Glutamine-enhanced bacterial killing by neutrophils from postoperative patients

APPLIED NUTRITIONAL

INVESTIGATION

Nutrition

Vol. 13, No. 10, 1997

Glutamine-Enhanced Bacterial Killing by Neutrophils From Postoperative Patients SATOSHI FURUKAWA, MD, HIDEAKI SAITO, MD, KAZUHIKO FUKATSU, MD, YOJIRO HASHIGUCHI, MD, TSUYOSHI INABA, MD, MING-TSAN LIN, MD, TOMOMI INOUE, MD, ILSOO HAN, MD, TAKEAKI MATSUDA, MD, AND TETSUICHIRO MUTO, MD From the Department of Surgery, Faculty of Medicine,

University of Tokyo, Tokyo, Japan

Date accepted: 7 February 1997

ABSTRACT Neutrophils play an important role in host defense by phagocytosing and destroying invading bacteria. A recent investigation revealed that glutamine (Gln) augmented the in vitro bactericidal activity of neutrophils from bum patients. However, it is unclear whether Gln enhances the function of neutrophils in postoperative patients. This study was designed to investigate the effect of Gln on the in vitro Escherichia c&i-killing activity of neutrophils from postoperative patients. Nine randomly selected patients were included in this study. On the morning of the first postoperative day, blood was drawn and neutrophils were isolated. Eight healthy volunteers served as controls. E. coli was opsonized with pooled normal serum. Neutrophils (5 X 106), together with opsonized E. coli (5 X 105), were incubated for 2 h at 37°C in Hanks’ balanced salt solution supplemented with 0, 100,500, or 1000 nmol/mL of Gln. The bactericidal function of neutrophils was determined by counting the number of viable bacteria. Tumor necrosis factor (TNF)-(Y, interleukin (IL)-lp, IL-8, and granulocyte elastase levels in the cell culture supematant were measured. Plasma C-reactive protein (CRP), cortisol, and amino acids were also analyzed. The plasma concentration of Gln was significantly lower in the postoperative patients than in the controls. Following culture with patient neutrophils, the number of viable E. coli decreased by 26% as the in vitro Gln concentration was increased from 500 to 1000 nmol/mL (P < 0.01). We defined the Gln lOOO/Gln 500 ratio of the number of viable bacteria as the number of viable E. coli at an in vitro Gln concentration of 1000 nmol/mL divided by the number of viable E. coli at an in vitro Gln concentration of 500 nmol/mL. A positive correlation was thus demonstrated between the plasma Gln level and the Gln lOOO/Gln 500 ratio of the number of viable bacteria in the patients (r = 0.69, P = 0.04). This finding indicated that as plasma Gln fell, there was an enhancement of neutrophil E. coli-killing activity by neutrophils in in vitro tests when the Gln concentration was increased from 500 to 1000 nmol/mL. Gln supplementation caused no appreciable changes in TNF-a, &l/3, R-8, or granulocyte elastase levels in cell culture supematants. A negative correlation was recognized between the patient plasma Gln level and the Gln lOOO/Gln 500 ratio of the cell culture supematant IL-8 level (r = -0.73, P = 0.025). In conclusion, Gln supplementation enhanced the in vitro bactericidal function of neutrophils from postoperative patients. Nutrition 1997;13:863-869. OElsevier Science Inc. 1997 Key words: glutamine, neutrophils, bacterial killing, Escherichia

INTRODUCTION Despite major advances in surgical techniques and perioperative care, infectious complications remain the major cause of postoperative morbidity and mortality.’ Currently, antibiotic therapy, adequate nutrition, and debridement of infected tissues are the major means of reducing infectious complications following surgery.* However, postoperative infectious complications still occur at a high incidence. Thus, new approaches are needed. Recently developed specific nutritional substrates have been shown to augment host immune function and improve survival.3 Glutamine (Gln) is one of these new substrates.3

coli

Host defense mechanisms are impaired following surgery.4 Neutrophils play a pivotal role in host defense by phagocytosing and destroying invading bacteria.5 Neutrophils isolated from patients after surgery,6s7 bums,s and trauma* show depressed bactericidal function. However, little information is available conceming means of augmenting the bactericidal activity of neutrophils. Gln has been shown to enhance the functions of lymphocytes and macrophages.9 Moreover, a recent study revealed that Gln augmented the in vitro bactericidal activity of neutrophils from bum patientslO However, it remains unclear whether Gln enhances the function of neutrophils in postoperative patients. This

Correspondence to: Satoshi Fumkawa, MD, Department of Surgery, Faculty of Medicine, University of Tokyo, 7-3-l Hongo, Bunkyoku, Tokyo, Japan.

Nutrition 13:863-869, 1997 OElsevier Science Inc. 1997 Printed in the USA. All rights reserved.

ELSEVIER

0899-9007/97/$17.00 PI1 SO899-9007(97)00271-2

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TABLE I. CLINICAL AND LABORATORY DATA

Preoperative Patient age/sex

Associated disease

Diagnosis

1. 46 M 2. 64 M 3. 66 M 4. 59 M 5. 71 M 6. 41 F

Arteriosclerosis obliterans Esophageal cancer Pharyngeal cancer Ulcerative colitis Pharyngeal cancer Breast cancer

7. 66 M 8. 75 M 9. 67 M

Arteriosclerosis (-) obliterans Rectal cancer (-) Sigmoid colon cancer

DM DM (y) DM (-) (-)

DM, LC

Operation

serum Alb (g/dL)

Operative Intraoperative Intraoperative time blood loss transfusion % IBW (min) (mL) (mL)

WBC on POD Postoperative 1 (/mm3) complications

Aortobifemoral bypass Subtotal esophagectomy Total laryngectomy Total colectomy

4.0

102.8

325

400

0

15 800

(-)

3.2

85.1

350

250

600

14 100

(-)

3.7

76.2

720

2000

840

22 900

(-)

3.5

91.2

330

1040

400

14 600

Middle pharyngectomy Modified radical mastectomy Femoropopliteal bypass Low anterior resection Sigmoidectomy

3.9

113.1

590

760

0

13 700

4.2

90.8

275

350

0

9900

(-)

3.8

104.4

210

250

0

9200

(-)

3.5

93.9

190

260

0

8400

(-)

3.5

93.6

220

500

0

10900

(-)

anastomotic leakage (-)

Alb, albumin; DM, diabetes mellitus; IBW, ideal body weight; LC, liver cirrhosis; POD, postoperative day; WBC, white blood cell count.

study was therefore designed to investigate the effect of Gln on the activity of neutrophils from pain vitro Escherichiu c&killing tients undergoing various kinds of major surgery. Production of interleukin-2 (IL-2) by concanavalin A-stimulated rat lymphocytes depends upon the Gln concentration.lt Moreover, the ability of murine peritoneal macrophages to secrete IL-l also depends on the Gln level.11 However, the effect of Gln on neutrophil production of cytokines has not been studied extensively. On the other hand, granulocyte elastase has been shown to

be a component of neutrophils, and this enzyme contributes to the nonoxidative bacterial killing capacity of neutrophils.5 Therefore, we measured the levels of tumor necrosis factor (TNF)-cq IL-l& B-8, and granulocyte elastase in cell culture supernatants to elucidate the mechanism by which Gln enhances the bactericidal function of neutrophils. PATIENTS AND METHODS Patients

We randomly selected nine patients who had undergone various kinds of operations. Informed consent was obtained from each patient. Clinical and laboratory data on the patients are shown in Table I. Patient ages ranged from 41 to 75 y. Diagnoses included arteriosclerosis obliterans, esophageal cancer, pharyngeal cancer, ulcerative colitis, breast cancer, rectal cancer, and sigmoid colon cancer. All but patient 2 could eat normally before the operation. Patient 2 was given a 1500 kcal/d elemental diet. The average preoperative serum albumin level and percent ideal body weight were 3.8 2 0.1 g/dL and 95 ? 4%, respectively. Associated diseases were diabetes mellitus in four patients and liver cirrhosis in one. The operations included aortobifemoral bypass, subtotal esophagectomy, total laryngectomy, total colectomy, middle pharyngectomy, modified radical mastectomy, femoropopliteal by-

pass, low anterior resection, and sigmoidectomy. Operative time ranged from 190-720 min with an average of 357 min. Intraoperative blood loss ranged from 250-2000 mL, averaging 646 mL. One patient (patient 4) undergoing total colectomy developed anastomotic leakage. There were no postoperative mortalities. We conducted the same measurements in eight healthy, male, fasting volunteers with an average age of 33 y. Blood Sampling and Isolation of Neutrophils

Blood was drawn at 0700 h on the first postoperative day (POD). No amino acid solution had been included in the postoperative parenteral nutrition regimens prior to the blood draw. At the time of blood drawing, at least 8 h had passed since the last administration of intravenous prophylactic antibiotics. The mean peripheral white blood cell count on POD 1 was 13 300/mm3, ranging from 8400-22 900/mm3. The percentage of neutrophils was counted using an automatic counter (Blood Cell Analyzer 8200, HITACHI, Tokyo, Japan) and varied from 52-81%. Neutrophils were obtained by a modification of the methods of Ogle et al.iO At 0700 h on POD 1, we drew 20 mL peripheral blood into a syringe containing 4 mL 6% dextran (Midori Juji, Osaka, Japan) and 0.6 mL heparin sodium (1000 U/mL) (Novo Nordisk A/S, Copenhagen, Denmark). Similarly, at 0700 h, we collected from the controls 40 mL peripheral blood in a syringe containing 8 mL 6% dextran and 1.2 mL heparin sodium (1000 U/mL). The controls had fasted for 8 h prior to the blood drawing. The syringe was placed upright for 45-60 min to allow for separation of red cells and plasma. The plasma layer was then removed through a bent needle and collected in a 50-mL centrifuge tube. The plasma layer was underlaid with 10 mL Ficoll (Pharmacia Biotech A/B, Uppsala, Sweden) and centrifuged at 500 X g for 30 min at 27°C. After centrifugation, the mononuclear layer was removed and the pellet containing the neutrophils and a

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few contaminating erythrocytes was resuspended. The erythrocytes were lysed with distilled water. The neutrophils were washed once with Hanks’ balanced salt solution (HBSS) (without calcium and magnesium; Nikken Biomedical Laboratory, Kyoto, Japan) supplemented with 0.1% gelatin (Iwaki Glass, Tokyo, Japan) and 10 mM HEPES (Cosmo Bio Co., Tokyo, Japan). The cells were then suspended with HBSS containing 0.1% gelatin and 10 mM HEPES. The neutrophils were >95% pure and viable as determined by microscopic observation. Live neutrophils were counted by trypan blue exclusion and adjusted to 1 X 107/mL. Preparation of Bacteria E. coli (American Type Culture Collection 25922) were cultured in a brain-heart infusion broth (Nissui Pharmaceutical Co., Tokyo, Japan) for 18 h at 37°C. The culture was centrifuged at 1700 X g for 10 min at 4°C to pellet the E. coli, then washed twice and resuspended in sterile normal saline. A lOO-PL suspension was serially diluted with sterile saline, plated on sheep blood agar plates (Nissui Pharmaceutical), and incubated for 18 h for determination of the bacterial concentration. The remainder was stored at 4°C until use. Just before use, the bacterial suspension was adjusted to 1 X 10’ colony-forming units (CFU) /mL. Opsonization of E. coli E. coli (1 X lo*), together with 1 mL pooled normal serum and 2 mL HBSS containing 0.1% gelatin and 10 mM HEPES, were cultured for 15 min at 37°C. The culture was centrifuged at 2500 X g for 10 min at 4°C to pellet the E. coli, then washed once and resuspended in HBSS supplemented with 0.1% gelatin and 10 mM HEPES. The final concentration of the opsonized E. coli was adjusted to 5 X lo6 CFUlmL. As our intent was to examine the bactericidal function of the neutrophils themselves, we opsonized the E. coli with pooled normal serum, rather than with subject serum. Measurement of Bactericidal Activity of Neutrophils Our preliminary examination showed the most appropriate neutrophil to E. coli ratio for assessing neutrophil bactericidal function to be 1O:l. Neutrophils (5 X 106), together with 5 X 10’ opsonized E. coli, were incubated in HBSS containing 0.1% gelatin and 10 mM HEPES supplemented with 0, 100, 500, or 1000 nmol/mL Gln in a final volume of 1 mL. After a 2-h incubation at 37”C, the reactive mixtures were vigorously vortexed and a 0. I-mL aliquot from each sample was added to 9.9 mL sterile saline. This dilution and one more 1:lOO serial dilution were plated on sheep blood agar plates and cultured for 18 h at 37°C. Viable E. coli counts were then determined. In handling a large number of samples at one measurement, isolated neutrophils may lose viability, thereby diminishing the validity of the experiment. Therefore, we determined viable bacterial counts in cell culture supematants of the samples on four separate days. Cell culture supematants were separated and stored at -20°C until the measurement of TNF-(Y, IL-l& IL-8, and granulocyte elastase levels, as described below. Assay of TNF-a, IL-Ifi IL-& and Granulocyte Elastase TNF-(r, IL-l& and IL-8 levels in the cell culture supematant were measured using commercially available enzyme-linked immunosorbent assay methods (TNF-c~ and IL-lp from Genzyme Immunobiologicals, Cambridge, MA, USA; IL-8 from TORAY, Tokyo, Japan). For each assay, a standard curve using recombinant cytokine was constructed. We measured granulocyte elastase levels in the cell culture supematant by chromogenic substrate assay using S-2484 (Chromogenix A/B, Molndal, Sweden).

Analyses of Plasma C-reactive Protein, Cortisol, and Amino Acids Blood samples were centrifuged at 1700 X g for 10 min at 4°C. The plasma was separated and stored at -20°C until analysis. Plasma C-reactive protein (CRP), cortisol, and amino acids were measured by turbidimetric immunoassay, radioimmunoassay, and high-performance liquid chromatography, respectively. Statistical Analysis Results are expressed as the mean ?SEM. To achieve a normal distribution, viable bacterial counts were transformed, if necessary, to a logarithmic scale before statistical analysis. An unpaired Student’s t test was used for the plasma amino acid concentrations of both patients and controls. Concerning the number of viable E. coli in the culture medium, a test with a one-way, repeatedmeasures analysis of variance model showed that significant difference(s) existed among the four in vitro Gln levels in the patient group. Therefore, a contrast test was carried out within the patient group to determine the Gln concentration at which viable bacterial counts differed from those at other Gln concentration(s). Linear regression analysis was also used. A P value of CO.05 was considered significant. RESULTS

Plasma Amino Acid Concentrations The plasma concentrations of the following amino acids on POD 1 were significantly lower in patients than in controls: threonine (P < O.OOOl), serine (P < 0.05), asparagine (P < O.OS), Gln (P < O.OOl), glycine (P < 0.05), alanine (P < 0.05), citrulline (P < O.OOl), valine (P < O.OOl), isoleucine (P < O.OOOl), leucine (P < O.Ol), omithine (P < O.OOl), lysine (P < O.OOOl), and histidine (P < 0.001) (Table II). Bactericidal Function of Neutrophils at Various in vitro Gin Concentrations Following culture with neutrophils from patients, the number of viable E. coli decreased by 26% as the in vitro Gln concentration was increased from 500 to 1000 nmol/mL, and this decrease was statistically significant (P < 0.01) (Table III). Gln supplementation caused no appreciable changes in the number of viable E. coli cultured with neutrophils from healthy volunteers. There was no difference in the number of viable E. coli between the patients and the volunteers at any of the Gln concentrations used. Correlation Between Plasma Concentrations of Gln and Bactericidal Function of Neutrophils There were no statistically significant correlations between plasma Gln levels and the number of viable E. coli at any of the supplementary Gln concentrations used, in either patients or controls. We defined the Gln lOOO/Gln 500 ratio of the number of viable bacteria as the number of viable E. coli at an in vitro Gln concentration of 1000 nmoYmL divided by the number of viable E. coli at an in vitro Gln concentration of 500 nmol/mL. Thus, a positive correlation was demonstrated between the plasma Gln level and the Gln lOOO/Gln 500 ratio of the number of viable bacteria in the patients (r = 0.69; P = 0.04) (Fig.1). White Blood Cell Count, Plasma CRP Values, and Plasma Corns01 Levels T ere were positive correlations between the peripheral white bloo $ cell count on POD 1 and the number of viable bacteria at all concentrations of Gln studied (Gln 0 nmol/mL, r = 0.71; 100 nmoYmL, r = 0.74; 500 nmol/mL, r = 0.72; 1000 nmol/mL, r = 0.67; and for all concentrations, P 5 0.05) (Fig.2). The mean

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TABLE II. PLASMA

AMINO ACID CONCENTRATIONS AND HEALTHY VOLUNTEERS Amino acid concentration

Amino acid Phosphoserine Tatnine Aspartate Threonine Serine Asparagine Glutamate Glutamine Proline Glycine Alanine Citrulline Valine Cysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Hi&dine Arginine Values are mean

t t ? 2 -c k 2 2 2 2 2 2 2 -c 2 2 2 5 2 2 5 2 k

(nmol/mL) Controls (n = 8)

Patients (n = 9) 11.8 57.1 12.1 79.0 91.3 36.5 61.8 506.9 119.8 191.8 312.9 21.4 181.1 32.9 19.6 47.3 111.9 59.5 77.5 45.4 129.6 53.3 57.2

IN PATIENTS

2.0 8.4 5.5 5.8 7.4 3.7 7.7 20.6 17.8 12.4 33.5 3.1 12.2 6.8 2.8 5.1 8.3 6.6 9.7 3.2 8.7 4.5 13.1

P value

10.8 54.2 8.9 125.4 113.4 47.2 70.1 616.9 169.5 241.1 416.7 37.5 261.0 17.4

+- 0.8 k 2.9 2 3.0 k 3.3 2 2.9 ? 3.0 ? 10.8 2 12.2 ? 17.1 _’ 13.9 2 23.4 k 2.0 k 13.0 2 3.7

26.0 83.7 152.2 70.5 64.6 80.0 188.3 77.3 88.3

2 0.9 2 4.3 k 7.3 _’ 7.0 k 2.7 k 7.2 ?z 6.6 2 3.1 ? 7.5

NS NS NS
I

I

I

600 500 400 Plasma Glutamine (nmol/ml) FIG. 1. Correlation between plasma glutamine (Gin) level and the Gln lOOO/Gln 500 ratio of the number of viable bacteria in the patients (n = 9; r = 0.69; P = 0.04). Gin lOOO/Gln 500 ratio of the number of viable bacteria equals the number of viable E. coli at an in vitro Gln concentration of 1000 nmol/mL divided by the number of viable E. coli at an in vitro Gln concentration of 500 nmol/mL.

at any of the Gln concentrations tested. The number of viable bacteria showed no significant correlation with either operative time or intraoperative blood loss. TNF-a, IL-I/3 IL-8, and Granulocyte Elastase Levels in Cell Culture Supenatants

+ SEM. NS, not significant.

CRP values in the patient (POD 1) and control groups were 12.2 and 0.1 mg/dL, respectively (P < 0.0001). In the patients, a positive correlation was recognized between the plasma CRP level and the number of viable E. coli at all Gin concentrations studied (Gln 0 nmol/mL, r = 0.74; 100 nmol/mL, r = 0.72; 500 nmol/mL, r = 0.72; 1000 nmol/mL, r = 0.68; and for all concentrations, P < 0.05) (Fig. 3). The mean plasma cortisol values in the patients and controls were 16.5 and 13.2 pg/dL, respectively. No correlations were recognized in either group between the plasma cortisol level and the number of viable E. cob plasma

TNF-a, IL-l& IL-8, and granulocyte elastase levels in the cell culture supematants did not differ significantly, in patients or controls, as the in vitro concentration was changed (Table IV). In the patients, a negative correlation was demonstrated between cell culture supematant granulocyte elastase levels and the number of viable bacteria (Gln 0 nmol/mL, r = -0.67; 100 nmollml, r = -0.85; 500 nmoYmL, r = -0.73; 1000 nmol/mL,

TABLE III. BACTERICIDAL

FUNCTION GLUTAMINE

OF NEUTROPHILS CONCENTRATIONS*

In vitro glutamine

Patients (n = 9) Controls (n = 8)

AT DIFFERENT

concentration

(nmol/mL)

0

100

500

1000

3.6 2 1.4 3.0 2 1.3

3.4 r 1.2 3.0 5 1.3

3.7 k 1.3 2.7 5 1.1

2.8 ” 0.9t 3.0 2 1.1

Values are mean t SEM. * X 16 colony-forming units of E. coli per milliliter of cell culture supematant. 7 P < 0.05versus in vitro glutamine

500 nmoVmL in the patients.

I

I

10000 15000 20000 Peripheral White Blood Cell Count (/mn?) FIG. 2. Correlation between peripheral white blood cell count on postoperative day one and the number of viable bacteria at an in vitro glutamine concentration of 0 nmol/mL (n = 9; r = 0.71; P = 0.03).

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0

Patients (n=9) HI.74 p.o.02

0 /

0

4

0

15’00

2ioo

2600

Cell Culture Supernatant Granulocyte

I

I

I

I

I

5

20

15 PIas:

CRP(

mg/dl

)

Elastase (t-g/ml)

FIG. 4. Correlation between cell culture supematant granulocyte elastase levels and the number of viable bacteria in the patients at an in vitro glutamine concentration of 1000 nmol/mL (n = 9; I = -0.78; P = 0.01).

FIG. 3. Correlation between plasma C-reactive protein (CRP) level and the number of viable bacteria in the patients at an in vitro glutamine concentration of 0 nmoVmL (n = 9; r = 0.74; P = 0.02). CFU, colony-forming units.

divided by the IL-8 level at an in vitro Gln concentration of 500 nmol/mL. A negative correlation was demonstrated between the plasma Gln level and the Gln lOOO/Gln500 ratio of the cell culture supematant IL-8 level (r = -0.73, P = 0.025) (Fig. 5).

and for all concentrations, P 5 0.05) (Fig. 4). In the patients, there was no relationship between the plasma Gln concentration and the cell culture supematant level of TNF-(Y, IL-lp, IL-8 or granulocyte elastase. We defined the Gln lOOO/Gln 500 ratio of the cell culture supernatant IL-8 level as the IL-8 level in the supernatant at an in vitro Gln concentration of 1000 nmol/mL

In the present study, the in vitro bactericidal function of neutrophils from postoperative patients was significantly greater at 1000 nmol/mL than at 500 nmoYmL of Gln. The bactericidal function of neutrophils after surgery has been shown to be depressed as compared with the preoperative level and to be lowest on POD 1.6.7Therefore, we examined the effect of Gln on the in vitro E. co&killing activity of neutrophils on POD 1. The serum Gln level decreases after surgery,iz trauma,i3 and bums.9 Our results confirmed this decreased serum Gln concentration in surgical illness. Depressed serum Gln in critically ill

DISCUSSION

r = -0.78;

TABLE IV. CELL

CULTURE SUPERNATANT TNF-a, IL-lp, IL-8, AND GRANULOCYTE ELASTASE LEVELS In vitro Gln concentration (nmol/mL)

Patients (n = 9)

Healthy volunteers (n = 8)

Pawsi

(n=9)

p=o:o25 TNF-(Y (pg/mL)

IL-l p (pg/mL)

IL-8 (pg/mL)

Granulocyte

elastase

0 100 500 1000 0 100 500 1000 0 100 500 1000 0

158? 53 132 f 33 169 2 57 141 + 41 405 2 107 405 % 99 397 ? 119 366 5 108 940 2 274 979 + 234 937 -t 233 792 2 202 1743 I 192

186 225 223 253 292 297 273 246 1163 1041 1089 1048 1827

2 5 t ? 2 + 2 t f 2 2 t -c

45 49 45 55 65 74 59 56 251 228 241 230 124

100 500 1000

1749 2 135 1762 2 132 1867 + 142

1638 f 105 1721 ? 124 1625 ? 121

t

400

I

I

600 500 Plasma Glutamine (nmol/ml)

(ng/mL)

Values are mean ? SEM. Gln, glutamine; IL, interleukin;

TNF, tumor necrosis factor.

FIG. 5. Correlation between plasma glutamine (Gln) level and the Gln lOOO/Gln 500 ratio of the cell culture supematant interleukin-8 (IL-8) level in the patients (n = 9; r = -0.73; P = 0.025). Gln lOOO/Gln 500 ratio of IL-8 level equals IL-8 level at an in vitro Gln concentration of 1000 nmoYmL divided by IL-8 level at an in vitro Gln concentration of 500 nmol/mL.

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patients might be partly explained by the increased consumption of Gln by lymphocytes,14 macrophages,i5 and intestinal epithelial cellsi Gln depletion results in immunosuppression.il Thus, it appears that Gln supplementation in critical illness enhances the function of lymphocytes and macrophages. Gln has, in fact, been shown to augment the in vitro activity of lymphocytes and macrophages.9 A recent study revealed that Gln augmented the in vitro bactericidal activity of neutrophils from bum patients. lo We demonstrated herein that the in vitro bactericidal function of neutrophils from postoperative patients was significantly greater, i.e., by 26%, at 1000 nmol/mL than at 500 nmoVmL of Gin. Thus, Gln clearly enhances the bactericidal function of neutrophils. The large SEM of the bacterial number shown in Table III may be attributable to the sample measurements having been done on separate days. To reduce the variability due to differences among individuals (between-subject), we applied a test with a one-way, repeated-measures analysis of variance model followed by a contrast test. This statistical method allowed intra-individual variation (within-subject) to be determined. We demonstrated that the number of viable E. coli decreased significantly as the in vitro Gln concentration was increased from 500 to 1000 nmol/mL. Following culture with neutrophils from patients, the number of viable E. coli decreased significantly as the in vitro Gln concentration was increased from 500 to 1000 nmol/mL, but not from 0 to 1000 nmol/mL. Therefore, we chose the Gln lOOO/Gln 500 ratio to interpret our results. The plasma Gln level of the postoperative patients was approximately 500 nmol/mL. On the other hand, the mean plasma Gln concentration of the patients receiving parenteral Gln supplementation (163 mmol * kg-’ - h-t) was approximately 950 nmol/mL.i7 Thus, we simulated in vivo Gln supplementation by using the Gln lOOO/Gln 500 ratio of the number of viable bacteria in this in vitro study. We demonstrated a positive correlation between the plasma Gln level and the Gln lOOO/Gln 500 ratio of the number of viable bacteria in postoperative patients. This finding indicated that as plasma Gln fell, neutrophil E. co&killing activity was enhanced in in vitro tests when the Gin concentration was increased from 500 to 1000 nmol/mL. In contrast, supplemental Gln had no significant effect on the bactericidal function of neutrophils from the controls. The mean age of our controls was lower than that of the patients. As age affects neutrophil function,” it would be worthwhile to study the effect of Gln on the bactericidal function of neutrophils from elderly, healthy volunteers. Opsonized E. coli are ingested and killed by neutrophils within phagocytic vacuoles, where they are exposed to an array of reactive oxygen metabolites and toxic lysosomal components.ig Thus, the bactericidal-enhancing effect of Gln in this study may have occurred at the level of phagocytosis, the formation of reactive oxygen metabolites, or degranulation. Ogle et al.i” showed that Gln had no effect on phagocytosis by neutrophils from bum patients. Therefore, Gln may enhance neutrophil bactericidal function by other mechanism(s) such as increasing the formation of reactive oxygen metabolites and/or promoting the degranulation of neutrophils. Neutrophils produce TNF-a, IL-l, and IL-8.2o Neutrophils release approximately 1 ng/mL IL-8 in response to stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine for 2 h.21 Moreover, neutrophils stimulated with 5 mg/mL lipopolysaccharide for 2 h have been shown to secrete 36 pg/mL TNF.22 Therefore, neutrophils can produce measurable amounts of cytokines with a 2-h incubation. However, in our study, Gln supplementaion produced no appreciable changes in TNF-(Y, IL-l/3, or IL-8 levels in cell culture supematants after a 2-h incubation. Further inves-

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tigation is needed to determine cytokine productions resulting from an incubation period of more than 2 h. Neutrophil activation by TNF-(Y, IL-l, or IL-8 in vitro increases the production of reactive oxygen metabolites.23 We did not determine reactive oxygen metabolite levels in cell culture supematants. Thus, the possibility remains that Gln supplementation increased the formation of reactive oxygen metabolites by neutrophils. However, the enhancement of oxidative burst production, if any, may not be directly mediated by TNF-cr, IL-lp, or IL-8 based on our results. We demonstrated a negative correlation, in the postoperative patients, between cell culture supematant granulocyte elastase levels and the number of viable bacteria. This finding indicated that as the cell culture supematant granulocyte elastase level rose, there was an enhancement of E. co&killing activity by neutrophils in in vitro tests. However, these granulocyte elastase levels did not differ significantly, in the patients, as the in vitro Gln concentration was changed. This observation suggests that the nonoxidative bacteria-killing capacity of neutrophils may not mediate the bactericidal-enhancing effect of Gln. Thus, the mechanisms underlying the enhancing effect of Gln on neutrophil bactericidal capacity are apparently not based on the increased production of the cytokines studied or granulocyte elastase. The energy source neutrophils use to produce reactive oxygen metabolites is believed to be glucose.24 There have been no studies investigating whether Gln is utilized by neutrophils. However, in a recent review, Vlessis and associatesz5 described new concepts in the pathophysiology of oxygen metabolism during sepsis. The progression of sepsis limits the aerobic oxidation of glucose. In contrast, aerobic oxidation of glutamate (and also Gln following transamination) is not inhibited in sepsis. Therefore, it appears that Gln is being utilized for aerobic metabolism in preference to glycolytic substrates. It was shown that glutaminase, the key enzyme in the utilization of Gln as an energy source, is located within the mitochondria of lymphocytes and macrophages.26 There are no reports, to the best of our knowledge, that neutrophils possess glutaminase. However, in light of the present results, it is tempting to speculate that Gln enhances the in vitro bactericidal function of neutrophils by serving as a neutrophil substrate. We showed that as the plasma Gln level fell, there was an elevation of the cell culture supematant IL-8 level in in vitro tests when the Gln concentration was increased from 500 to 1000 nmol/mL. Since IL-8 is a neutrophil chemoattractant, the increased IL-8 level at an infected site results in more neutrophils migrating to the lesion in vivo. This migration of neutrophils is advantageous for host defense. In the patients who had undergone various operations, there was a positive correlation between the peripheral white blood cell count on POD 1, and the number of viable bacteria in neutrophilkilling assays at all Gln concentrations. In addition, we observed in the patients positive correlations between the plasma CRP level and the number of viable bacteria at all Gln concentrations studied. These findings indicate that as the surgical stress increased, there was a decrease in the E. coli-killing capacity of neutrophils in in vitro tests. Thus, it is important to prevent this reduction in bactericidal activity under severe surgical stress. Ziegler et ali7 reported that bone marrow transplantation patients receiving parenteral Gln had a lower incidence of infection and had a shorter hospital stay than patients receiving Gln-free parenteral nutrition. Our results support the beneficial effects of Gln on host defense against infection. Further trials are needed to clarify the immuneenhancing effect of Gln supplementation in postoperative patients. In conclusion, Gln supplementation enhanced the in vitro bactericidal function of neutrophils from postoperative patients.

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(For an additional perspective, see Editorial Comment on page 914.)