Clin. e.\p. Immunoi (1992) 88, 269 274
Intracellular hydrogen peroxide production by peripheral phagocytes from diabetic patients. Dissociation between polymorphonuclear leucocytes and monocytes M. NORITAKE, Y. KATSURA*. N. SHTNOMIYA*. M. KANATANI, Y. UWABE, N. NAGATA & S. T S U R U * Third Department of Internal Medicine, and * Department of Microbiology, National Defence Medicat Cotlege. Tokorozawa. Saitama, Japan
(Accepted for publication } 2 December 1991)
SUMMARY Although the standard assays for reactive oxygen species have been ba.sed on the tneasurement or those released into Ihe extracellular environment, the microbieidal capacity to the engulfed microorganisms is mainly dependent on those released into the intracellular cnvironrnent. such as phagosomes. We studied intracellular oxidative activities of individual phagoeytes by dichlorofluorescein (DCFH) oxidation assay to investigate the relationship between the reactive oxygen species released intraoellularly and the impaired microbicidal capacity in diabetic patients. Time courses of intracellular production of hydrogen peroxide by polymorphonuclear leucocytes (PMNL) and monocytes were observed at the resting condition and after the stimulation with phorbol tnyristate acetate {PMA; 160 nM) by How cytometry. Thirty-four patients with non-insulin-dependcnt diabetes mellitus (NIDDM) and 23 age-matehed healthy volunteers were subjected to the studies. PMNL from patients with NIDDM showed a significantly decreased capacity to produce hydrogen peroxide after the stimulation ( / ' < 0 0 5 at 15 min, / ' < 0 0 I at 30 and 45 min). By contrast- intraeellular hydrogen peroxide production by monocytes at the resting condition and an early stimulatory phase (8 min after the stimulation) was significantly (/'O-) producing capacity of peripheral blood phagocytes to further investigate the tiieehanism ofthe impaired bactericidal activity in diabetic subjeets. Although H^O; is weakly antimicrobial by itself, it participates in the well established H:.O2myeloperoxidase-halide anti-microbial system [9]. To our knowledge, this is a first report of intracellular H:O; production by phagocytes in diabetic patients using tlow cytometry.
20 r
MATERIALS AND METHODS Reagents DCFH-DA was purchased from Eastman Kodak (Rochester, NY). FACS lysing solution and PE-conjugated monoclonal anti-CDi4 antibody (Leu-M3) were obtained from Becton Dickinson Immunocytometry Systems (Mountain View, CA). Phorbol myristate acetate (PMA) and EDTA were purchased Irom Sigma Chemical Co. (St Louis. MO). Subjects We studied 34 men with non-insulin-dependent diabetes mellitus (NIDDM) (mean age 480 + 9 0 years s.d.: range 23-65). Patients with renal failure, infections, malignancies or taking drugs known to influence the leucocytes function were eliminated from the studies. Seventeen were taking insulin and 12 were under treatment with oral hypoglycaemie agents. The average haemoglobin Ai, (HbAi.) level was 10 4 + 1 -H"/., (mean±s.d,; range 70-13 5%, normal range 4 0 - 6 0 % ) , the fasting plasma glucose levels on theday of study 10 4 + 16 mM (mean ± s.d.; range 5 4-16 2 mM), plasma cholesterol 5-34 ±0-83 mM (mean + s.d.. range 3 49-7-70 mM) and plasma triglyceride I 60 + 0 58 niM (mean + s.d., rangeO 67-3 27 niM). Twenty-three healthy men (mean age47 6 ± 8 2 years s.d.; range 23-62 years) were used as control subjeets. The blood was drawn in the morning before insulin administration, while the subjects were in the fasting state. Flow cytometric a.'i.say of intraccUutar o.xidation of DCFH Intracellular H;O> release in phagoeytes was measured by the DCFH oxidation assay according to the method of Bass et at. [3] with some modification. Briefly. 0 5 ml of freshly drawn heparinized peripheral blood was diluted 10-fold with Dulbeeeo's PBS contaitiing 5 mM glucose and 0 1% gelatin (PBS-G). After sampling an aliquot for control, cell suspensions were preincubated for 15 min with 5 /iM DCFH-DA in a water bath at 37 C. After washing twice with PBS to remove the untrapped dye. the cells were treated with EDTA (final concentration 2 mM) and PMA (final concentration 160 nM). Aliquots were sampled before and after Ihe stimulation at defined intervals and .stored in ice-cold eonditions. Finally, cells were washed and analysed by a FACS (FACSean. Becton Dickinson Immunocytometry Systems) using a 488 nm argon ion laser alter washing and treating them with FACS lysing solution for the simultaneous lysis of erythroeytes and partial fixation of leucocytes. Data of polymorphontielear leucocytes (PMNL) were acquired using selective gating based on light scatter properties (forward light scatter and side scatter), and peak channel of log intensity of DCF fluorescence was measured along with the time course;
0 Time ( m i n )
Fig, 1. Time courses of intracellular H^O: productions by PMAstiniulated PMNL from 2.1 heaithy subjects (D) and 34 diabetic subjects
Intracellular hydrogen peroxide production by peripheral phagocytes from diabetic patients. Dissociation between polymorphonuclear leucocytes and monocytes M. NORITAKE, Y. KATSURA*. N. SHTNOMIYA*. M. KANATANI, Y. UWABE, N. NAGATA & S. T S U R U * Third Department of Internal Medicine, and * Department of Microbiology, National Defence Medicat Cotlege. Tokorozawa. Saitama, Japan
(Accepted for publication } 2 December 1991)
SUMMARY Although the standard assays for reactive oxygen species have been ba.sed on the tneasurement or those released into Ihe extracellular environment, the microbieidal capacity to the engulfed microorganisms is mainly dependent on those released into the intracellular cnvironrnent. such as phagosomes. We studied intracellular oxidative activities of individual phagoeytes by dichlorofluorescein (DCFH) oxidation assay to investigate the relationship between the reactive oxygen species released intraoellularly and the impaired microbicidal capacity in diabetic patients. Time courses of intracellular production of hydrogen peroxide by polymorphonuclear leucocytes (PMNL) and monocytes were observed at the resting condition and after the stimulation with phorbol tnyristate acetate {PMA; 160 nM) by How cytometry. Thirty-four patients with non-insulin-dependcnt diabetes mellitus (NIDDM) and 23 age-matehed healthy volunteers were subjected to the studies. PMNL from patients with NIDDM showed a significantly decreased capacity to produce hydrogen peroxide after the stimulation ( / ' < 0 0 5 at 15 min, / ' < 0 0 I at 30 and 45 min). By contrast- intraeellular hydrogen peroxide production by monocytes at the resting condition and an early stimulatory phase (8 min after the stimulation) was significantly (/'O-) producing capacity of peripheral blood phagocytes to further investigate the tiieehanism ofthe impaired bactericidal activity in diabetic subjeets. Although H^O; is weakly antimicrobial by itself, it participates in the well established H:.O2myeloperoxidase-halide anti-microbial system [9]. To our knowledge, this is a first report of intracellular H:O; production by phagocytes in diabetic patients using tlow cytometry.
20 r
MATERIALS AND METHODS Reagents DCFH-DA was purchased from Eastman Kodak (Rochester, NY). FACS lysing solution and PE-conjugated monoclonal anti-CDi4 antibody (Leu-M3) were obtained from Becton Dickinson Immunocytometry Systems (Mountain View, CA). Phorbol myristate acetate (PMA) and EDTA were purchased Irom Sigma Chemical Co. (St Louis. MO). Subjects We studied 34 men with non-insulin-dependent diabetes mellitus (NIDDM) (mean age 480 + 9 0 years s.d.: range 23-65). Patients with renal failure, infections, malignancies or taking drugs known to influence the leucocytes function were eliminated from the studies. Seventeen were taking insulin and 12 were under treatment with oral hypoglycaemie agents. The average haemoglobin Ai, (HbAi.) level was 10 4 + 1 -H"/., (mean±s.d,; range 70-13 5%, normal range 4 0 - 6 0 % ) , the fasting plasma glucose levels on theday of study 10 4 + 16 mM (mean ± s.d.; range 5 4-16 2 mM), plasma cholesterol 5-34 ±0-83 mM (mean + s.d.. range 3 49-7-70 mM) and plasma triglyceride I 60 + 0 58 niM (mean + s.d., rangeO 67-3 27 niM). Twenty-three healthy men (mean age47 6 ± 8 2 years s.d.; range 23-62 years) were used as control subjeets. The blood was drawn in the morning before insulin administration, while the subjects were in the fasting state. Flow cytometric a.'i.say of intraccUutar o.xidation of DCFH Intracellular H;O> release in phagoeytes was measured by the DCFH oxidation assay according to the method of Bass et at. [3] with some modification. Briefly. 0 5 ml of freshly drawn heparinized peripheral blood was diluted 10-fold with Dulbeeeo's PBS contaitiing 5 mM glucose and 0 1% gelatin (PBS-G). After sampling an aliquot for control, cell suspensions were preincubated for 15 min with 5 /iM DCFH-DA in a water bath at 37 C. After washing twice with PBS to remove the untrapped dye. the cells were treated with EDTA (final concentration 2 mM) and PMA (final concentration 160 nM). Aliquots were sampled before and after Ihe stimulation at defined intervals and .stored in ice-cold eonditions. Finally, cells were washed and analysed by a FACS (FACSean. Becton Dickinson Immunocytometry Systems) using a 488 nm argon ion laser alter washing and treating them with FACS lysing solution for the simultaneous lysis of erythroeytes and partial fixation of leucocytes. Data of polymorphontielear leucocytes (PMNL) were acquired using selective gating based on light scatter properties (forward light scatter and side scatter), and peak channel of log intensity of DCF fluorescence was measured along with the time course;
0 Time ( m i n )
Fig, 1. Time courses of intracellular H^O: productions by PMAstiniulated PMNL from 2.1 heaithy subjects (D) and 34 diabetic subjects
