Proc. NatI. Acad. Sci. USA Vol. 76, No. 11, pp. 5896-5900, November 1979

Medical Sciences

Enhancement of hexose uptake in human polymorphonuclear leukocytes by activated complement component C5a (hexose transport/leukocyte metabolism)

CHARLES E. MCCALL, DAVID A. BASS, SUE COUSART, AND LAWRENCE R. DECHATELET Departments of Medicine and Biochemistry, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103

Communicated by Paul B. Beeson, July 25,1979

The polymorphonuclear leukocyte (PMNL) ABSTRACT depends on glucose as a source of energy for motility, chemotaxis, phagocytosis, and bactericidal activity. Activated complement (CMa) at low concentrations stimulates carrier-mediated carbohydrate transport in PMNLs as measured by the uptake of 2-deoxy-D[3H]glucose. Human PMNLs were preincubated at 370C for 15 min with zymosan-activated human serum or various purified preparations of human CMa. A concentrationdependent increase in deoxyglucose transport (>700% of control) into PMNLs occurred with all test substances. Reaction was linear for 30 min, and uptake of deoxyglucose followed saturation kinetics. C5a caused a decrease in the Km for deoxyglucose, from 0.53 to 0.11 mM, without altering the Vmax (44 nmol/30 min per 5 X 106 PMNLs in control and 46.6 with C5a). The optimal concentration of C5a for enhanced carrier-mediated transport of deoxyglucose was similar to that which promoted optimal chemotaxis. Activated serum from C5-deficient mice had little or no effect on deoxyglucose transport whereas that from normal syngeneic mice enhanced deoxyglucose transport. C5a did not enhance deoxyglucose transport into isolated erythrocytes, platelets, or lymphocytes. The deoxyglucose within the cell was primarily in the phosphorylated form, and hexokinase activity was not increased in PMNLs stimulated with C5a, indicating that hexokinase was not rate limiting and that enhanced transport was the mechanism of the C5a activity. Insulin at physiologic concentration (10 ng/ml) had no effect on deoxyglucose transport in PMNL and did not act as a competitive inhibitor of C~a. This insulin-like bioactivity could be detected with the amount of C5a that would be present after activation of 0.1-0.5% of the C5 in 1 ml of serum. This suggests that uptake of [3Hjdeoxyglucose by PMNLs might serve as a highly sensitive test for activation of the fifth component of complement.

C5a-induced lysosomal enzyme release by PMNL has been demonstrated when PMNL are attached to surfaces or when they are incubated with cytochalasin B (4, 5). C5a also stimulates the respiratory burst in PMNL (6). Abrupt neutropenia follows complement activation in vivo or infusion in vitro of zymosan-activated serum (ZAS) (7). This effect is probably mediated by C5a (8, 9). This neutropenic activity of C5a occurs in humans and closely correlates with the ability of C5a to increase PMNL adhesiveness and PMNL aggregation in vitro (10). Chenoweth and Hugh (11) recently demonstrated that PMNL-related biologic activities of both C5a and Des-Arg-C5a are expressed only after interaction of the glycoprotein with a specific receptor on the surface of human PMNL (11). Remarkably similar concentrations were required for binding of the molecule and for the biologic activities of chemotaxis and lysosomal enzyme secretion. C5a may interact in inflammation with PMNLs by sequestering PMNLs in the microvasculature (at the site of inflammation), attracting PMNLs into tissue, inducing secretion of lysosomal enzymes, and activating oxidative metabolism in PMNLs which is required for their optimal microbicidal activity (12). We report herein another biologic activity of C5a, that of enhanced hexose transport into PMNLs. Because PMNLs depend on glucose for their sole source of energy for motility, phagocytosis, and optimal microbicidal activity, C5a may aid in the preparation of PMNLs for their physiologic role in host defense.

Human C5a, a glycoprotein with a molecular weight of approximately 11,000, is formed during activation of complement by either the classic or the alternative pathway (reviewed in ref. 1). A number of diverse biological activities associated with the C5a molecule have been defined. Among the systemic effects of intact C5a are smooth muscle contraction, vasoconstriction of arterioles, increased vascular permeability, and anaphylactic shock in guinea pigs. Thus, the intact molecules have the spasmogenic properties of an anaphylatoxin. In human serum, this spasmogenic activity is rapidly ablated by a carboxypeptidase B-like enzyme which selectively removes the COOHterminal arginine, thus forming the physiologic end product in man, Des-Arg-C5a (2). Of paramount importance is that this derivative maintains biological activities which probably play a central role in inflammation in humans. The effects of C5a and Des-Arg-C5a on polymorphonuclear leukocytes (PMNL) are perhaps the most extensively studied bioactivities of these molecules. C5a and Des-Arg-C5a are chemotactic for PMNL, with the former being approximately 10 times more active than the latter (2, 3). C5a- or Des-Arg-

METHODS Pooled human serum from control donors was stored at -700 C in small portions prior to use. Preparations of C5a. ZAS was obtained by incubation of stored serum with zymosan (25 mg/ml) for 30 min at 37°C. The zymosan then was removed by centrifugation. This preparation was not treated with E-aminocaproic acid to inhibit carboxypeptidase B activity; accordingly, the activated C5 present would be predominantly or solely Des-Arg-C5a (2). A partially purified preparation of human C5a was obtained through the courtesy of Stephen Kunkel and Peter Ward (University of Connecticut Medical School). This preparation was prepared from ZAS and isolated by Sephadex G-75 chromatography. It was stored in acetate buffer (pH 1-3) and diluted 1:4 in Dulbecco's phosphate-buffered saline (Pi/NaCI) with an adjustment to pH 7.4 prior to use. A more highly purified preparation of C5a was obtained through the courtesy of Henry Showell and Elmer R. Becker (University of Connecticut Medical School). It contained five

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviations: PMNL, polymorphonuclear leukocyte; ZAS, zymosan-activated human serum; Pi/NaCl, Dulbecco's phosphate-buffered saline. 5896

Proc. Natl. Acad. Sci. USA 76 (1979)

Medical Sciences: McCall et al. bands on polyacrylamide gel electrophoresis and a protein concentration of 400 ,g/ml. It was stored at -70'C prio toui.s.. and was diluted 1:100 to 1:1000 in Pi/NaCl before 'each assay. Blood Cell Isolation. PMNLs. Heparinized (10 units/ml) venous blood was sedimented for 30 min at room temperature in 40-ml test tubes containing Plasmagel (HTI, Buffalo, NY) at a ratio of 3-4 ml of blood per ml of Plasmagel. The leukocyte-rich plasma was centrifuged at 100 X g for 10 min, washed with Pi/NaCI (pH 7.4) (GIBCO), and centrifuged again at 100 X g. Erythrocytes were lysed by addition of 6 ml of distilled H20 for 20 sec, and isotonicity was established by addition of 2 ml of 3.5% NaCI. Cells were suspended in Pi/NaCl and counted in a hemocytometer. Cell suspensions then adjusted to 5 X 106 PMNL per ml. Differential counts indicated 85-95% PMNLs. Lymphocytes. Heparinized blood was allowed to sediment as for PMNLs. The leukocyte-rich plasma was then centrifuged at 400 X g. The cells were then diluted in Pi/NaCl and layered over Isolymph (Gallard Schlesinger, Carle Place, NY) at a ratio of 4 ml of cell suspension to 3 ml of Isolymph. This was then centrifuged at 400 X g for 30 min at 20'C. The plasma and interface layers were removed and centrifuged at 400 X g. Cells were washed with Pi/NaCl and again centrifuged at 400 X g. Cells were then suspended in Pi/NaCl and counted in a hemocytometer. The suspension was adjusted to 5 X 106 lymphocytes per ml. Differential counts indicated 90-95% lymphocytes, with the contamination consisting of both PMNLs and monocytes. Platelets. Heparinized blood was centrifuged at 400 X g for 10 min. The upper two-thirds of platelet-rich plasma was removed and centrifuged at 1200 X g at room temperature for 10 min. Platelets were then washed with P1/NaCl, centrifuged again, suspended in Pi/NaCl, and counted in a Coulter Counter. The suspension was adjusted to 5 X 106 platelets per ml. Differential counts indicated 99% platelets. Erythrocytes. Heparinized blood was centrifuged for 10 min at room temperature at 100 X g. The buffy coat was removed and the erythrocyte pellet was then resuspended to twice the original volume in 0.9% NaCI and centrifuged at 400 X g for 20 min. Erythrocytes were then washed once in Pi/NaCl, centrifuged, resuspended in Pi/NaCl, and counted in a Coulter Counter. The suspension was adjusted to 5 X 106 erythrocytes per ml. Differential counts indicated 99% erythrocytes. [3HJDeoxyglucose Transport. 2-Deoxy-D-[3H]glucose [hereafter referred to as [3H]deoxyglucose; specific activity, 50 mCi/mmol (1 Ci = 3.7 X 101s becquerels); New England Nuclear] was diluted in H20 to a concentration of 10 ,Ci/ml and stored frozen at -20°C. Triplicate samples containing 5 X 106 PMNLs and specific substances to be studied were preincubated in a final volume of 3.0 ml for 15 min at 37°C in a shaking water bath. The reaction was initiated by the addition of 1 uCi of [3H]deoxyglucose and stopped after 30 min by immersion of the tube in an ice-water bath. The samples were centrifuged at 100 X g for 10 min at 4°C, and the pellets were then washed once with 5 ml of cold Pi/NaCl and recentrifuged. The pellets were resuspended to 3.0 ml with deionized H20 and sonicated for 2 min in a bath sonicator. A 1-ml aliquot was added to 10 ml of Aquasol, and the radioactivity was measured in a Beckman liquid scintillation counter. Values were expressed as cpm/5 X 106 PMNLs. Phosphorylation of Deoxyglucose. PMNLs (2 X 107) were preincubated with or without C5a (200,l of a 1:4 dilution of the partially purified C5a) at 370C in a shaking water bath for 15 min. The reaction was started by the addition of 10 gCi of [3H]deoxyglucose and incubated for 30 min in a total volume

5897

14 J

z

Fig. 1. Effect of ZAS on uptake of [3H]deoxyglucose in human PMNLs. Varying amounts of ZAS were used and uptake was allowed to proceed for 30 min. The curve represents 1 of 10 separate experiments; each point represents the mean of triplicate assays.

x I',

x

E a

adjusted to 3.0 ml with Pi/NaCI; the reaction was stopped by chilling in ice. The samples were centrifuged at 100 X g for 10 min at 40C. Thepellets were then washed with 5 ml of P1/NaCI and recentrifuged. The final pellets were resuspended in 2.0 ml of H20 and sonicated in a bath sonicator. Samples were boiled for 5 min in a water bath (with a marble on top of each tube to prevent evaporation). Then denatured protein was removed by centrifugation at 27,000 X g for 10 min and the entire supernatant was applied to a small column of Dowex-1-Chresin. The column was washed with a total of 40 ml of deionized water to elute free glucose and fractions (2 ml each) were collected. The column was then washed with 40 ml of 0.05 M HCI to remove glucose phosphate and again 2.0-ml fractions were collected. Each 2.0-ml fraction was added to 15 ml of Aquasol and the radioactivity was measured in a liquid scintillation spectrometer. A similar separation was performed using a parallel sample incubated in the absence of C5a. [1-14C]Glucose and [1-'4C]glucose 6-phosphate standards were separated on the same column to determine the elution profiles of the pure compounds. Hexokinase Assay. PMNLs were incubated in Pi/NaCl with ZAS or C5a for 1 hr at 370C, disrupted by sonication, and centrifuged for 15 min at 27,000 X g at 4VC. Hexokinase activities in the supernates were determined by spectrophotometric assay. Each cuvette contained 1.2 ml of 0.05 M triethanolamine.HCI at pH 7.5, 1.2 ml of glucose (100 mg/ml), 0.20 ml of 0.1 M MgCI2, 0.20 ml of NADP (10 mg/ml), 0.10 ml of ATP (10 mg/ml), and 1.25 units of purified glucose-6phosphate dehydrogenase. The reaction was initiated by the addition of sonicates, and the increase in absorbance at 340 nm was measured on a Beckman DU spectrophotometer with a Gilford recorder. Each measurement was performed at three levels of protein to ensure linearity and results were expressed as nmol of NADPH formed per min per 5 X 106 PMNLs. Chemotaxis. Chemotaxis was assayed by the migrationunder-agarose technique (13). 81g0-J

Po -

106 0

x

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0

FIG. 2. Effect of C5a on transport in human PMNLs. Varying amounts of a partially purified preparation of C5a were used and incubations were carried out for 30 min. The curve represents one of three closely agreeing experiments; each point represents the mean of triplicate assays. The 500 values are corrected for uptake in the absence of C5a.

lrt '_

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Medical Sciences: McCall et al.

5898

/

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FIG. 3. Effect of highly purified C5a on [3H]deoxyglucose transport into human PMNLs. Varying amounts of C5a were used and incubations were carried out for 30 min. The curve represents one of two experiments with similar results; each point represents the mean of triplicate assays. Differences in transport between this preparation and that used in Fig. 2 are due to differences in dilution of the stock solution. The values are corrected for uptake in absence

Proc. Natl. Acad. Sci. USA 76 (1979) J 60 z E 50 o X LOl

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0

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10 15 20 25 30 Time, min

FIG. 5. Time course of the uptake of [3Hldeoxyglucose in the presence of 10pgl of crude C5a. The curve represents one of three closely agreeing experiments; each point represents the mean of triplicate assays.

of C5a.

Statistical Analysis. When indicated, the Student t test was used for statistical analysis. Double reciprocal plots for Km and Vmax were obtained by the method of least squares.

RESULTS C5a-Carrier-Mediated Transport of [3Hpleoxyglucose. The effects of varying concentrations of ZAS on uptake of [3H]deoxyglucose are shown in Fig. 1. The dose-response curve reached a maximum between 10 and 50 Ail of ZAS in a reaction volume of 3 ml containing 5 X 106 PMNLs. Concentrations above 100 ,l did not further enhance transport, and 1 ml of ZAS suppressed it. This suppression could be accounted for by the competitive inhibition by glucose in the serum, which could be removed by dialysis (data not shown). Based on the quantity of C5 in human serum and the amount of Des-Arg-C5a that might be produced by maximal activation (0.4 ,uM) (2), between 0.4 and 2 pM Des-Arg-C5a enhanced deoxyglucose transport. The effect of the partially purified preparation of C5a (crude C5a) is depicted in Fig. 2. The dose-response curve reached a maximum at 50 1l. The actual quantity of C5a in this preparation could not be determined, but the data further substantiate that the bioactivity is saturable. To determine further the specificity of the reaction for C5a, the more highly purified preparation of C5a was used; the results are shown in Fig. 3. A dose-response curve was again obtained. Because this purified material was available in limited quantity, the remainder of the experiments were performed with the crude preparation of C5a or ZAS (putative Des-ArgC5a) unless otherwise specified. The effect of this preparation of C5a on uptake of [3H]deoxyglucose appeared to be less than that observed with the crude C5a; this was because the purified C5a preparation was diluted more, prior to addition to the reaction tubes. To establish C5-specificity for enhanced deoxyglucose transport in PMNLs, the effect of ZAS from normal D1OB2/

NSN mice and C5-deficient D1OB2/OSN mice (Jackson Laboratory) on [3H]deoxyglucose transport in PMNLs was determined. A concentration of ZAS (0S l) that was supraoptimal for uptake of deoxyglucose was used; the uptake followed for 60 min (Fig. 4). ZAS from the C5-deficient mice had no effect on deoxyglucose uptake in human PMNL at 30 min and a slight effect at 60 min, whereas ZAS from normal mice significantly enhanced uptake of deoxyglucose. The C5-deficient ZAS'was also not chemotactic (data not shown). A time course of [3H]deoxyglucose transport with PMNLs plus crude C5a (10 Al) is illustrated in Fig. 5. The uptake was essentially linear for 30 min. Because deoxyglucose is irreversibly phosphorylated after it enters the cell, the quantities of phosphorylated and nonphosphorylated deoxyglucose in the PMNL were determined. There was a significant increase in phosphorylated deoxyglucose in PMNLs treated with C5a (Fig. 6). Little or no change in free deoxyglucose was observed. The possibility that C5a might activate hexokinase, which is required for phosphorylation of deoxyglucose and therefore might account for increased phosphorylation, was examined. There were no differences between hexokinase levels in control PMNLs and PMNLs treated with either C5a or fMLP in three experiments (each assay performed in triplicate; data not shown). In order to determine the alteration that might be responsible for the enhanced transport of deoxyglucose, double reciprocal plots of velocity and substrate concentration were constructed as described by Lineweaver and Burk (14). In this experiment, unlabeled as well as radiolabeled deoxyglucose was added in varying concentrations so that the same specific activity was maintained in all tubes. The apparent Km for deoxyglucose in PMNLs alone was 0.53 mM; it fell to 0.11 mM in the presence of an optimal quantity of C5a (Fig. 7). In contrast, Vx was not significantly altered by C5a: 46.6 nmol/30 min per 5 X 106 PMNLs with C5a and 44 without. Deoxy-

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into human PMNLs. The curve represents one of two experiments; each point represents the mean of triplicate assays. The values are corrected for uptake in the absence of ZAS.

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FIG. 6. Phosphorylation of [3H]deoxyglucose in human PMNLs. The curves show the nonphosphorylated and phosphorylated deoxyglucose in human PMNLs with (- *) and without (- - --0) C5a. Reactions were carried out for 60 min.

Medical Sciences: McCall et al. -C5a

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FIG. 7. Double-reciprocal plots of deoxyglucose transport into human PMNLs. The slopes were obtained by the method of least squares; for each line, r > 0.8.

Comparative Effects of Deoxyglucose Transport and Chemotaxis. Because other bioactivities of C5a, such as secretion of lysosomal enzymes and chemotaxis closely correlate with the uptake of C5a by its specific receptor, we compared the effects of enhanced deoxyglucose transport with chemotaxis, by using the migration-under-agarose technique (13). With lower concentrations of C5a, there was a distinct parallel between the chemotactic response and enhanced transport of deoxyglucose (Fig. 8). In contrast, supraoptimal concentrations of C5a inhibited chemotaxis whereas the enhanced transport of deoxyglucose reached a plateau. This effect of C5a on chemotaxis has been reported and is referred to as deactivation or desensitization (15). The kinetics of glucose transport did not conform to the definition of desensitization or deactivation. Effect of Insulin on Deoxyglucose Transport. Concentrations of insulin between 1 fg/ml and 1 ,gg/ml were used to study the effect of insulin on deoxyglucose transport into PMNLs. There was no effect on deoxyglucose transport at 30 min by physiologic or pharmacologic concentrations of insulin under conditions such that crude C5a showed a 400% stimulation (Table 1). Moreover, when PMNL were preincubated for 5 min with insulin at 1 ttg/ml prior to addition of C5a, there was no competitive inhibition of enhanced deoxyglucose transport by C5a (data not shown). Cell Specificity. Because the preparations of PMNL were not pure and contained some contamination with erythrocytes, platelets, eosinophils, monocytes, and lymphocytes, the specificity of deoxyglucose transport into various isolated blood cells was studied. ZAS at optimal concentrations for PMNL did not enhance deoxyglucose transport in erythrocytes or platelets (Table 2). There was a modest increment in deoxyglucose transport in the lymphocyte preparations which might have been due to contamination with 5-10% PMNL and monocytes. We have preliminary data indicating that C5a also stimulates deoxyglucose transport into other phagocytes (unpublished data). Insulin in a supraphysiologic concentration (1 ktg/ml) had

None 17,108 1 fg/ml 17,200 1 pg/ml 17,700 1 ng/ml 16,100 1 1ugIml 21,200 None + C5a 70,200 * Shown as cpm/5 X 106 PMNLs after 30-min incubation. Data represent mean of triplicate samples in one of two experiments, each with similar results. no effect upon the uptake of deoxyglucose in any of the blood cells.

DISCUSSION C5a has insulin-like activity for PMNLs as defined by an increase in the transport of radiolabeled hexose, deoxyglucose. Deoxyglucose is used as a measure for glucose transport because it enters phagocytes at the same site as glucose by a specific, saturable mechanism with characteristics compatible with carrier-facilitiated transport (16). The insulin-like activity in ZAS is due to C5a derivatives; this was demonstrated by using C5a preparations of different purities and by demonstrating that ZAS from C5-deficient mice, in contrast to ZAS from

normal mice, had no effect on deoxyglucose transport. An increased affinity for the deoxyglucose transport receptor is suggested because the Km decreased without a significant change in Vmax. This differs from. the effect of insulin on adipocytes: enhanced glucose transport by insulin is associated with an increase in V.. with no appreciable change in Km (16). The concentration-dependent increase in deoxyglucose uptake induced in PMNLs by C5a, however, is similar to the effect of insulin on isolated adipocytes in that the transport is saturable, suggesting a receptor-ligand interaction at the cell membrane (17). No activation of hexokinase by C5a occurred for up to 60 min, and most of the, transported deoxyglucose was recovered in the phosphorylated form, indicating that hexokinase was not rate limiting. C5a had no effect on deoxyglucose transport into erythrocytes and platelets and little effect in lymphocyte preparations. This is compatible with the observation that specific C5a receptors have been described on intact human PMNLs but not on erythrocytes or lymphocytes (11). Thus, there appears to be cell specificity that is either confined to PMNL or to PMNL and other contaminating phagocytes such as eosinophils or monocytes. Preliminary data suggest that C5a affects eosinophils in a similar fashion (unpublished observations). The concentration of C5a producing 50% saturation of binding sites correlates with the concentration required for 50%

[ 3H ]Deoxyglucose

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Table 2. Cell specificity of transport of [3H]deoxyglucose*

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Table 1. Effect of insulin on transport of [3H]deoxyglucose in PMNLs Insulin added Transport*

8r 7

Proc. Natl. Acad. Sci. USA 76 (1979)

)(~ ~ \ Chemotaxis

I' Is_' 20 25I_ "50 _1 00 1i_5 10I_ C5a, glI FIG. 8. Comparative effects of C5a on [3H]deoxyglucose transport and chemotaxis in human PMNLs. Deoxyglucose transport was terminated at 30 min, whereas chemotaxis was measured after 3 hr. 5

Transportt Erythrocytes Platelets Lymphocytes

Cells 20,600 4,800 1,700 Cells +C5a 86,600 3,500 1,900 Cells + insulin (1 jug/ml) 20,500 2,300 1,400 * Representative of one experiment for each cell type, performed in triplicate. t cpm/5 X 106 PMNL after 30-min incubation.

5,600 12,400 7,300 each assay

5900

Medical Sciences: McCall et al.

maximal response in stimulation of chemotaxis or secretion of lysosomal enzymes (11). These reported values were: 3-7 nM for C5a binding, 2 nM for chemotaxis, and 3 nM for enzyme release. From our data, assuming the total activation of CS in human serum by zymosan to Des-Arg-C5a we estimate the concentration of Des-Arg-C5a for 50% maximal response in the stimulation of deoxyglucose transport to be 5-10 nM. We also demonstrated a close correlation between stimulation of chemotaxis and deoxyglucose transport by putative Des-Arg-C5a. The combined data suggest that stimulation of deoxyglucose transport, like that of chemotaxis and lysosomal enzyme secretion, will correlate with binding of specific quantities of C5a to its membrane receptor. Insulin had no effect upon deoxyglucose transport within a wide range of concentrations. This corroborates other reports that insulin does not enhance glucose transport in PMNLs, despite the fact that PMNLs have a receptor specific for insulin (18, 19). Because both PMNLs and erythrocytes have receptors for insulin, it appears that these receptors cannot activate the carrier-mediated transport mechanism for glucose into the cell (18, 20). It is tantalizing to speculate that a complement-derived product such as C5a might have developed phylogenetically as an "insulin" for phagocytes. Although PMNLs depend on glucose for energy for locomotion, phagocytosis, and microbicidal activity, the physiologic significance of the enhancement of deoxyglucose transport by C5a cannot be determined from this investigation. Other substances with insulin-like activity could be present in the serum or tissues, or PMNLs might accumulate enough glucose for energy requirements through passive diffusion. PMNLs contain glycogen stores (21); when studied in vitro, PMNLs can respond to chemotactic stimuli and phagocytize in the absence of exogenous glucose (19, 22). However, whether the glycogen stores are adequate to provide the energy required for sequential margination, diapedesis, chemotaxis, phagocytosis, and bacterial killing in vivo is unknown. Monocytes, for example, lose their ability to kill certain tumor cells by oxidative mechanisms after 2 hr of incubation in glucose-free medium (23). It is possible that CSa equips PMNLs with glucose prior to requirement for chemotaxis, phagocytosis, or killing. The transported glucose could be stored as glycogen to be utilized later as needed. We have observed that deoxyglucose transport is increased in PMNLs isolated from patients with acute bacterial infections and polycythemia vera (24). We do not know whether this enhanced transport may follow attachment of C5a to the surface of the PMNL or whether there is an increase in transport sites for glucose in these "activated" PMNLs. Because intravascular complement activation occurs during acute bacterial infection (25), it is possible that the enhanced uptake of deoxyglucose reflects attachment of CSa to PMNLs during infection. This would be compatible with the hypothesis that, by enhancing the energy reservoirs of phagocytes, the insulin-like activity of C5a may help to prepare phagocytes for their defensive roles in vivo. To date, assays for C5a activity have been rather cumbersome and difficult to quantitate when based on chemotactic responses of PMNLs or the ability of CSa to aggregate PMNLs (11, 26). The radiolabel assay used in this study is highly sensi-

Proc. Natl. Acad. Sci. USA 76 (1979)

tive, detecting the amount of Des-Arg-C5a that would be present after activation of 0.1-0.5% of the CS in 1 ml of serum (estimated to be 0.4-2 pM Des-Arg-C5a). This suggests that the uptake of [3H]deoxyglucose by PMNLs might serve as a highly sensitive and reproducible assay for detecting cleavage of the fifth component of complement to its CSa derivative. We are indebted to a number of individuals at the University of Connecticut Medical School who collaborated in this project. They include Drs. Joseph O'Flaherty, Peter Ward, Elmer Becker, Henry Showell, and Stephen Kunkel. We also gratefully acknowledge the hard work of Ms. Janet Terry and Ms. June Heflin in the preparation of this manuscript. This research was supported by U.S. Public Health Service Grants A109169, HL16769, and A110732 and a grant from the National Foundation-March of Dimes. 1. Muller-Eberhard, H. J. (1978) Hosp. Pract. 13,65-76. 2. Fernandez, H. N., Henson, P. M., Otani, A. & Hugh, T. E. (1978) J. Immunol 120,109-115. 3. Snyderman, R., Phillips, J. K. & Mergenhagen, S. E. (1971) J. Exp. Med. 134, 1133-1139. 4. Henson, P. M. (1971) J. Immunol. 107,1547-1603. 5. Goldstein, I. M., Brai, M., Osler, A. G. & Weissmann, G. (1973) J. Immunol. 111, 33-39. 6. Goetzl, E. G. & Austen, K. F. (1974) J. Clin. Invest. 53, 591596. 7. McCall, C. E., DeChatelet, L. R., Brown, D. & Lachmann, P. (1974) Nature (London) 249,841-844. 8. O'Flaherty, J. R., Craddock, P. R. & Jacob, H. S. (1978) Blood

51,731-739. 9. Craddock, P. R., Fehr, J., Dalmasso, A. P., Brigham, K. L. & Jacob, H. S. (1977) J. Clin. Invest. 59, 879-888. 10. O'Flaherty, J. T., Kreutzer, D. L. & Ward, P. A. (1978) Am J. Pathol. 90, 537-550. 11. Chenoweth, D. E. & Hugh, T. E. (1978) Proc. Natl. Acad. Sci. USA 75,3943-3947. 12. DeChatelet, L. R. (1978) J. Reticuloendothel. Soc. 24,73-83. 13. Bass, D. A., DeChatelet, L. R. & McCall, C. E. (1978) J. Immunol. 121,172-178. 14. Lineweaver, H. & Burk, D. (1934) J. Am. Chem. Soc. 56, 658-666. 15. Ward, P. A. & Becker, E. L. (1968) J. Exp. Med. 127, 653671. 16. Olefsky, J. M. & Kobayashi, M. (1978) Metabolism Suppl. 12, 27, 1917-1928. 17. Olefsky, J. M. (1978) Blochem. J. 172,137-145. 18. Fussganger, R. B., Kahn, C. R., Roth, J. & DeMeyts, P. (1976) J.

Biol. Chem. 251,2761-2769. 19. Stossel, T. P., Mason, R. J., Hartwig, J. & Vaughan, M. (1972) J. Clin. Invest. 51,615-624. 20. Gambhir, K. K., Archer, J. A. & Bradley, C. G. (1978) Diabetes

27,701-708. 21. Stossel, T. P., Murad, F., Mason, R. J. & Vaughan, M. (1970) J.

Biol. Chem. 245,6228-6231.

22. Carruthers, B. M. (1967) Can. J. Physiol. Pharmacol. 45,269280. 23. Nathan, C. F., Silverstein, S. C., Baukner, L. H. & Cohn, Z. A. (1979) J. Exp. Med. 149,100-113. 24. Bibi, S. S., DeChatelet, L. R. & McCall, C. E. (1977) J. Infect. Dis.

135,949-951. 25. Frank, M. M. & Atkinson, J. P. (1975) Dis. Mon. 1-55. 26. Craddock, P. R., Hammerschmidt, D. E., White, J. G., Dalmasso, A. P. & Jacob, H. S. (1977) J. Clin. Invest. 60,261-264.

Enhancement of hexose uptake in human polymorphonuclear leukocytes by activated complement component C5a.

Proc. NatI. Acad. Sci. USA Vol. 76, No. 11, pp. 5896-5900, November 1979 Medical Sciences Enhancement of hexose uptake in human polymorphonuclear le...
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