The Effect of 2-deoxygiucose on Guinea Pig Polymorphonuclear Leukocyte Phagocytosis 1 LAURENCE A. BOXER,' ROBERT L. BAEHNER AND JACQUELINE DAVIS Department of Pediatrics, Division of Pediatric Hematology-Oncology, James Whitcomb Riley Hospital for Children and the Indiana University School of Medicine, Indianapolis, Indiana 46202
ABSTRACT The effects of 2-deoxyglucose (DOG), an inhibitor of glycolysis, on guinea pig polymorphonuclear leukocytes (PMN) obtained from peritoneal exudates was examined. ATP levels in PMN were reduced by 40% by one hour following an incubation with 2-deoxyglucose. When complement (C3) coated I4Cstaphylococcus aureus, C3 coated lipopolysaccharide-paraffin oil droplets (LPSPO), 14C-pneumococcusopsonized with IgG, or albumin coated paraffin oil droplets opsonized with IgG were added to cell suspensions containing DOG, the phagocytizing rate was 1,310 2 55 cpm/5 X lo6cells/l5 minutes, 6 2 pg paraffin oil (PO)/ lo7 cells/minute, 2,250 f 175 cpm/l X lo6 cells/2O minutes or 0.037 +- 0.01 mg PO/107cells/minute compared to control values of 5,970 +- 275 cpm/5 x lo6cells/ 15 minutes, 35 -+ 3 pg PO/107cells/l5 minutes, 4,510 f 200 cpm/l x lo6cells/20 minutes and 0.067 0.01 mg PO/107 cellslminute. In parallel studies the phagocytic index for latex was 0.74 f 0.28 in DOG compared to control of 2.36 1.13 and the phagocytic rate of albumin coated paraffin oil droplets was 0.029 ? 0.01 mg PO/107 PMN/minute in DOG compared to control of 0.048 mg PO/107cells/minute. When ATP levels were maintained by the simultaneous addition of 5 mM glucose or pyruvate to media containing DOG, latex ingestion was improved to 1.15 0.3 with glucose and 1.59 -+ 0.64 with pyruvate and albumin coated particles to 0.045 f 0.01 mg PO/107 PMN/minute with pyruvate. There was no improvement in the uptake of either the C3 dependent particles or IgG coated Pneumococci in media containing DOG and glucose and/or pyruvate. Following the removal of DOG from the extracellular medium and the addition of pyruvate or glucose, phagocytosis of C3 dependent LPS-PO was restored to normal values. Neither the binding of C3 or IgG coated particles to the PMN nor the lateral movement of glycoprotein utilizing concanavalin A capping was affected by DOG. Thus, the presence of DOG in the PMN containing adequate amounts of ATP will selectively and reversibly inhibit those surface events required for phagocytosis of C3 and IgG bound particles but not latex particles or albumin particles which non-specifically bind to PMN.
Phagocytosis by polymorphonuclear leukocytes (PMN) is an energy requiring process served primarily by glycolytic oxidation of glucose (Sbarra and Karnovsky, '59). Particle uptake by guinea pig PMN is significantly inhibited by metabolic blockade of the glycolytic pathway, but not by interference with oxidative phosphorylation (Sbarra and Karnovsky, '59; Karnovsky, '62). More recently, investigators have explored the involvement of oxidative J. CELL. PHYSIOL., 91: 89-102.
phosphorylation in energy metabolism in both guinea pig and human PMN (Temelin and Frei, '70; Marchand et al., '72).In addition, mitochondria isolated from human Received Apr. 5, '76. Accepted Aug. 19, '76. 'Supported by NIH Grants PHS ROI A1 10892-03, A1 35806-01 and grants from the James Whitcomb Riley Memorial Association. Address reprint requests to: Laurence A. Boxer, M.D., Division of Pediatric Hematology-Oncology, JamesWhitcomb Riley Hospital for Children, 1100 West Michigan Street, Indianapolis, Indiana 46202.
LAURENCE A. BOXER, ROBERT L. BAEHNER AND JACQUELINE DAVIS
leukocytes were found to be capable of oxidative phosphorylation (Foster and Terry, '67). This system was found to contribute approximately 20% of the total ATP in these PMN (Jemelin and Frei, '70). Previous studies were performed on PMN in which the normal energy requirements could be met by glycolysis. Study of PMN with defective glycolysis due to phosphoglycerate kinase deficiency suggested that oxidative phosphorylation was required for phagocytosis in those cells since cyanide inhibited the uptake of opsonized bacteria (Baehner et al., '71). To further study the contribution of oxidative phosphorylation in IMN with limited energy capacity, we have blocked glycolysis with 2-deoxyglucose and observed the ATP level and the effect on phagocytosis. To distinguish Krebs cycle from glycolytic oxidation involvement in ATP generation, substrates normally dependent upon Krebs cycle for oxidation (pyruvate, alanine, succinate and citrate) were studied in cells previously exposed to 2-deoxyglucose. Finally, to assess whether 2-deoxyglucose might directly impair phagocytosis apart from its effect on ATP levels, the rate of uptake of particles with differing opsonic requirements was studied in PMN incubated with both 2deoxyglucose and either Krebs cycle or glycolytic substrates to maintain ATP levels. MATERIALS AND METHODS
Preparation of PMN Guinea pigs were injected intraperitoneally with 20 ml 12% sodium caseinate (Difco Laboratories) 16 to 18 hours prior to harvesting. Greater than 90% of the cells obtained in this manner were PMN as determined by Wright stained smears. Cells were centrifuged at 250 X g for ten minutes at 4°C and washed three times with Krebs Ringer phosphate buffer (KRP), pH 7.4. The final supernatant was discarded and the cells were suspended in KRP. An aliquot was taken for cell count and for viability studies using trypan blue exclusion. Trypan blue exclusion tests con-
sistently revealed greater than 90% viability in all PMN suspensions.
Preparation of 14CStaphylococcus aureus and I4CStreptococcus pneumoniae, type I1 The respective organisms were inoculated into 50 ml nutrient broth and incubated at 37°C in a Dubnoff metabolic shaker for three hours until log phase growth was established. Then, each suspension was incubated overnight at 37°C in the presence of 250 microcuries 14C linoleic acid (New England Nuclear) bound to 10 ml defatted human serum albumin. On the following day the bacteria were centrifuged at 16,000 X g at 4°C for 15 minutes; a pellet of live bacteria was then resuspended in KRP plus 10 mM glucose and 1% bovine serum albumin (KRPGA) and incubated in an 80°C water bath for 60 minutes to kill the bacteria. Following another 16,000 X g centrifugation for 15 minutes at 4"C, the concentration of bacteria was adjusted by suspending the pellet to 85% transmission at 590 mp on a Coleman junior spectrophotometer. This percent transmission yielded a concentration of Staphylococcus aureus3 x 108/ml. Streptococcus pneumoniae was resuspended to a concentration of 2 X lo7bacterialml. The suspensions were then stored at -30°C in aliquots of 12 ml until needed. At the time of use of 14C Staphylococcus aureus equal volumes of bacterial suspensions were combined with equal volumes of fresh frozen type AB non-immune serum previously stored at -70°C and incubated at 37"C, which leads to complement fixation through activation of the alternative complement pathway (Scribner and Fahrney, '76). Then the bacteria were pelleted at 16,000x g for 15 minutes and resuspended in saline to a concentration of 1 x lo8bacteria/ml. Determination of rate and extent of uptake of opsonized I4CStaphylococcus aureus Six 50 ml siliconized Erhlenmeyer flasks
EFFECT OF 2-DEOXYGLUCOSE ON PMN PHAGOCYTOSIS
each containing 20 ml of 5 X lo6 PMNlml were incubated at 37°C in a Dubnoff metabolic shaker for three hours. The control flask was supplemented with 5 mM glucose. The other five flasks received 5 mM 2deoxyglucose. Flasks three through six were supplemented with either 5 mM pyruvate, alanine, succinate or citrate. After three hours, 1 ml aliquots were placed in 12 X 75 mm plastic tubes (Falcon Plastics) containing 1 x 10814C Staphylococcus aureus and 1ml saline. Phagocytosis was determined by the method of Root (Root et al., '72) by tumbling 5 X lo6PMN with 1 ml Staphylococcus aureus (1 X lo8 bacteria) at 37°C. At successive time intervals of 0,5,10, 15 and 30 minutes, ingestion was halted by the addition of 1 ml ice cold saline containing M sodium fluoride. Tubes were centrifuged immediately at 4°C for 200 x g for five minutes and washed twice. The pellets were dried overnight and digested the next day with 0.2 ml of 0.5 normal sodium hydroxide. The contents were transferred to vials containing 10 ml Buehler's solution and counted in a liquid scintillation counter for ten minutes. Each time point was measured in triplicate and the results were averaged.
Determination of uptake of IgG 14C Streptococcus pneumoniae Phagocytosis over 20 minutes was determined in an analagous fashion to the above method by tumbling 1 X lo7 14C Streptococcus pneumoniae with 1 X lo6PMN in 0.9 ml of KRP and 0.1 ml of type I1 specific antisera containing 336 ngrams nitrogen/ml of monospecific IgG previously heat inactivated at 56°C for 30 minutes along with the various supplemental metabolites. The type I1 purified IgG had been previously found capable of opsonizing type I1 Streptococcus pneumoniae (Provisor et al., '76). Phagocytosis of latex particles and lipopolysaccharide-parafin oil droplets PMN phagocytosis of latex particles (0.81 p in diameter) (Dow Chemical Com-
pany, Midland, Michigan) was quantitated by a modification of a previously described method (Penny et al., '66). A suspension of 1 X lo7 PMN previously preincubated with desired metabolite in 0.8 ml of KRP were incubated with 0.2 ml of previously dialyzed latex particles for 15 minutes at 37°C. The number of latex particles ingested by 100 neutrophils was recorded and based on the average number of particles ingesterhell, the phagocytic index (PI) was determined. Ingestion of C 3 coated lipopolysaccharide paraffin-oil or bovine serum droplets albumin coated paraffin-oil droplets by PMN was analyzed as previously described (Stossel et al., '72; Stossel, '73). In some experiments the albumincoated oil droplets were incubated at 37°C for 15 minutes with rabbit anti-bovine antiserum which contained 4 mg of antialbumin antibody/ml (Cappel Laboratories, Inc., Downington, Pennsylvania) according to a previously described method (Boxer and Stossel, '74).
Determination of PMN ZgG and C3 receptors The C 3 receptor the PMN was identified by modifying a previously described method (Gelfand e t al., '76). Salmonella typhii 0901 were directly fluoresceinated and then incubated in non-immune human serum which leads to complement fixation through activation of the alternative complement pathway (Gelfand e t al., '76). PMN obtained from guinea pig peritoneal exudates were incubated for one hour at 37°C in Krebs-Ringer phosphate, pH 7.4, with 5% bovine serum albumin containing either 5 mM glucose, 5 mM 2-deoxyglucose, or 5 mM deoxyglucose and 5 mM pyruvate. Following the incubation in the sugars, 4 X lo6 PMN/ml were removed from each of the tubes and 0.1 ml of the PMN were incubated with an equivalent concentration and volume of C 3 coated fluoresceinated Salmonella typhii for 90 minutes at 37°C in a shaking water bath. A drop of this preparation was placed under a coverslip and inspected under W light.
LAURENCE A. BOXER, ROBERT L. BAEHNER AND JACQUELINE DAVIS
160 PI co
40 20 J
G 2-P A S C
G 2 - P A S C
G 2-P A S C
Fig. 1 Mean f SEM of ATP content of PMN incubated with glucose (G), 2-deoxyglucose (2-DO), 2-deoxyglucose plus pyruvate (P), 2-deoxyglucose plus citrate (CP). At mM substrate concentration was used for all experiments. Length of incubation is indicated on the abscissa.
The total number of celldfield under bright field illumination was determined as well as the number of cells with three or more adherent bacteria. In a similar fashion Streptococcus pneumoniae were directly fluorescienated. The Streptococcus pneumoniue were added to an equivalent volume of heat-inactivated (56°C for 30 minutes) rabbit type I1 specific antisera for 30 minutes. The bacteria were washed with cold Krebs-Ringer phosphate, pH 7.4, three times and finally suspended in Krebs-Ringer phosphate, pH 7.4, at a final concentration of 4 x lo* fluoresceinated bacteria/ml. To determine the PMN IgG receptor the opsonized Streptococcus pneumoniae were incubated with the PMN for 120 minutes at 37°C following which the number of PMN containing three or more IgG coated bacteria were enumerated.
Distribution of fluorescein-conjugated concanavalin A on PMN In order to assess capping PMN previously treated for one hour with either 5 mM glucose, 2-deoxyglucloseand glucose, or 2deoxyglucose and pyruvate were incubated with fluorescein-conjugated concanavalin A (FTIC-con A, Miles Laboratory, Elkhart, Indiana) before or after exposure to colchicine according to a previous method (Oliver et al., '75). Determination of 14C02oxidation from 14C substrates The rate of I4CO2evolution from I4Clabeled substrates was determined by a method previously described (Baehner et al., '70). One microcurie of gl~cose-l-'~C (New England Nuclear) was added to each flask and additional cold substrate (glucose, pyruvate, succinate acid, sodium citrate or
EFFECT OF 2-DEOXYGLUCOSE ON PMN PHACOCYTOSIS
OXIDATION O F K R E B S C Y C L E S U B S T R A T E S 1
14c-u-alanine 14C-6-citrate Fig. 2 Effect of 5 mM 2-deoxyglucose on "CO, evolution from PMN incubated with pyruvate, alanine, succinate and citrate. Data expressed as mean f SEM. Three separate experiments were performed in triplicate.
L-alanine) was added to the respective radioactive material to achieve a final concentration of 5 mM in each incubation flask. At a final concentration of 5 mM, 2-deoxyglucose was added to the incubation mixture 15 minutes prior to the addition of 14C substrate and the incubations were carried out at 37°C in a Dubnoff metabolic shaker for 45 minutes. The reaction was stopped by the addition of 0.2 ml 1 N H2S04. The flasks were placed on a Fisher rotator for an additional 30 minutes. They were then uncapped and contents of the center well were removed and placed in 10 ml
Buehler's solution and counted for ten minutes in a liquid scintillation counter.
Determination of A TP ATP levels were determined enzymatically by a reviously described method (Adam, ' 6 5 . PMN suspensions were incubated at 37°C at a concentration of 1 x lo8 PMN/ml. Appropriate flasks were prepared: one contained 5 mM glucose; all others contained 5 mM 2-deoxyglucose. Flask three through six were supplemented additionally with either 5 mM pyruvate, alanine, succinate or citrate. One ml ali-
LAURENCE A. BOXER, ROBERT L. BAEHNER AND JACQUELINE DAVIS
o Glucose 2 deoxyglucose
+ pyruvate 8000
alanine succinate A citrate
MINUTES Fig. 3 Uptake of I4C Staphylococc~saweus by PMN incubated with substrates as indicated in figure 1. Results are expressed as mean f SEM. Five separate experiments were performed with each time point measured in triplicate.
quots were taken from all flasks at 0,60 and 180 minutes. The aliquots were added to 1 ml ice cold 6% perchloric acid and neutralized with 5 M K & 0 3 and centrifuged at 4°C at 1,000 X g for 20 minutes. Assays were performed on the same day and values were calculated and expressed as nmoles ATP/108 PMN. Identical studies were performed on flasks supplemented with 1 mM KCN. No effect of 1 mM KCN was observed on the enzymatic assay of ATP.
of time, the ATP fell significantly ?tialperiod