Immunology 1978 35 141

The effect of chemotactic factors and agents which influence neutrophil movement on anaerobic glycolysis and hexose monophosphate shunt activity

R. ANDERSON, ANNEMARIE GLOVER & A. R. RABSON Department of Immunology, School ofPathology of the University of the Witwatersrand and the South African Institute for Medical Research, Johannesburg, South Africa

Received 6 October 1977; acceptedfor publication 31 October 1977

Summary. The effects of two chemotactic factors, endotoxin activated serum (EAS) and casein and a number of drugs known to affect intracellular cyclic nucleotide levels and various forms of neutrophil movement, on neutrophil anaerobic glycolysis and hexose monophosphate shunt (HMPS) activity were assessed. EAS caused stimulation of glycolysis. HMPS activity and NBT reduction, but casein was without effect on glycolysis and NBT reduction and inhibited HMPS activity. Drugs known to increase intracellular cAMP levels caused a depression of HMPS activity whereas those reported to elevate cGMP had a variety of effects. Glycolysis was not affected by any of these agents. These results indicate a lack of relationship between cyclic nucleotide effects on cell motility and neutrophil glycolysis and HMPS activity.

chemoattractant casein, indicated that stimulation of glycolysis was not essential for cell motility. It has also been shown that resting activity of the hexose monophosphate shunt (HMPS) is stimulated by the interaction of PMN with purified chemotactic factors (Goetzl & Austen, 1974) and that ascorbic acid, an agent which stimulates neutrophil motility, promotes a dose dependent stimulation of HMPS (Goetzl, Wasserman, Gigli & Austen, 1974). However, neutrophil immobilizing factor (NIF) which prevents both random and directed motility of PMN does not prevent the stimulation of the HMPS by an active chemoattractant (Goetzl, Wasserman, Gigli & Austen, 1973) and methylene blue a potent stimulator of HMPS has no effect on neutrophil migration. These findings suggest that HMPS stimulation is not alone sufficient to enhance migration. To assess further the relationship between glycolysis and HMPS activity to cell motility, the effects on these pathways of a number of agents known to alter cell locomotor responses were investigated.

INTRODUCTION Inhibitor studies have firmly implicated glycolysis as being the major energy source for cell motility (Carruthers, 1966). Furthermore certain chemotactic factors, C5a and kallikrein cause stimulation of polymorphonuclear leucocyte (PMN) glycolytic activity (Goetzl & Austen, 1974). Initial studies using the



Adenosine 3',5'-cyclic monophosphoric acid Correspondence: Dr R. Anderson, S.A.I.M.R., P.O. Box 1038, Johannesburg 2000, South Africa. (cAMP), N6, 02-dibutyryl adenosine 3',5'-cyclic 0019-2805/78/0700-0141 S02.00 © 1978 Blackwell Scientific Publications 141


R. Anderson, Annemarie Glover & A. R. Rabson

monophosphoric acid (d'cAMP), histamine, isoproterenol, acetylcholine, carbamyl choline and ascorbic acid were obtained from the Sigma Chemical Company (St Louis, Missouri). Guanosine 3',5'-cyclic monophosphoric acid (cGMP) was supplied by Boehringer Mannheim (South Africa), prostaglandin A, and El by Dr John Pike (Upjohn Company, Kalamazoo, Michigan), levamisole hydrochloride by Ethnor Laboratories (Johannesburg) and propranolol by I.C.I. (South Africa).

Neutrophil preparation For all investigations pure neutrophil suspensions were used. Peripheral blood from normal volunteers was collected into preservative-free heparin (Panheparin-Abbott 5 units/ml) and was separated on Hypaque-Ficoll gradients. Mononuclear cells were discarded, the resultant pellet was resuspended in physiological saline and sedimented with a 25 % volume of 3 % gelatin (Difco, Detroit, Michigan) for 45 min. The neutrophil-rich layer was collected, centrifuged at 250 g for 10 min and the resultant cell pellet treated with 0-83 % ammonium chloride at 40 for 10 min to lyse residual erythrocytes. The remaining cells which consistently contained greater than 90% viable PMN were washed in phosphate buffered saline and resuspended to a final volume of 2x 107 PMN/ml. Preparation of chemotactic factors (a) Endotoxin activated serum (EAS). Fresh serum activated by 100 ,ug/ml of bacterial lipopolysaccharide (E. coli 0127: B8 Difco). The mixture was incubated for 30 min at 370 followed by a four-fold dilution with M150. (b) Casein. Denatured casein (Casein nach Hammerstein-Merck) at a concentration of 5 mg/ml, prepared by alkali treatment with subsequent restoration to pH 7-2. Both the above chemoattractants have consistently been shown to attract substantial numbers of PMN through 5 pm pore size filters in Boyden chambers, the casein being the weaker chemotactic agent, attracting 50-60% as many PMN as the EAS.

Assay of glycolytic activity The extent of glycolysis was measured by lactate production using recognized procedures (Hohorst, 1962), (Boehringer Mannheim, Biochemical Test

Combination). Pure neutrophil suspensions were resuspended to a final concentration of 2 x 107/ml in

0-15 M phosphate buffered saline (PBS) containing 10 mM glucose. Each assay tube (performed in triplicate) utilized 6 x 106 PMN (0 3 ml) in a final volume of 0-5 ml. The remaining 0-2 ml contained the various agents (leucoattractants or drugs) under investigation. Tubes were incubated at 370 for varying time intervals and the reaction was terminated and the system deproteinized by the addition of 10 ml of cold 0-6 N perchloric acid. Tubes were mixed well, centrifuged at 2000g for 10 min and aliquots (0-2 ml) from each tube were assayed for lactic dehydrogenase and NAD at 25° for 60 min and the change in absorbance at 340 nm (AE340 nm) was assessed spectrophotometrically. Results were expressed as pg lactate/6 x 10 PMN/ unit time. The variability between identical samples was 3-1 %. Experiments were performed at 1, 3, 5, 10, 20, 40 and 60 min to investigate the effects on glycolysis of casein (at a final concentration of 100 pg/ml), serum (5 %) and EAS (5 %). The control system contained PMN only. The effects of d'cAMP, histamine, levamisole and propranolol on EASstimulated glycolysis were also investigated at the same time intervals. In a further series of experiments the effects of the leucoattractants and drugs at fixed time intervals (60 min) were assessed. To control for the effects of macromolecular materials in the EAS preparations which could produce false positive results, by inducing phagocytosis, the effects of endotoxin alone and endotoxin-treated heatinactivated serum (56° for 40 min) were studied. Drug effects on glycolysis were investigated in the presence and absence of 5 % EAS.

Assay of hexose monophosphate shunt (HMS) activity The extent of HMPS activity was measured according to the method of Wood, Katz & Landau (1963), by potassium hydroxide (KOH) absorption of 14CO2 produced from glucose radiolabelled in the Cl position. (Obtained from New England Nuclear, Boston, Massachusetts as D-Glucose 1-'4C.) Pure neutrophil suspensions (2 x 107) were resuspended in glucose free 0-15 M PBS. Each assay was performed in duplicate and utilized 4 x 106 PMN in 0-2 ml, and 0-6 ml of radiolabelled glucose containing 0 06 pCi. The remaining 0-2 ml PBS, contained the various agents (leucoattractants and drugs) under investigation. The test apparatus was a 10 ml glass scintillation vial (Packard) which served as the outer chamber


Chemotatic agents which influence anaerobic glycolysis stoppered with a tightly fitting perforated McCartney top. Placed inside the outer chamber was a 2 ml autoanalyser cup which served as the inner chamber. The radiolabelled glucose was placed in the outer chamber and 0-06 ml of 1 N KOH in the inner chamber. The outer chamber was stoppered and the apparatus allowed to stand in a 370 water bath for a few minutes. The reaction was initiated by the introduction of the cell suspension to the outer chamber by injection through the cap with a long-needled syringe, and was terminated after varying time intervals by the addition of 2 ml of 2 N HCI. The chambers were allowed to stand for 60 min to permit release of 14C02 and absorption by the KOH, 0 5 ml of which was then transferred to scintillation vials containing 10 ml of acid instagel (Packard), (55 ml of 17 N HCI/litre instagel) and activity assessed on a Tri-Carb liquid scintillator for 5 min. Results were expressed as corrected mean counts per minute (c.p.m.). Background controls without neutrophils contained radiolabelled glucose and appropriate additives, and counts obtained were subtracted from the corresponding experimental values. The variability between identical samples for this technique was 71 %. In an initial series of experiments the effects of chemotactic stimulation by casein and EAS at final concentrations of 100 ug/ml and 10% respectively (concentrations which stimulate chemotaxis maximally) and phagocytosis (by the ingestion of Candida albicans) were assessed at varying time intervals (1, 3, 5, 10, 20, 40 and 60 min). In doseresponse experiments a fixed time interval (60 min) was selected and the effects of d'cAMP (10-5 M10-2 M), histamine (10-6 M_10-3 M), levamisole (10-6 M_10-3 M) and propranolol (10-6 M_10-3 M) on EAS stimulated neutrophils were investigated. In further kinetic experiments, the effects of the same drugs and also ascorbic acid were assessed at varying time intervals at fixed drug concentrations (d'cAMP 10-3 M, histamine 2-5 x 10-5 M, levamisole 10-3 M, propranolol 104 M). The effects of the leucoattractants, cyclic nucleotides and various drugs were also tested at fixed time intervals (60 min) and at fixed concentrations which have previously shown to produce maximal effects on PMN motility. Endotoxin- and heat-inactivated serum controls were also included in these experiments. Cyclic nucleotide and drug effects were evaluated in the presence of 10% EAS. A similar series of experiments was performed to assess the effects of the same agents on K

neutrophil phagocytosis. Phagocytosis was initiated by the addition of 0-1 ml Candida albicans containing 8 x 106 organisms/ml to give a final cell: particle ratio of 1: 2. For opsonization 0X1 ml of autologous serum was added to the reaction mixture. Using this system 90%-100% ingestion is evident at 20 min.

Semi-quantitative nitroblue tetrazolium test (NBT) This test was performed according to the method of Sher, Anderson, Rabson & Koornhof (1974). Pure neutrophils were mixed with equal volumes of NBT solution (1 mg/ml) and the appropriate additives. Tubes were incubated at 37° for 30 min and smears of the mixtures were made on glass slides after incubation. These were fixed in methanol and stained for 5 min in dilute 10% haematoxylin. Slides were evaluated microscopically and results expressed as percentage of NBT (reduced)-positive PMN. RESULTS Glycolysis The effects of EAS, serum and casein on the kinetics of lactate production are shown in Fig. 1. As can be seen, EAS caused a marked stimulation of glycolysis


, 20 Ci




30 Time (min)




Figure 1. The effects of PBS (A), casein (0), serum (x ) and EAS (0) on the kinetics of neutrophil glycolysis.

at all time intervals whereas no such stimulation was mediated by serum alone or casein. Dibutyryl cAMP, histamine, levamisole and propranolol were without effect on glycolysis at all time intervals tested (results not shown). The results of experiments investigating the effects of the leucoattractants, and the effects of the various drugs on EAS stimulation are shown in Table 1. Only EAS stimulated glyco-


R. Anderson, Annemarie Glover & A. R. Rabson

Table 1. Effects of casein, EAS and controls (a) and EAS, in the presence of various drugs on neutrophil glycolysis (b)

Final concentrations of drugs and leucoattractants added to 6 x 10-6 PMN

Mean lactate production pg/6 x 106 PMN/60 min with standard deviation

(a) PBS only Casein (100 pg/ml) Serum(6%) Endotoxin alone Inactivated EAS EAS (6 %) (b) EAS + 10-3 M d'cAMP EAS+10-3McAMP EAS + 10-3 M cGMP EAS + 10-3 M Isoproterenol EAS +2-5 x 10-5 M Histamine EAS + 10pg PGE, EAS+10 gPGA1 EAS + 10-3 M Levamisole EAS + 10-4 M Propranolol EAS + 10-3 M Acetylcholine EAS + 10-3 M Carbamylcholine

9-8 ±1-2 10-2 ±0-98 12-2i±-3 11 9 ± 04 17-4 ± 3-1 39-3 ±4-1 38-6 +33 39-1 3-6 40-2 ±40 40-1 ± 3-2 38-7 ±3-6 41-2 ± 3-5 400±4-1 39-6 ± 3-7 38-3 ± 4-0 39-6 ± 3-8 40-1 ± 3-6


~c0 10 0

6 6

2O 0~5 4 a:


3 2




40 30 Time (min)



Figure 2. The effects of chemotactic and phagocytic stimulation on neutrophil HMPS activity. Resting (0), casein (O) and EAS (O) effects are shown in the lower graph and the effect of phagocytosis of C. albicans in the upper graph.

lysis. Endotoxin alone produced an insignificant increase in lactate production and serum alone and endotoxin-treated heat-inactivated serum gave slight stimulation. Cyclic nucleotides and the other drugs tested had no effect on the EAS stimulation of glycolysis. HMPS The effects of phagocytic and chemoattractant stimulation on HMPS activity are shown in Fig. 2. Phagocytosis caused a considerable increase in HMPS activity whereas the effects of chemotactic stimulation varied with the leucoattractant. EAS promoted a delayed stimulation which was only evident at 60 min whereas casein mediated a considerable inhibition of the HMPS which was present at all time intervals tested. Results of experiments utilizing serum were corrected for serum inhibition of HMPS activity presumably due to a competitive effect of serum glucose. The results of six separate (mean and standard error) dose-response experiments for d'cAMP and histamine are shown in Fig. 3 and for levamisole and propranolol in Fig. 4. As can be seen all four agents mediated a progressive dose dependent inhibition of HMPS activity. Fig. 5 indicates the kinetics of EAS associated HMPS activity in the presence and absence of the same four drugs. Inhibition is evident throughout, but is greatest at 60 min.



E C-

0 0 a a 0



i0-4 i0o ' o-2 Molar concentration Figure 3. The effects of varying dibutyryl cAMP and histao io-5

mine concentration on neutrophil HMPS activity. (a) Histamine; (b), Dibutyryl cAMP.

Chemotatic agents which influence anaerobic glycolysis 3

The effects of ascorbate (5 x 10- M) on HMPS activity in the absence of EAS or phagocytosis are shown in Fig. 6. Ascorbate promoted considerable stimulation of HMPS activity which is evident at all time intervals. Table 2 shows the effects of various drugs on EAS stimulated HMPS activity. Those drugs which elevate intracellular cAMP depressed HMPS activity, and levamisole and propranolol had similar effects. Acetylcholine, cGMP and carbamyl choline however, had no effect, and ascorbate enhanced HMPS activity in the presence of EAS. The effects on HMPS activity associated with the phagocytosis of Candida albicans, of the leucoattractrants casein and EAS as well as the various agents which were previously shown to depress HMPS activity are shown in Table 3. Casein and EAS had no effect on neutrophil HMPS activity associated with phagocytosis whereas all other agents tested caused inhibition.

(a )

2 _


E x


CL d


3 _

0 .0



0 10-6





Molar concentration

Figure 4. The effects of varying concentrations of levamisole and propranolol on neutrophil HMPS activity.

NBT reduction As can be seen in Table 4 casein had no statistically significant effect on NBT reduction whereas EAS mediated significant stimulation.

Table 2. Effects of various drugs which promote elevation of intracellular cyclic nucleotide levels on EAS stimulated HMPS activity

Drug cAMP d'cAMP Histamine Isoproterenol

Prostaglandin Al Prostaglandin E, cGMP Acetylcholine

Carbamylcholine Levamisole

Propranolol Ascorbic Acid

Concentration Ix 10-3 M 1 x 10-3 M 2-5 x 10-5 M 1 x 10-4 M 10 pg/mI

lOpg/ml 1 x

10-3 M 1 x 10-3 M Ix 10-3 M Ix 10-3 M Ix 10-4 M 5 x 10-3 M

Control mean and standard deviation

Experimental mean

2913 ±708 2032 ±449 2913 ±708 2913 ± 708 2913 ± 708 2913 ±708 2913 ±708 2913 ±708 2913 ±708 2608 ±626 3123 ±395 2203 ±442

2968 ±589 1073 ± 135 1929 ±515 2079 ±508 1462 ± 612 1642 ±416 2779 ±769 2727 ±506 2890 ±750 1367 ±425 1380 ±675 3312 ±500

and standard deviation

% Inhibition of control 7-3 47 34 29 50 44 4-6 6-3 -

48 56 +50


P value


The effect of chemotactic factors and agents which influence neutrophil movement on anaerobic glycolysis and hexose monophosphate shunt activity.

Immunology 1978 35 141 The effect of chemotactic factors and agents which influence neutrophil movement on anaerobic glycolysis and hexose monophosph...
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