it seems likely that one or more of these components is altered in diabetes. To directly address host defense impairments that may lead to infectious complications of this disease, it is imperative that the functional responsiveness of individual immune and/or inflammatory cell types in diabetes be definitively established.

It has long been recognized that neutrophilic polymorphonuclear leukocytes (PMNs) play an essential role in resistance to infectious agents because reduced PMN function is associated with increased susceptibility to bacterial infection [3 4 5 ]. Not surprisingly, therefore, decreased PMN function has been implicated in impaired host defense in diabetes. Previous studies have described diminished PMN function [e.g., defective chemotaxis, bacterial killing, superoxide production, leukotriene (LT) release, and lysosomal-enzyme secretion] as well as altered basal levels of intracellular calcium and superoxide in diabetes [6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 ]. Despite these observations, however, normal or even enhanced functional responsiveness of diabetic-subjects’ PMNs has also been reported [11 , 22 , 24 25 26 27 28 29 30 31 ]. Thus, the nature and extent of PMN dysfunction in diabetes remain to be resolved.

The reported disparities regarding PMN function in diabetes may be explained at least in part by the heterogeneity of diabetic subjects, PMN stimuli, and/or PMN functional assays used in these studies. For instance, in many investigations of PMN function in subjects with diabetes, the level of glycemic control (either acutely or chronically) has not been reported. Additionally, the types and amounts of PMN stimuli employed have been highly variable. Finally, diverse end points and/or assays of PMN responsiveness (e.g., chemotaxis, phagocytosis, bacterial killing, oxidant or superoxide production, and the release of various lysosomal enzymes) have been assessed. Each of these variations in study design could have contributed to the conflicting conclusions about the (patho)physiologic responses of isolated PMNs in diabetes.

The present study was designed to address some of the issues outlined above, i.e., to comprehensively and systematically assess PMN function in relation to the level of hyperglycemia in diabetic subjects. Thus, the objective of this cross-sectional study was to determine whether there is a relationship between the extent of hyperglycemia and lysosomal-enzyme secretion or LTB4 synthesis in isolated diabetic-subject PMNs stimulated with diverse agonists. The selected agonists included agents which modulate PMN activation by distinct signal transduction mechanisms: a calcium ionophore (A23187), complement-dependent stimulation [serum-opsonized zymosan (OpZ)], agonists acting via G-protein-coupled receptors (GPCR) [i.e., formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF)], and protein kinase C-dependent stimulation with phorbol myristate acetate (PMA). Interestingly, deficits in the in vitro responsiveness of diabetic-subject PMNs occurred only with stimuli that initiate signal transduction through GPCR; it is significant that the degree of in vitro responsiveness of diabetic-subject PMNs was significantly inversely correlated with the extent of in vivo glycemic control. Thus, there is a concomitant agonist-dependent and hyperglycemia-related impairment of PMN function in diabetes. Taken together, these findings may help to explain some of the earlier divergent observations regarding the functional responsiveness of PMNs from diabetics. Moreover, these results provide an essential foundation for future studies designed to elucidate alterations in the intracellular signal transduction mechanism(s) responsible for PMN dysfunction and associated impairments of host defense in diabetes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
fMLP, PMA, and A23187 were obtained from Peninsula Laboratories (Belmont, CA), Sigma-Aldrich Fine Chemicals, Inc. (St. Louis, MO), and Calbiochem (San Diego, CA), respectively. Stock solutions of each agonist were prepared in dimethyl sulfoxide, and aliquots were stored at -70°C. Immediately prior to use, final dilutions of fMLP and PMA were made in freshly prepared Hanks’ balanced salt solution (HBSS; see below), whereas A23187 was further diluted in dimethyl sulfoxide. OpZ was prepared as previously described [32 ]. In brief, zymosan (Sigma-Aldrich Fine Chemicals, Inc.) was activated by boiling and subsequent washing in water; serum was prepared from an aliquot of blood obtained at the time of venipuncture for PMN isolation. After incubation of washed zymosan with serum for 20 min at 37°C, the OpZ was washed twice in sterile pyrogen-free saline (0.9% NaCl); after the final wash, the OpZ was resuspended in HBSS. 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (16:0-alkyl-PAF) was purchased from Bachem Bioscience (Philadelphia, PA). The concentration of stock PAF solutions prepared (and stored at -20°C) in phased chloroform was determined by quantification of inorganic phosphorus after digestion with perchloric acid [33 ]. On the day of use, 16:0-alkyl-PAF was prepared from a stock solution after removal of solvent by evaporation under a stream of nitrogen gas at 37°C followed by resuspension in pyrogen-free saline containing 0.25% human serum albumin (crystalline; Bayer Pharmaceuticals; Kankakee, IL) for at least 60 min prior to use in the bioassay.

HBSS (pH 7.35) was composed of 0.14 M NaCl, 5.4 mM KCl, 0.5 mM NaH2PO4, 1.0 mM Na2HPO4, 0.81 mM MgSO4, and 5 mM glucose. All cell isolation and incubation media were prepared with endotoxin-free water. Disposable and sterile plasticware was used throughout PMN isolation and assay procedures.

Human subjects
Healthy, nonsmoking male and female volunteers who participated in this study either were normal subjects or were diabetic subjects (having either type 1 or type 2 diabetes mellitus) who were receiving insulin therapy (Table 1 ). All subjects were in apparent good health for at least 2 weeks before participation in this study and were receiving no medications (other than insulin); some of the diabetic subjects had previously developed and been treated for complications of their diabetic disease (e.g., neuropathy, retinopathy, or myocardial infarction). Seven subjects (two healthy and five type-1 diabetic) were studied on multiple occasions. This study was approved by the University of Texas Health Science Center committee (Institutional Review Board) that reviews the use of human subjects in research. Informed consent was obtained from all subjects prior to their enrollment and participation in this study.


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  		Table 1. Demographics of Normal and Diabetic Subjects

 
FPG and HgA1c determinations
Fasting plasma glucose (FPG) levels in plasma samples were derived from the same blood used to isolate PMNs and were determined by a colorimetric enzymatic (glucose oxidase) assay according to the manufacturer’s specifications (Glucose HK Reagent; Chiron Diagnostics, East Walpole, MA) with an automated system (VP Systems; Abbott Laboratories; North Chicago, IL). The sensitivity of this assay was 0.36 mg of glucose/dL. Glycosylated-hemoglobin (HgA1c) levels in these same blood samples were measured by a spectrophotometric assay, based on latex immunoagglutination inhibition methodology, with assay kits and an automated analyzer (DCA 2000 hemoglobin A1c reagent kit and DCA 2000 analyzer; Bayer Corporation, Elkhart, IN). For both FPG and HgA1c determinations, a reference calibration curve was used to quantitatively estimate the levels of glucose and HgA1c.

PMN isolation
PMNs were isolated from venous blood by a previously described procedure, with only slight modification [34 ]. In brief, blood was drawn from the antecubital vein of fasting donors into 1/7 volume of acid citrate dextrose (0.065 M citric acid, 0.085 M trisodium citrate, and 5 mM dextrose). Erythrocytes were sedimented with 1% dextran (Dextran T500; Pharmacia Fine Chemicals; Piscataway, NJ) for 30 min at room temperature, and PMNs were isolated on a discontinuous gradient of Ficoll-Hypaque (Pharmacia Fine Chemicals). After 20 s of hypotonic lysis of residual erythrocytes at 4°C, PMNs were washed and resuspended in HBSS at a density of 2 x 107/mL and were maintained on ice until use, which was accomplished within 1 h after completion of PMN isolation. Isolated cell preparations routinely contained >98% PMNs.

PMN stimulation
Isolated PMNs were diluted to a concentration of 5 x 106/mL in HBSS containing human serum albumin (0.25%) and Ca2+ (1.4 mM) and equilibrated at 37°C for 10 min prior to exposure to various agonists [i.e., 1 µM fMLP, 2.5 µM A23187, 40 particles of OpZ/cell, 30 nM PMA, or 1 µM 16:0-alkyl-PAF (final concentrations)]. In PMN suspensions stimulated with fMLP or PAF, cytochalasin B (5 µg/mL final concentration) was introduced 2 min prior to the addition of the agonist. At the end of the incubation period (30, 20, 20, 10, or 5 min for OpZ, A23187, PMA, fMLP, and PAF, respectively), the assay tubes were placed in an ice-water bath prior to centrifugation (2,400xg, 10 min) at 4°C to obtain cell-free supernatants. In every experiment, negative controls (PMNs incubated for the appropriate interval with only the corresponding diluent for each agonist) were processed in parallel. The cell-free supernatants were stored at -70°C until assayed for lysosomal enzymes or LTB4 levels.

Lysosomal-enzyme assays
The cell-free supernatants obtained after PMN stimulation were assayed for ß-glucuronidase and lysozyme activities as previously described [34 ]. Briefly, lysozyme activity was determined on the basis of the rate of lysis of suspensions of Micrococcus lysodeikticus (formerly Micrococcus luteus) (Sigma-Aldrich Fine Chemicals, Inc.); using a suspension of 0.2 mg of M. lysodeikticus/mL in 0.1 M phosphate buffer, pH 6.2, 1 U of activity was defined as a net decrease in turbidity of 1.0 per 20 s at 450 nm with a 1-cm light path at 37°C and expressed as units per 5 x 106 PMNs. Using 0.1 M acetate buffer, pH 4.5, ß-glucuronidase activity was spectrophotometrically (540 nm) determined utilizing phenolphthalein glucuronic acid (Sigma-Aldrich Fine Chemicals, Inc.) as a substrate; 1 U of enzymatic activity was defined on the basis of the quantity of substrate hydrolyzed over a 20-h period at room temperature and expressed as nanomoles per 20 h per 5 x 106 PMNs. For each enzyme, the secretion of activity into cell-free supernatants was expressed as the percentage of total PMN lysosomal-enzyme activity that was released from parallel PMN samples after treatment with Triton X-100 [0.1% (v/v) final concentration].

LTB4 immunoassay
LTB4 levels in the cell-free supernatants obtained after PMN stimulation were determined by an enzyme-linked immunosorbent assay (ELISA) with immunoassay kits used according to the manufacturer’s recommendations (Cayman Chemical Co., Inc., Ann Arbor, MI). Tracer quantities of [3H]LTB4 (Amersham Life Sciences, Arlington Heights, IL) were included in each sample prior to storage at -70°C. Subsequently, LTB4 levels determined by ELISA were corrected for recovery of [3H]LTB4; the level of recovery of [3H]LTB4 in PMN supernatants was 84.0% ± 0.24% (mean ± SE; n=158 samples). The sensitivity of the ELISA for LTB4 was ~7 pg/mL.

In pilot experiments, LTB4 levels were determined in PMN supernatants fractionated by solid-phase extraction. By following the recommendations of the ELISA kit manufacturer, PMN supernatants were extracted into ethanol and further fractionated by solid-phase extraction on C18 reversed-phase columns (Extract Clean; Alltech Associates, Deerfield, IL). When this purification procedure was used, ELISA quantification of LTB4 in cell-free supernatants obtained from stimulated PMNs yielded results that were virtually identical to those obtained with unfractionated samples. Therefore, in all subsequent studies, unfractionated samples were used for determination of LTB4 levels.

Data management and statistical analysis
In each experiment, the results derived from negative controls (indicative of the nonspecific response of unstimulated PMNs; see above) were subtracted to derive net (specific) secretion of LTB4, lysozyme, or ß-glucuronidase after stimulation with a given agonist. Data are presented as the means ± SE of results obtained for separate subjects in a given group (i.e., normal subjects or subjects with type 1 or type 2 diabetes). In those instances in which a subject was studied on more than one occasion, all experimental results for the individual subject (i.e., FPG, HgA1c, and PMN functional responses) were averaged prior to their inclusion in data analysis.

Descriptive statistics (means ± SE) were used to establish measures of central tendency and variability. Significant differences between diabetic and nondiabetic (normal) groups were determined by Student’s t-test or, when more than two groups were compared, by one-way analysis of variance. Spearman’s correlation coefficients (r values) were used to determine the relationships between measures of glycemic control (FPG or HgA1c) and estimates of PMN functional responsiveness (net enzyme secretion or LTB4 production). Effects of covariates (e.g., subject age or total PMN enzyme activity) on correlations were considered by computing partial correlation coefficients. Possible covariate effects on means (e.g., PMN functional responses) were assessed by analysis of covariance. Residuals from all analyses were verified to have a near-normal distribution. Standard transformations were considered, and confirmation that there were no effects due to unequal variances of subject groups was obtained. SAS software (SAS Institute, Cary, NC) was used for all statistical analyses, and P values of <=0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lysosomal-enzyme secretion from PMNs isolated from diabetic and normal subjects
The total enzymatic activities of ß-glucuronidase and lysozyme of PMNs isolated from diabetic and normal subjects were not significantly different. For ß-glucuronidase, the total enzymatic activities (means ± SE) were 215 ± 17 and 242 ± 14 nmol/20 h/5 x 106 PMNs for diabetic-subject (n =18) and normal-subject (n =20) PMN preparations, respectively; similarly, the total lysozyme activities of diabetic- and normal-subject PMNs were 1.74 ± 0.10 and 1.80 ± 0.06 U/5 x 106 PMNs, respectively. Importantly, there were no significant relationships between measures of in vivo glycemic control (either FPG or HgA1c) and total ß-glucuronidase or lysozyme in the PMNs obtained from both groups of subjects.

After stimulation with fMLP or 16:0-alkyl-PAF, the average level of secretion of lysosomal enzymes from diabetic-subject PMNs was significantly reduced (by ~20–30%) in comparison with that of PMNs isolated from normal subjects (Table 2 ). In contrast, diabetic-subject PMNs displayed normal responses to all other PMN agonists; diabetic-subject-PMNs’ responsiveness to A23187, OpZ, or PMA was not significantly different from that of normal-subjects’ PMNs (Table 2) . With all PMN stimuli, there were no significant differences between the averaged responses of PMN from subjects with type 1 and type 2 diabetes (Fig. 1 ). Furthermore, the release of ß-glucuronidase or lysozyme from unstimulated diabetic-subject PMNs (negative controls) was not significantly different from the corresponding results observed with normal PMNs. Finally, statistical adjustments for total enzyme activity within each PMN preparation did not change the extent or significance of the comparative results of diabetic and normal subjects in terms of lysosomal enzyme secretion, regardless of the agonist (partial correlation coefficients are not shown). Thus, there was an agonist-specific (fMLP- or PAF-specific) impairment of lysosomal-enzyme secretion in diabetic-subject PMNs.


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  		Table 2. Lysosomal Enzyme Secretion by Normal and Diabetic PMN

 


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  		Figure 1. fMLP-induced lysosomal-enzyme secretion from PMNs isolated from normal- and diabetic (type-1 and type-2) subjects. After PMN stimulation with fMLP (1 µM final concentration), lysosomal-enzyme activity in the cell-free supernatant was estimated as a percentage of the total enzyme activity. In each experiment, net (specific) enzyme secretion was calculated by subtraction of the enzyme activity of the buffer-treated negative control. Each point represents the results derived from a single subject; when a subject was studied on more than one occasion, results for that individual were averaged and are presented here as a single point. For each group, the mean value for net enzyme secretion ± SE is also indicated. Asterisks denote significant differences (P <=0.001) when compared with corresponding lysosomal-enzyme secretion by PMNs isolated from normal subjects (pairwise comparison of means by using the pooled variability from one-way ANOVA).

 
LTB4 production by diabetic- and normal-subjects’ PMNs
In most experiments, agonist-induced synthesis and release of LTB4 were determined in parallel with lysosomal-enzyme secretion. Maximum LTB4 production by both diabetic- and normal-subject PMNs occurred after stimulation with A23187; although the average amount of LTB4 released from A23187-stimulated diabetic-subject PMNs was somewhat less than that of normal PMNs (96.3±5.70 vs. 107.02±8.59 ng of LTB4/5x106 PMNs, respectively), this difference was not significant. While the average amount of LTB4 produced by diabetic-subject PMNs stimulated with OpZ appeared to be larger than that of normal-subject PMNs, this difference was also not significant (Table 3 ). There was little or no production of LTB4 by either diabetic- or normal-subject PMNs after stimulation with 30 nM PMA (Table 3) . Finally, there was no significant difference in the amounts of LTB4 released from unstimulated normal- and diabetic-subject PMNs. In contrast to all of the above, there was a significant reduction (~50–65%) in the average amount of LTB4 released from diabetic-subject PMNs after either fMLP or PAF stimulation (Table 3) .


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  		Table 3. Secretion of LTB4 by Normal and Diabetic PMNs

 
When the extent of lysosomal-enzyme (either ß-glucuronidase or lysozyme) release was compared with the level of LTB4 produced by diabetic-subject PMNs stimulated with fMLP or PAF, there were significant correlations (r = 0.584–0.904; P <= 0.002). Thus, agonist-specific attenuation of LTB4 production occurred in concert with agonist-selective decreases in lysosomal-enzyme secretion. In contrast, there was no relationship between lysosomal-enzyme release and LTB4 production in diabetic-subject PMNs stimulated with A23187, OpZ, or PMA. Interestingly, with PMNs from normal subjects, there were no significant correlations between lysosomal-enzyme release and LTB4 production with any stimulus.

Relationship between in vitro functional responsiveness of PMN and in vivo glycemia
More important than the averaged PMN functional responses for the diabetic population under study was the issue of PMN responsiveness relative to measures of glycemic control given the considerable variation in individual diabetic subjects in comparison to that of normal (nondiabetic) subjects. Thus, for normal subjects, FPG levels ranged from 75.0–115.0 mg/dL, while in diabetic subjects they ranged from 104–324 mg/dL. HgA1c levels ranged from 4.6–6.0% and from 7.4–12.5% for normal and diabetic subjects, respectively. It is not surprising that, among all subjects, FPG and HgA1c were significantly correlated (r = 0.860; P <= 0.0001).

The extent of fMLP- or PAF-induced lysosomal enzyme secretion (ß-glucuronidase or lysozyme) was inversely and significantly correlated with the extent of in vivo glycemia (Fig. 2 and 3; Table 4 ). Consistent with lysosomal-enzyme release, the synthesis of LTB4 by fMLP- and PAF-stimulated PMN was also inversely related with circulating levels of glucose (Fig. 3 ; Table 4 ). Thus, as the level of circulating glucose was increased (either acutely or chronically), the secretion of lysosomal enzymes and the production and release of LTB4 were decreased. Interestingly, for both fMLP and PAF stimulation, the significant inverse correlations between the long-term index of blood glucose control (HgA1c) and PMN functional responsiveness were somewhat greater than those for the corresponding short-term index of blood glucose control (FPG) (Table 4) . With all other PMN stimuli (A23187, OpZ, and PMA), there was no relationship between LTB4 production or lysosomal-enzyme secretion and either measure of glycemic control (FPG or HgA1c) (Fig. 3) . Importantly, the significance and extent of all relationships between glycemic control and PMN functional responsiveness were unaffected by subject age or gender (partial correlation coefficients are not shown).



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  		Figure 2. Relationship between in vivo glycemic control (HgA1c) and ex vivo PMN secretion of lysozyme after fMLP, PAF, A23187, OpZ, or PMA stimulation. In each experiment, lysozyme activity in the cell-free supernatant was determined as a percentage of the total enzyme activity. Net enzyme secretion was calculated by subtraction of the enzyme activity released in unstimulated (control) cells processed in parallel. Each point represents the results derived from a single subject; when a subject was studied on more than one occasion, results for that individual were averaged and are presented here as a single point. For A23187, OpZ, PMA, PAF, and fMLP, Spearman’s correlation coefficients were 0.17 (P =0.32), 0.29 (P =0.09), 0.05 (P =0.76), -0.59 (P =0.003), and -0.59 (P <=0.0001), respectively.

 

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  		Table 4. Relationship between Glycemia and fMLP or PAF-induced Release of Lysosomal Enzymes or LTB4

 


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  		Figure 3. Relationship between in vivo blood glucose and ex vivo PMN secretion of lysosomal enzymes and LTB4 after fMLP stimulation. For each fasting subject, an aliquot of blood was utilized to derive plasma for subsequent determination of glucose (in milligrams per deciliter); PMNs were isolated from the remainder of the blood sample. After stimulation of isolated PMNs, lysosomal-enzyme activity in the cell-free supernatant was determined as a percentage of the total enzyme activity. Net (specific) enzyme or LTB4 secretion was calculated by subtraction of the enzyme activity or LTB4 level in the cell-free supernatant of buffer-treated (negative-control) cells processed in parallel. Results are the mean ± SE; n = the number of individual subjects. Asterisks indicate significant differences compared with corresponding lysosomal-enzyme secretion by PMNs isolated from subjects with an FPG of <90 mg/dL (pairwise comparison of means using the pooled variability from one-way analysis of variance). *, P <= 0.05; **, P <= 0.01; ***, P <= 0.001.

 
In addition to the above cross-sectional comparisons between subjects, strong correlations were also observed in results obtained from repeated studies within a single diabetic subject (PMN functional responses were monitored on five different occasions with intervals of at least 8 weeks’ duration each). For example, the extent of ß-glucuronidase release was inversely correlated with FPG, which varied between 45 and 221 mg/dL (r = -0.89; P = 0.042). While these observations must be confirmed and extended with data for PMNs from additional diabetic subjects, it is encouraging to find that there is also a consistency in the relationship between PMN functional responsiveness and glycemic control within an individual diabetic subject.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study has documented that there is no intrinsic or generalized PMN defect in patients with diabetes. Rather, there is an agonist-specific impairment in the functional responsiveness of the diabetic-subject PMNs that is related to the extent of elevated in vivo blood glucose concentrations. Thus, as indices of blood glucose levels (FPG and HgA1c) are increased, there is a progressive decrease in PMN functional responsiveness, as reflected by significant reductions of lysosomal-enzyme secretion and LTB4 production, but only after stimulation of PMNs with agonists that initiate signal transduction via GPCRs (i.e., fMLP and PAF).

Our findings provide insight into conflicting published data regarding the function of diabetic-subject PMNs (see Introduction). Thus, in many previous studies, the range and extent of glycemia often were not taken into account in the diabetic study populations. This problem was further compounded by the use of PMN stimuli with responses which were unaffected by hyperglycemia. Notwithstanding, it is important to point out that irrespective of the PMN stimulus utilized in the present study, the functional responsiveness of PMNs from diabetic subjects in a state of good glycemic control was comparable to that of PMNs isolated from nondiabetic subjects. In view of the preceding, it is now clear why some earlier investigations led to the conclusion that the in vitro behavior of diabetic-subject PMNs was indistinguishable from that of normal-subject PMNs while other studies found reduced diabetic-subject-PMN responsiveness.

There are additional considerations relative to the present observations of reduced lysosomal-enzyme secretion by diabetic-subject PMNs. For instance, given that neutrophil-derived oxidants can inactivate secreted PMN enzymes in vitro [35 , 36 ], it is conceivable that excessive production of oxidants by diabetic-subject PMNs could explain our findings. However, in separate preliminary studies, we have observed that fMLP-stimulated diabetic-subject PMNs produced less O2- than did normal-subject PMNs and noted that this impaired respiratory burst in diabetic-subject PMNs also correlated with a lack of glycemic control; moreover, irrespective of indices of glycemia, diabetic-subject PMNs generated normal amounts of O2- in response to PMA stimulation [37 ]. Therefore, it seems unlikely that products of the respiratory burst in diabetic-subject PMNs are responsible for reductions in secretion of lysosomal enzymes by these cells.

Another consideration in the present report relates to the inclusion of cytochalasin B (CB) in PMN assays with fMLP and PAF. This agent, which enhances fMLP- and PAF-induced functional responses, is also known to inhibit selected isoforms of glucose transporter proteins (e.g., GLUT1) that facilitate the passive transport of glucose into diverse cell types, including the PMN [38 ]; thus, binding of CB to glucose transporters noncompetitively blocks the binding and transmembrane uptake of glucose [39 ]. While CB treatment may have altered PMN glucose transport in the present study, it seems unlikely that the differences between normal- and diabetic-subject PMNs can be explained on the basis of this agent inasmuch as all cell preparations were treated in an identical manner. Moreover, as outlined above, similar hyperglycemia-related differences in normal- and diabetic-subject PMNs have also been observed relative to fMLP-induced superoxide production (an assay performed in the absence of CB). It is noteworthy that both fMLP and PAF may be involved in the translocation of GLUT1 in transfected Chinese hamster ovary cells [40 ]. If this proves to be the case in PMNs, reduced responsiveness to these stimuli could also affect the overall energy status and related function of these cells.

Alterations in the release of LTB4 from diabetic-subject PMNs have been previously described [41 , 42 ]. Thus, release of LTB4 from A23187-stimulated PMNs from subjects with type 1 diabetes was found to be inversely correlated with plasma glucose levels (but not with levels of HgA1c) [41 ]. On the other hand, a contrasting study found that A23187-stimulated PMNs obtained from subjects with type 2 diabetes released larger amounts of LTB4, an effect that was positively correlated with glycated hemoglobin [42 ]. Interestingly, the basal levels of LTB4 in unstimulated diabetic-subject PMNs were also significantly increased. Nevertheless, our results are inconsistent with both of these previous reports. That is, irrespective of glycemic indices, both the basal and A23187-stimulated levels of LTB4 produced by PMNs from diabetic subjects were comparable to those observed with normal-subject PMNs (Table 3) . Nevertheless, the same diabetic-subject PMN preparations had significantly impaired fMLP- and PAF-induced LTB4 synthesis, the net increase of which was inversely correlated with plasma glucose or glycated hemoglobin (Table 4 ; Fig. 3 ). The basis for differences between our findings and those of the two preceding independent studies remains to be elucidated.

The decreased production of LTB4 by fMLP- or PAF-stimulated diabetic-subject PMNs could be a consequence of either reduced LTB4 precursor availability or dysfunctional enzymes that are involved in the release of arachidonic acid and its subsequent metabolism via 5-lipooxygenase. However, given that diabetic-subject PMNs produced normal levels of LTB4 after stimulation with A23187 (Table 3) , it seems unlikely that there are deficits in either arachidonate availability or the enzymatic machinery required for LTB4 synthesis in diabetic-subject PMNs. Rather, these findings suggest an inadequate activation of metabolic enzymes after the stimulation of diabetic-subject PMNs via GPCR. In any case, the reduced production of LTB4 by diabetic-subject PMNs has substantial pathophysiologic ramifications because LTB4 serves as an autocrine activator of PMNs [43 ]. Thus, the reduced production of LTB4 by diabetic-subject PMNs may contribute to the overall reduction in PMN functional responsiveness. Furthermore, because LTB4 acts synergistically with other PMN agonists to enhance PMN activation [44 , 45 ], a reduction in LTB4 production by PAF- or fMLP-stimulated diabetic-subject PMNs would further reduce the critical functions of these essential inflammatory cells.

In keeping with the preceding notion, we have observed that there is a significant relationship between the extents of LTB4 production and lysosomal-enzyme secretion by diabetic-subject PMNs. However, since degranulation responses were estimated as a percentage of the total PMN enzyme activity of either ß-glucuronidase or lysozyme, it was important to compare the total amounts of enzyme activity between normal- and diabetic-subject PMNs. This point is especially critical because the activities of lysosomal enzymes in diabetic-subject PMNs have also been inconsistent in published reports. For instance, total myeloperoxidase (MPO) activity in PMNs from patients with type 2 diabetes was found to be significantly lower than that of normal-subjects’ PMNs [46 ]. In contrast, Oberg et al. observed that the MPO content of diabetic-subject PMNs was comparable to that of PMNs from healthy subjects despite the fact that the activities of cathepsin G, elastase, and lysozyme were higher in the former [47 ]. The intracellular lysozyme content of PMNs from type 1 diabetics was reported to be lower while the content of ß-glucuronidase was similar to that of PMNs of controls [29 ]. Furthermore, elastase levels of PMNs from type 1 diabetics and controls have been found to be comparable [48 ]. In the present study, the total activities of lysozyme and ß-glucuronidase in isolated diabetic-subject PMNs were the same as those of normal-subject PMNs. Therefore, a coordinated reduction in degranulation of and LTB4 production by diabetic-subject PMNs is indicative of a generalized decrease in the functional responsiveness of these cells.

The results of the present study not only confirm and extend but also conflict with data from previous investigations relative to diabetic-subject-PMN functional responsiveness after fMLP stimulation. For instance, fMLP-stimulated aggregation of diabetic-subject PMNs has been reported to be either normal [21 ] or increased [49 ]. Reductions of fMLP-induced chemotaxis and chemiluminescence by diabetic-subject PMNs have also been described [6 , 50 ]. Very recent investigations have measured a reduction in membrane fluidity in fMLP-activated PMNs from subjects with insulin resistance [51 ]. These latter findings are intriguing given observations that elevated levels of insulin interfere with MPO-dependent production of reactive oxygen metabolites in fMLP-stimulated PMNs from nondiabetic subjects [52 ]. In any case, and as outlined above, inconsistencies in experimental findings with fMLP may be explained, in part, on the basis of glycemic control, an often unreported variable. Given the results obtained in the present investigations, it will be important to (re)assess other functional responses of diabetic-subject PMNs, with careful attention to the influence of this highly unpredictable clinical parameter in diabetic subjects.

The in vitro functional responsiveness of isolated PMNs presumably is a reflection of the in vivo physiologic capabilities of these cells, which include adherence, chemotaxis, phagocytosis, superoxide and/or oxygen-derived radical production, microbial killing, degranulation, pro-inflammatory cytokine gene expression, and synthesis of pro-inflammatory lipid mediators [53 ]. Nonetheless, whether the hyperglycemia-related in vitro dysfunction of diabetic-subject PMNs observed in the present study will be reflected in adverse clinical consequences remains to be determined. Clearly, individuals with inherited defects in phagocyte function (e.g., chronic granulomatous disease) develop severe and recurrent bacterial and fungal infections [3 4 5 ]. However, our results indicate that in contrast to the sustained PMN deficits that occur in genetically directed disorders, the extent of impairment of diabetic-subject PMNs will oscillate in relation to glycemic control. Therefore, persistently poor glycemic control would have a progressively deleterious effect on PMN function, thereby impairing host defense and predisposing affected individuals to an increased incidence and severity of infections. In this regard, it will be important to determine the extent of reversibility of diabetic-subject-PMN dysfunction with improved glycemic control. That is, despite the relatively short life span of the PMN in the circulatory system, are hyperglycemia-associated impairments returned toward normal as glycemic control is acutely improved? Another, equally important aspect of diabetic-subject PMN (patho)physiology of possible clinical relevance is the issue of agonist-stimulated enhancement of PMN survival (delayed apoptosis) in response to GPCR activation [54 55 56 57 58 ]. Indeed, a recent report describes impairments in delayed apoptosis in diabetic-subject PMNs [59 ]. An abbreviated survival of stimulated diabetic-subject PMNs coupled with hyperglycemia-associated decreased functional responsiveness would likely compound PMN ineffectiveness relative to combating infectious disease processes.

It should be stressed that in association with hyperglycemia, diabetic-subject-PMN responsiveness was differentially impaired in our studies. Thus, PMN responses to some agonists (i.e., OpZ, PMA, and A23187) were unaffected by hyperglycemia whereas the responses to other agonists (i.e., fMLP and PAF) were significantly decreased in a glucose-dose-dependent manner (Tables 2 and 3) . With each PMN stimulus and functional response, there are complex, sequential intracellular signaling steps involved in stimulus-response coupling. Therefore, the in vitro functional assays described herein (LTB4 and lysosomal-enzyme release) reflect outcomes that are considerably downstream from sites of cell membrane-agonist interaction. In related preliminary studies, we have observed similar GPCR agonist-selective, hyperglycemia-related deficits in other PMN functional responses [e.g., cytosolic–ionized-calcium or intracellular-calcium-ion (Ca2+)i mobilization] (data not shown). In combination, these observations support the hypothesis that hyperglycemia is associated with the selective alteration of signal transduction after PMN activation via GPCR.

In view of the preceding points, it is somewhat surprising that there has been little attention given to altered signaling or biochemical events in diabetic-subject PMNs. A relatively recent report described decreased inorganic-phosphate (Pi) turnover and [Ca2+]i mobilization in diabetic-subject PMNs stimulated with fMLP [60 ]; however, it is noteworthy that the resting [Ca2+]i concentration in these studies was quite high (>250 nM). In this regard, the issue of basal [Ca2+]i in diabetic-subject PMNs is also controversial inasmuch as both normal [61 ] and increased [13 , 62 ] levels have been described. Very relevant to the present studies, Gawler et al, reported a decrease in hepatic expression of the {alpha}-subunit of Gi in an experimental model of diabetes [63 ]. Because this G-protein subunit is essential in many signal transduction events, such a loss would effectively decrease the function of second-messenger systems, thereby preventing downstream functional responses associated with G-protein activation. These findings, together with the results of the present study documenting selectively impaired responses to fMLP and PAF, support the need for future studies to assess alterations in GPCR-initiated intracellular signal transduction in diabetic-subject PMNs. Given our observations of comparable responsiveness of diabetic- and normal-subject PMNs to PMA stimulation, it seems unlikely that altered signaling events in diabetic-subject PMNs are dependent on activation of the conventional protein kinase C. Clearly, additional studies must directly address the entire signal transduction cascade of events in diabetic-subject PMNs.

The results of the present study hopefully will foster further investigations to elucidate the basis for the relationship between PMN impairment(s) and hyperglycemia. That is, glucose per se may not be directly responsible for altered PMN function. In preliminary studies, we have observed that a brief (10-min) in vitro exposure to 15 or 25 mM glucose does not significantly affect normal- or diabetic–subject-PMN functional responses induced by any agonist, including fMLP and PAF (data not shown). Nevertheless, both depressed and enhanced responses have been observed for normal- and diabetic-subject PMNs exposed to excessive exogenous glucose for longer durations [64 65 66 67 68 69 70 71 72 ]. Thus, a sustained exposure to hyperglycemia or other pathophysiologic events associated with hyperglycemia likely are integral to impaired PMN functional responsiveness in diabetes. For instance, advanced glycation end (AGE) products are known to stimulate cells, including vascular endothelial cells and peripheral-blood monocytes, via a specific cell surface receptor(s) for AGE products [73 ]. The presence of AGE receptors on PMNs has not been reported; however, enhanced chemotaxis and priming of PMNs with glycoxidized collagen have recently been described [74 ]. Alternatively, and on the basis of relatively recent observations of nondiabetic-subject PMNs [75 76 77 78 ], it is conceivable that hypertonic, hyperosmolar, or cell-volume-dependent alterations might adversely and selectively affect diabetic-subject PMN responses after stimulation via GPCR. Additional investigations are clearly required to assess these important possibilities.

In summary, we have documented agonist-specific decreases in diabetic-subject PMN responsiveness that are associated with hyperglycemia. These findings hopefully will foster future efforts to better understand the function of these important acute inflammatory cells in both health and diabetic disease. Notwithstanding, hyperglycemia also develops during a variety of other common, adverse clinical scenarios (e.g., after traumatic head wounds, extensive burn injuries, and even minor surgical procedures), as well as during normal aging. Importantly, poor prognosis and life-threatening clinical sequelae are directly related to the extent of hyperglycemia in these patients [79 80 81 82 83 84 85 86 ]. Thus, an improved understanding of hyperglycemia-related deficits in host defense will be of immense benefit in advancing the prevention of these complications as well.


   ACKNOWLEDGEMENTS
 
These studies were supported in part by grants from the South Texas Health Research Center, the U.S. Public Health Service (grant HL22555), and the Juvenile Diabetes Foundation International. The authors acknowledge the assistance of Ms. Shelley Meyers, a summer student who was supported by the Minority Scientist Development Program of the NIGMS (GM55386).

Received January 2, 2001; revised March 13, 2001; accepted March 15, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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