JOURNAL OF CELLULAR PHYSIOLOGY 168:26-33 (1996)
Supplemental 1-Arginine HCI Augments
Bacterial Phagocytosis in Human
Polymorphonuclear Leukocytes
FREDERICK L. MOFFAT, JR.,* TIERAN HAN, ZHI-MINC LI, MICHAEL D. PECK,
WENCHE JY, YEON S. A H N , ARTHUR J. CHU, AND LlLLY Y.W. B O U R C U I G N O N
Veterans Administration Medical Center, Miami, Florida 33 7 36 (F.L.M., T.H.), and the
Sylvester Comprehensive Cancer Center (F.L.M., T.H., W.J., Y.S.A., A.J.C., L.Y.W.B.),
Departments ofsurgery (F.L.M., T.H., Z.-M.L., M.D.P.), Medicine (W.J., Y.S.A.), and
Anatomy and Cell Biology (A.J.C.,L. Y. W.B.), University of Miami School
of Medicine, Miami, Florida 33 136
That L-arginine (L-Arg) augments the host response to acute bacterial sepsis suggests that this amino acid intervenes early in the immune response, perhaps via
the nitric oxide synthetase (NOS) pathway. The effect of L-Arg supplementation
o n in vitro phagocytosis of fluorescein-labeled,heat-killed Staphylococcus aureus
by peripheral blood neutrophils (PMNs) from 1 2 normal human volunteers was
studied. Separated PMNs were incubated for 2 h with labeled bacteria, with and
without supplemental L-Arg, D-arginine, glycine, and/or the NOS inhibitors Lcanavanine, aminoguanidine, or L-NG-nitroargininemethyl ester. PMNs were
fixed and extracellularfluorescencequenched with crystal violet. By flow cytometry and confocal microscopy, L-Arg supplementation was shown to result in a
highly significant increase in PMN bacterial phagocytosis, the maximal effect
being seen with L-Arg 380 pM and falling off with higher concentrations. This
augmentation was completely abrogated by NOS inhibitors in molar excess, but
inhibitors alone did not suppress phagocytosis below that of unsupplemented
controls. Neither D-arginine nor glycine affected phagocytosis; the L-Arg effect
was stereospecific and not related to utilization of L-Arg as an energy source.
L-Arg supplementation significantly enhances bacterial phagocytosis in human
neutrophils, perhaps by effects on cytoskeletal phenomena, and this appears to
be mediated through NOS activity. Phagocytosis by nonspecific immune cells
which intervene early in the response to sepsis is critically important, and beneficial effects of L-Arp. on the clinical course of sepsis may be due at least in part
to augmentation ofuphagocyte function. Q 1996 Wiley-Liss, Inc.
The immunostimulative activity of the semiessential
amino acid L-arginine (L-Arg) continues to receive
much attention. Early rodent studies showed that LArg enhances cell-mediated immunity (CMI) and Tlymphocyte responses to mitogenic stimulation (Alexander et al., 1980; Barbul et al., 1980a,b, 1981, 1985;
Barbul, 1986; Freund et al., 1978,1979) and augments
thymic weight and thymic lymphocyte counts (Barbul
et al., 1980a,b). The mitogenic response of peripheral
blood lymphocytes from healthy human volunteers is
similarly augmented by L-Arg (Barbul et al., 1981). LArg also enhances skin allograft rejection and abrogates posttraumatic suppression of CMI (Barbul et al.,
1985; Barbul, 1986).
In addition t o its importance as a structural amino
acid and its role in a variety of physiological functions
(Barbul, 1986), L-Arg is the substrate for production of
reactive nitrogen species such as nitric oxide (NO * ) by
neutrophils (PMNs) (Kaplan et al., 1989; Lopez Farre
et al., 1991; McCall et al., 1989; Schmidt et al., 1989;
Wright et al., 1989), macrophages (Drapier and Hibbs,
0 1996 WILEY-LISS, INC.
1988;Drapier et al., 1988;Hibbs et al., 1987), and many
other mammalian cells. L-Arg has been shown to prevent tumor induction or retard tumor growth in at least
24 animal tumor systems (Barbul, 1986; Drapier et al.,
1988; Reynolds et al., 1988, 1990; Tachibana et al.,
1985; Takeda et al., 1975; Ye et al., 1992) through production of reactive nitrogen species by mononuclear
cells. L-Arg is required by mononuclear phagocytes for
cytostatic and cytocidal activity against several intracellular pathogens (Green et al., 1990; Hibbs et al.,
1988; James and Glaven, 1989); NO * generation from
Arg via the nitric oxide synthase (NOS) enzymatic
pathway appears to play a role in these microbicidal
effects.
Experimental evidence suggests that L-Arg may be
Received September 30, 1994; accepted January 5, 1996.
*To whom reprint requestdcorrespondence should be addressed
at University of Miami School of Medicine, Sylvester Comprehensive Cancer Center, Surgical Oncology (310T), Room 3550, 1475
N.W. 12th Avenue, Miami,FL 33136.
ARGININE AND NEUTROPHIL PHAGOCYTOSIS
important in the host response to acute, severe bacterial sepsis. Madden et al. (1988) demonstrated that LArg, administered every 12 h for 4 days, enhances survival in a rat caecal ligation model of fecal peritonitis,
even when the first dose of L-Arg is withheld until 3 h
after induction of sepsis. Fukatsu et al. (1995) demonstrated that NOS inhibition is deleterious to survival
in an in vivo murine model of gram-negative bacterial
sepsis. There is also indirect clinical evidence of the
association between L-Arg and host immune response,
such as the observation by Freund et al. (1978) that
low serum L-Arg correlates with immune suppression
in critically ill, septic patients. Marked reduction in
serum L-Arg is also a predictor of mortality in patients
with severe bacterial infections (Alexander et al., 1980;
Freund et al., 1979).
Madden et al. (1988) ascribed the observed survival
benefit of L-Arg in rat faecal peritonitis to immunomodulatory effects on T cells. However, lymphocyte response t o antigen challenge requires several days to
become fully manifest. That significant effects on survival should be observed in a model of fulminant polymicrobial peritonitis suggests that L-Arg must also
intervene a t a much earlier point in the host response
to sepsis. It is postulated that L-Arg enhances antibacterial function in nonspecific immune cells such
as mononuclear phagocytes. We have tested this hypothesis in a study of the effects of L-Arg and several
competitive inhibitors of NOS on bacterial phagocytosis in human PMNs.
MATERIALS AND METHODS
Isolation of P M N s from peripheral venous
blood samples
Fresh venous blood drawn into a heparinized tube
from normal volunteers was centrifuged at 200 g for
10 min and the plasma removed. The blood cells were
diluted with 10 ml 1:l Hanks balanced salt solutionl
phosphate-buffered saline (HBSSPBS), poured over 3
ml Histopaque-1077R(Sigma Chemical Co., St. Louis,
MO), and centrifuged a t 400g for 20 min. The aqueous
fraction containing the mononuclear leukocytes was diluted with two volumes of HBSSPBS plus 1.5 ml 6%
dextran in isotonic saline. After 90 min at O"C, the
suspension was centrifuged at 200 g for 10 min and
residual erythrocytes lysed with distilled water. The
PMNs remaining (98% pure by differential staining
and microscopy, 95% viable by trypan blue exclusion)
were resuspended in HBSSPBS counted, and PMN
concentration adjusted to 5 x 106 to 1 x 107/ml.
Test chemicals
L-arginine hydrochloride, Gglycine hydrochloride,
D-arginine hydrochloride, and the NOS inhibitors Lcanavanine (CA), aminoguanidine (AG), and L-p-nitroarginine methyl ester (NAME) were obtained from
Sigma Chemical Co.
27
boiling for 20 min and then labeled with fluorescein
isothiocyanate (FITC) (Sigma) by sitting for 20 h in
the presence of the fluor at room temperature. Final
bacterial concentration was adjusted to 108/ml.
PMNs in phenol red-free RF'MI-1640 (Sigma) (100
pl aliquots) witldwithout supplemental L-Arg, glycine,
and/or NOS inhibitors were put on ice in 1 x 8 strip
ELISA wells (Corning Glass Works, Coming, NY).
FITC-labeled S. aureus (50 pl) was added and samples
incubated in 5% COJ95% air a t 37°C for 2 h. PMNs
were fixed with 30 ~ 1 2 %
paraformaldehyde and extracellular and PMN surface-adherent FITC fluorescence
quenched by addition of 30 pl crystal violet (1 g/L in
0.15M NaC1). Phagocytosis was assayed using an Epics
Profile I1 flow cytometer (Coulter, Hialeah, FL), the
argon laser set to emit an excitation wavelength of 488
nm a t 15 mW operating power, and green (FITC) fluorescence measured between 515 and 560 nm on 50,000
PMNs per sample. PMNs were gated by their forward
and sideways light scatter characteristics, the window
setting for phagocytosis-related fluorescence being between channels 1 and 1,023 (only 5% fluorescence observed in negative controls a t these settings). The data
were stored in list mode and analyzed on a Bernoulli
20 MB disk for percent PMNs fluorescing (PPF) and
mean fluorescence per PMN (MFP). Total PMN fluorescence (TPF) was calculated as the product of PPF
and MFP for each sample.
The results obtained for these three parameters under each experimental condition for each sample were
then standardized, reporting the control result as unity
and test results as the quotient of the raw test result
divided by the raw control result.
Supernatant L-citrulline/NOp measurement
Supematant L-citrulline concentration was assayed
colorimetrically as described by Boyde and Rahmatullah (1980). Specimens were deproteinized and the supernatants collected. A chromogenic solution (5 mg thiosemicarbazide in 50 ml diacetyl monoxime solution (5
mg/ml) and 100 ml acid-ferric solution) was added to
0.1 ml of supernatant; the specimen was mixed and
then boiled for 5 min. After cooling, absorbance a t 530
nm was read on a double beam spectrophotometer. Dilutions of a citrulline standard were run concomitantly.
Nitrite was also measured colorimetrically, as described by Keller et al. (1990a). One milliliter of sample
supernatant was mixed with 100 ~ 1 7 0 %
sulphosalicylic
acid, vortexed for 30 min, centrifuged, and then mixed
with 800 ~ 1 5 %
aqueous NH&l buffer (pH = 9.0),200 pl
10%NaOH, and finally 500 pl modified Griess reagent.
f i r 10 min incubation at 60°C and 5 min a t 4"C,
absorbance a t 546 nm was read. Dilutions of a NaNO,
standard were run simultaneously.
Confocal microscopy
After 2 h incubation with FITC-S. aureus and fixation with 2% paraformaldehyde, PMNs were washed
Flow cytometric assay for PMN phagocytosis
twice and rendered permeable by standing for 30 rnin
This has been described by Cantinieaux et al. (1989). in 90% ethyl alcohol. After washing twice with 0.1%
Briefly, S. aureus 502A incubated for 18 h in trypticase glycine in PBS, mouse anti-ankyrin was added and insoy broth at 37°C was centrifuged (3,000 rpm for 7 min) cubated 30 min. After washing twice with 0.1% glycine
and washed three times in HBSSPBS and the optical in PBS again, rhodamine-conjugated rabbit antimouse
density adjusted to 0.3 a t 650 nm (3-5 x lo8 colony- IgG was added and cells incubated for another 30 min.
forming units per milliliter). Bacteria were killed by After washing twice with PBS for a final time, 5 p1
28
MOFFAT ET AL.
samples of PMNs were mounted on coverslides with 5
pl antifading reagent.
PMNs were viewed by epifluorescent microscopy on
an inverted Nikon microscope equipped with an Arl
Kr laser scanning device (Multiprobe 2001; Molecular
Dynamics, Sunnyvale, CA) with the laser excitation
wavelength set at 488 nm. The confocal laser scanning
images were collected (emission wavelengths 530 nm
for FITC and 590 nm for rhodamine) and recorded at
0.6 nm increments along the z-axis. The image data
were analyzed on a Silicon Graphics computer (Mountain View, CA) using Imagespace (version 3.10) software. Pictures were taken using a 35 mm camera.
Statistical methods
Statistical significance was set at the 0.05 (two-tailed)
level. Repeated measures analysis of variance (ANOVA)
was used to test the effect of increasing concentrations
of supplemental Arg on the parameters being studied.
Student’spaired t-test with the Bonferroni correction was
used for two-group statistical comparisons.
RESULTS
Flow cytometry and supernatant
citrullindnitrite
The effect of Arg on PMN phagocytosis was studied
in PMNs isolated from peripheral blood samples from
12 normal volunteers. PMNs were suspended in RPMI1640 (which contains L-Arg a t a concentration of 95
pM, which is within the range of L-Arg concentration
in human serum), and incubated with FITC-labeled S.
aureus, with and without supplemental concentrations
of L-Arg and/or the NOS inhibitors CA, AG, and NAME.
Supernatant citrulline and nitrite concentrations were
measured. There were no differences in postincubation
PMN viability (by trypan blue exclusion) between any
of the L-Arg or NOS inhibitor-treated cells studied in
these experiments.
Flow cytometry histograms are shown in Figure 1.
FITC fluorescence of PMNs supplemented with L-Arg
380 pM is shifted to the right, with a higher and
broader peak than that for unsupplemented control
PMNs.
The effect of increasing supplemental L-Arg concentrations on PMN phagocytosis is shown in Figure 2A.
L-Arg supplementation resulted in highly significant
(P < 0.001, repeated measures ANOVA) overall increases in PPF, MFP, and TPF. The peak increase was
seen in PMNs supplemented with L-Arg 380 pM (i.e.,
a total L-Arg concentration of 475 pM),the effect being
gradually lost as supplemental L-Arg concentration
was increased further. In two-group comparisons to unsupplemented controls, PPF of PMNs supplemented
with L-Arg 190 and 380 yM was significantly increased.
MFP and TPF of samples incubated with 95,190,380,
760, and 1,520 pM supplemental L-Arg were also augmented significantly. L-Arg 95 pM, the lowest concentration of supplemental L-Arg tested, resulted in a n
11% increase in PPF over unsupplemented controls
(95% confidence interval: 3-19%), a 25% increase in
MFP (6-44%) and a 46% increase in TPF (15-77%).
For L-Arg 380 pM, the concentration giving the maximal enhancement in phagocytosis over unsupplemented controls, the respective increases were as fol-
Fig. 1. Flow cytometry histograms of unsupplemented and L-Arg
380 FM-supplemented PMNs (upper and lower panels, respectively). FITC fluorescence of L-Arg-supplementedPMNs was significantly more intense, as evidenced by an increase in peak height as
well as a shift of the peak to the right.
lows: PPF, 23% (95% confidence interval: 15-31%);
MFP, 33% (12-55%); and TPF, 72% (37-106%).
Extra samples were incubated with 190 or 380 yM
concentrations of the amino acid glycine, employed as
a nutritionallenergy substrate control. There was no
difference in phagocytosis between glycine-supplemented PMNs and unsupplemented controls (data not
shown). The effect of L-Arg was therefore not related
to its nutrient properties.
There was an almost fourfold L-Arg concentration-dependent increase in supernatant citrulline concentration
(Fig. 2B) (repeated measures ANOVA, P < 0.001),suggesting that the effect of L-Arg on PMN phagocytosis may
be mehated through the NOS pathway. L-Arg supplementation above 380 p M resulted in progressive reduction in phagocytosis to control levels while supernatant
citnrlline continued to increase; with excessive NOS-mediated production of NO from L-Arg, the enhancement
in bacterial phagocytosis is lost.
Supernatant nitrite levels did not rise significantly
with increasing concentrations of supplemental L-Arg.
These results were corroborated by two other laboratories, thereby ruling out technical difficulties with the
-
ARGININE AND NEUTROPHIL PHAGOCYTOSIS
29
Fig. 2. The effect of increasing supplemental L-Arg concentration on
PMN bacterial phagocytosis as measured by flow cytometry. A: The
overall effect of L-Arg on percent PMNs fluorescing (PPF),mean fluorescence per PMN (MFP), and total PMN fluorescence (TPF) (the
product of PPF and MFP) was highly significant (repeated measures
ANOVA). B Whereas L-Arg-mediated enhancement of phagocytosis
peaked at 380 pM, supernatant citrulline levels continued to increase
across all tested Arg concentrations ( P < 0.001, repeated measures
ANOVA). Supernatant nitrite concentration was increased only with
3,040
supplemental L-Arg ( P = 0.03 as compared to unsupplemented controls).
assay. A further experiment in which intracellular nitrite was assayed by sonication of PMNs after 2 h
phagocytosis revealed no differences between groups
(data not shown).
The addition of 10 mM CA, AG, and NAME to L-Arg
380 pM-supplemented PMNs abrogated the stimulatory effect of L-Arg on phagocytosis (Fig. 3), suggesting
that augmentation of phagocytic activity by L-Arg supplementation is mediated by the NOS pathway. It is
noteworthy that 10 mM CA, AG, or NAME without
supplemental L-Arg did not significantly impair PPF,
MFP, or TPF as compared to control samples.
Confocal microscopy
Confocal microscopy images of PMNs are shown in
Figure 4. FITC fluorescence was much more intense in
L-Arg-supplemented than in unsupplemented PMNs,
PMNs incubated with D-arginine 380 @
PMNs
I,incubated with NAME alone, or PMNs incubated with both
supplemental L-Arg and NAME. Intracellular FITC
fluorescence of PMNs incubated with NAME or L-Arg
+ NAME was comparable to that of unsupplemented
controls, consistent with the findings on flow cytometry.
DISCUSSION
PMNs generate NO from Arg (Kadota et al., 1991;
Keller et al., 1990b; Kubes et al., 1991; McCall et al.,
1991; Mehta et al., 1990; Mulligan et al., 1991;
Salvemini et al., 1989; Ward and Mulligan, 19911, albeit in lower quantities than macrophages (Kaplan et
al., 1989). PMN NO. production can be stimulated by
-
MOF'FAT ET AL.
30
ways (Schmidt et al., 1989; Ding et al., 1988;Iyengar et
al., 1987).Oxygen radicals and NO are highly reactive
with one another; generation of either tends to blunt
the biological effects of the other, while the use of an
inhibitor of one pathway often results in a larger net
biological effect of the other pathway (McCall et al.,
1991; Schmidt et al., 1989; Wright et al., 1989). This
may explain the observation of Keller et al. (1990b)that
isolated PMNs activated by formylated polypeptides,
lipopolysaccharide, LTBl, and phorbol esters do not
produce nitrite. In some circumstances, NO and oxygen radicals react with one another to form peroxynitrites, which in turn have been implicated in tissue
injury phenomena related to inflammation (Mulligan
et al., 1991; Ward and Mulligan, 1991). Thus, it is apparent that the biological control and net effects of
these two pathways are complicated and subject to
many influences.
As compared to macrophages, less is known about
the relevance of the NOS pathway to immune competence of PMNs, especially in the context of acute bacterial sepsis. Kaplan et al. (1989) observed that PMNs
treated with the NOS inhibitor NG-monomethyl-L-arginine (NMA)show marked inhibition in formylated polypeptide-stimulated chemotaxis which can be reversed
by addition of L-Arg or dibutyryl cyclic guanosine
monophosphate. While NMA did not inhibit microbial
killing, the effect of L-Arg on this function was not
studied. However, that chemotaxis was impaired by
NMA while bacterial killing remained unaffected implies that these two functions are modulated differently
by NOS products. In contrast, Mulligan et al. (1991)
and Ward and Mulligan (1991) reported that NMA did
not inhibit PMN accumulation in the lungs in a rat
model of IgG immune complex-induced acute pulmonary injury, suggesting that chemotaxis in vivo continues to respond to other stimuli, such as C5a.
LArg is known to have other effects on PMN function
and metabolism. In an in vivo superfusion experiment,
Kubes et al. (1991) observed that L-Arg abrogates NOS
inhibitor-induced augmentation of PMN adhesiveness
through effects on the leukocyte adhesion protein
CDllKD18. Lopez Farre et al. (1991) reported that LArg
M in HEPES buffer blocks endothelin- and
formylated polypeptide-induced increases in PMN cytoplasmic calcium, and this effect is reversed by NOS
inhibition. They proposed that L-Arg provides a negaFig. 3. The effect of L-canavanine (CA), aminoguanidine (AG), or tive feedback mechanism for intracellular calcium flux
P-nitroarginine methyl ester (NAME) 10 mM either alone or in the a t this very low concentration.
on PMN bacterial phagocytopresence of supplemental L-Arg 380
The experiments reported herein clearly demonsis. In all cases, NOS inhibitors alone or with L-Argresulted in statistically significant (P < 0.05) reduction of phagocytosis as compared strate that L-Arg supplementation enhances bacterial
to L-Arg 380 bM-supplementedPMNs. There were no significant dif- phagocytosis in PMNs in vitro, even a t the lowest conferences in PPF, MFP, or TPF between NOS inhibitor-supplemented centration tested (95 pM). The concentration of L-Arg
PMNs and unsupplemented controls.
in standard unsupplemented RPMI-1640 tissue culture
medium, 95 pM, is in the middle of the reported ranges
for mean serum L-Arg concentration in normal humans
formylated polypeptides, leukotriene B4 (LTB4), or (54-112 pM) (Alexander et al., 1980; DAndrea et al.,
platelet activating factor (Schmidt et al., 1989). These 1993; Rosenlund, 1993) and surgical patients (50- 125
PMN activators and phorbol esters also stimulate the pM) (Daly et al., 1988; Vente et al., 1989; Yamaguchi
respiratory burst in PMNs, through which radical oxy- et al., 1993). Sigal et al. (1992) showed that in humans
gen intermediates (peroxides, superoxides, etc.) are receiving total parenteral nutrition (TPN) with high Lgenerated (Schmidt et al., 1989; Keller et al., 1990b). Arg supplementation, mean serum L-Arg concentration
to 228
I t 50
Production of NO and oxygen radicals by PMNs and increases from a baseline of 49 5 16 @
macrophages, while often stimulated by the same acti- vM. The supplemental L-Arg concentrations tested in
vators, proceeds by separate and independent path- our study of PMN bacterial phagocytosis bracketed
-
-
ARGININE AND NEUTROPHIL PHAGOCYTOSIS
31
Fig. 4. Confocal microscopy of PMNs under experimental conditions.
Images labeled with unprimed letters show rhodamine fluorescence
only, and those with primed letters show only FITC fluorescence (i.e.,
phagocytosis).A Unsupplemented controls. B: D-arginine 380
C:
L-arginine380
D NAME 10 mM. E Larginine 380 pM + NAME
10 mM. There is no difference in intracellular FITC fluorescence between unsupplemented PMNs and PMNs incubated with D-arginine,
attesting to the stereospecificity of the L-Arg effect on phagocytosis,
as with other immunomodulatory effects of this amino acid. PMNs
supplemented with L-arginine380 pM had much greater intracellular
FITC fluorescence; this effect was abrogated by the addition of 10
mM NAME. Intracellular FITC fluorescence of PMNs incubated with
NAME alone was comparable to that seen in unsupplemented PMNs.
these levels; the stimulative effect of L-Arg on this
PMN function was seen a t L-Arg concentrations well
below those achievable in patients on L-Arg-supplemented TPN.
Supplemental L-Arg-mediated augmentation of
phagocytosisis concentration-dependent to 380 yM,but
with further increases in supplemental L-Arg concentration this functional enhancement is progressively
lost. In contrast, the supernatant concentration of the
NOS metabolite citrulline is maximal at the highest
Arg concentrations. Thus, higher supplemental L-Arg
concentrations appear to result in overproduction of
NOS pathway byproducts, with the result that L-Argmediated augmentation of bacterial phagocytosis is
progressively attenuated and ultimately disappears
with increasing L-Arg concentration. Finally, L-Arginduced augmentation of bacterial phagocytosis is abrogated by addition of NOS inhibitors in molar excess.
Taken together, these findings suggest that augmentation of phagocytosis by L-Arg is mediated through
the NOS pathway and that there is an optimal level of
NO generation above which phagocytic enhancement
is progressively lost.
Albina et al. (1989a,b) and Takema et al. (1991) demonstrated concentration-dependent L-Arg effects, both
positive and negative, on rodent macrophage functions.
Macrophages incubated overnight in medium with deficient or excessive (1.2 mM) L-Arg concentrations manifest reduced phagocytosis, superoxide generation, cytotoxicity, tumoricidal activity, and protein synthesis.
Macrophages incubated in the presence of very high LArg concentrations become nonviable more quickly
than cells incubated in standard medium (Albina et
al., 1989a). These phenomena are due to progressive
impairment of metabolic processes in the macrophages
because of excessive generation of NO in medium containing very high concentrations of L-Arg (Albina et
al., 1989a; Drapier and Hibbs, 1988). The metabolic
impairment in these macrophages is similar to that
induced in tumor cells by NO . Thus, generation of
large quantities of NO * by mononuclear phagocytes is
ultimately deleterious to the function of these same
cells.
Our observations regarding L-Arg supplementation
and PMN phagocytosis are consistent with the findings
of Albina et al. (1989a,b) in rodent macrophages. In our
experiments, high L-Arg concentrations had no apparent effect on PMN viability, probably because of the
short incubation times utilized. However, PMNs incubated with supplemental L-Arg concentrations above
380 pM demonstrated a concentration-dependent loss
of phagocytic enhancement, probably due to interference with PMN metabolic processes by excessive NO
generation. Neither L-Arg 1,520 or 3,040 p M nor the
NOS inhibitors suppressed phagocytosis as compared
to unsupplemented controls. These observations are
analogous to and consistent with those reported by
Kaplan et al. (1989) in a study of PMN chemotaxis.
Whereas supernatant citrulline concentration increased progressively as supplemental L-Arg concentration increased, supernatant nitrite levels changed
only marginally (Fig. 2B). This may be explained by
NO scavenging by oxygen radicals stimulated by the
cell isolation procedure (Keller et al., 1990b; Mulligan
et al., 1991); the resultant peroxynitrites are degraded
to nitrate, which is not measured by the assay.
Both phagocytosis and chemotaxis involve cell movement and therefore interactions with the cytoskeleton.
The mechanism(s) by which L-Arg andor L-Arg-mediated NO production alter cytoskeletal function remains unknown. At least two leukocyte cell membrane
receptors, CD44 (also designated gp 85, Pgp-1, Hermes,
ECMRIII) and CD45 (gp 180, T-200, Ly-5, leukocyte
common antigen), are presently known to relate directly to the cytoskeleton. These receptors are connected to cytoskeletal elements (actin and myosin,
m.
m.
-
-
-
-
-
-
32
MOFFAT ET AL.
(1986) A lymphoma plasma membrane-associated protein with ankyrin-like properties. J. Cell. Biol., 102:2115-2124.
Bourguignon, L.Y.W., Walker, G., and Huang, H.S. (1990) Interactions between a lymphoma membrane-associated guanosine 5'-triphosphate-binding protein and the cytoskeleton during receptor
patching and capping. J. Immunol., 144~2242-2252.
Boyde, T.R.C., and Rahmatullah, M. (1980) Optimization of conditions
for the colorimetric determination of citrulline, using diacetyl monoxime. Anal. Biochem., 107:424-431.
Cantinieaux, B., Hariga, C., Courtoy, P., Hupin, J., and Fondu, P.
(1989) Staphylococcus aureus phagocytosis. A new cytofluorometric
method using FITC and paraformaldehyde. J. Immunol. Methods,
121:203 -208.
Daly, J.M., Reynolds, J., Thom, A., Kinsley, L., Dietrick-Gallagher,
M., Shou, J., and Ruggieri, B. (1988) Immune and metabolic effects
of arginine in the surgical patient. Ann. Surg., 208512-523.
DAndrea, G., Welch, K.M.A., Cananzi, A.R., Zamberlan, F.,Alecci,
M., and Grunfeld, S. (1993) Platelet amino acid profile in control
subjects. Thromb. Res., 70:107-108.
Ding, A.H., Nathan, C.F., and Stuehr, D.J. (1988) Release of reactive
nitrogen intermediates from mouse peritoneal macrophages. J. Immunol., 141:2407 -2412.
Drapier, J.-C., and Hibbs, J.B., Jr. (1988) Differentiation of murine
macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondria1 iron-sulfur enzymes in the macrophage effector cells. J. Immunol.,
140:2829-2838.
Drapier, J.-C., Wietzerbin, J., and Hibbs, J.B., Jr. (1988) Interferongamma and tumor necrosis factor induce the Larginine-dependent
cytotoxic effector mechanism in murine macrophages. Eur. J. Immunol., 18:1587-1592.
Freund, H.R., Ryan, J.A., and Fischer, J.E. (1978) Amino acid derangements in patients with sepsis: Treatment with branched chain
amino acid-rich infusions. Ann. Surg., 188:423-430.
Freund, H.R., Atamian, S., Holroyde, J., and Fischer, J.E. (1979)
Plasma amino acids as predictors of the severity and outcome of
sepsis. Ann. Surg., 190:571-579.
Fukatsu, K, Saito, H., Fukushima, R., Inoue, T., Lin, M.-T., Inaba,
T., and Muto T. (1995) Detrimental effects of a nitric oxide synthase
in a murine sepsis
inhibitor (N-w-nitro-L-arginine-methyl-ester)
model. Arch. Surg., 130:410-414.
Hibbs, J.B., Jr., and Nacy, C.A. (1990)
Green, S.J., Meltzer, M.S.,
ACKNOWLEDGMENTS
Activated macrophages destroy intracellular Leishmania amastigotes by a n Larginine-dependent killing mechanism. J. Immunol.,
This work was supported by American Cancer Soci144:278-283.
ety Clinical Oncology Career Development Award 93- Hibbs, J.B., Jr., Vavrin, Z., and Taintor, R.R. (1987) L-arginine is
required for expression of the activated macrophage effector mecha216 (F.L.M.).
nism causing selective metabolic inhibition in target cells. J. Immunol., 138:550-565.
LITERATURE CITED
Hibbs, J.B., Jr., Taintor, R.R., Vavin, Z., and Rachlin, E.M. (1988)
Nitric oxide: A cytotoxic activated macrophage effector molecule.
Albina, J.E., Caldwell, M.D., Henry, W.L., Jr., and Mills, C.D. (1989a)
Biochem. Biophys. Res. Commun., 157:87-94.
Regulation of macrophage functions by Larginine. J. Exp. Med.,
Iyengar, R., Stuehr, D., and Marletta, M.A. (1987) Macrophage syn169:1021- 1029.
thesis of nitrite, nitrate and N-nitrosamines: Precursors and role
Albina, J.E., Mills, C.D., Henry, W.L., Jr., and Caldwell, M.D. (198913)
of the respiratory burst. Proc. Natl. Acad. Sci. U S A . , 84~6369Regulation of macrophage physiology by L-arginine: Role of the
6373.
oxidative Larginine deiminase pathway. J. Immunol., 143:3641James, S.L., and Glaven, J. (1989) Macrophage cytotoxicity against
3646.
schistosomula of Schistosoma mansoni involves arginine-dependent
Alexander, J.W., MacMillan, B.G., Stinnett, J.D., Ogle, C.K., Bozian,
production of reactive nitrogen intermediates. J. Immunol.,
R.C., Fischer, J.E., Oakes, J.B., Moms, M.J., and Krummel, R.
143:4208-4212.
(1980) Beneficial effects of aggressive protein feeding in severely
Kadota, K, Yui, Y., Hattori, R., Uchizumi, H., and Kawai, C. (1991)
burned children. Ann. Surg., 192:505-517.
A new relaxing factor in supernatant of incubated rat peritoneal
Barbul, A. (1986) Arginine: Biochemistry, physiology and therapeutic
neutrophils. Am. J. Physiol., 260:H967-H972.
implications. JPEN, 10:227-238.
Barbul, A,, Wasserkrug, H.L., and Efron, G. (1980a) Immunostaining Kalomiris, E.L., and Bourguignon, L.Y.W. (1989) Lymphoma protein
kinase C is associated with the transmembrane glycoprotein, GP85,
action of arginine in humans. Surg. Forum, 31r82-84.
and may fundion in GP85-ankyrin binding. J. Biol. Chem.,
Barbul, A., Wasserkrug, H.L., Seifter, E., Rettura, G., Levenson, S.M.,
264:8113-8119.
and Efron, G. (1980b) Immunostimulatory effects of arginine in
Kaplan, S.S., Billiar, T., Curran, R.D., Zdziarski, U.E., Simmons, R.L.,
normal and injured rats. J. Surg. Res., 29:228-235.
and Basford, R.E. (1989) Inhibition of chemotaxis with N"-monoBarbul, A,, Sisto, D.A., Wasserkrug, H.L., and Efron, G. (1981) Argimethyl-Larginine: A role for cyclic GMP. Blood, 74~1885-1887.
nine stimulates lymphocyte immune responses in healthy humans.
Keller, R., Geiges, M., and Keist, R. (1990a) L-arginine-dependent
Surgery, 90:244-251.
reactive nitrogen intermediates as mediators of tumor cell killing
Barbul, A., Fishel, R.S., Shmazu, S., Wasserkrug, H.L., Yoshimura,
by activated macrophages. Cancer Res., 50:1421- 1425.
N.N., Tao. R.C., and Efron, G. (1985) Intravenous hyperalimentation with high arginine levels improves wound healing-and immune Keller, R., Keist, R., Erb, P., Aebischer, T., De Libero, G., Balzer,
M., Groscurth, P., and Keller, H.U. (199Ob) Expression of cellular
function. J. Surg. Res., 38:328-334.
effector functions and production of reactive nitrogen intermediates:
Bourguignon, L.Y.W., Suchard, S.J., Nagpal, M.L., and Glenney, J.R.
A comparative study including T lymphocytes, T-like cells, neutro(1985) A T-lymphoma transmembrane glycoprotein (gp180) is
phil granulocytes, and mononuclear phagocytes. Cell. Immunol.,
linked to the cytoskeletal protein, fodrin. J. Cell. Biol., 101:477131:398-403.
481.
Bourguignon, L.Y.W., Walker, G., Suchard, S.J., and Balazovich, K. Kubes, P., Suzuki, M.,and Granger, D.N. (1991) Nitric oxide: An
chiefly) through the linker proteins fodrin and ankyrin
(Bourguignon et al., 1985, 1986, 1990; Kalomiris and
Bourguignon, 1989; Lokeshwar and Bourguignon,
1992).Whether L-Arg or NO alter expression of these
or other receptors, intervene in receptor-cytoskeleton
interactions, or have a direct effect on cytoskeletal function warrants investigation.
A less likely possibility relates t o the role of argininespecific ADP-ribosyltransferase in ADP-ribosylation of
actin in PMNs, which requires L-Arg as an ADP-ribose
acceptor molecule. ADP-ribosylation of actin may be
important in phagocytosis, secretion, and migration of
PMNs from humans and several animal species (Obara
et al., 1989; Terashima et al., 1992). However, this
mechanism does not appear to involve the NOS effector
pathway. Given that NOS inhibitors abolished the LArg-mediated enhancement of phagocytosis reported
here, it is unlikely that this L-Arg effect is related to
arginine-specific ADP-ribosyltransferase activity.
L-Arg supplementation significantly enhances bacterial phagocytosis in human PMNs. That L-Arg in supraphysiological concentrations augments phagocytic
function is evidence that the observed beneficial effect
of this amino acid in in vivo sepsis models is due to
modulation of the earliest, nonspecific phase of the immune response to bacterial infection. The L-Arg effect
appears to be mediated by NO as L-Arg-mediated enhancement of phagocytosis is abrogated by NOS inhibitors. While it is possible that more than one metabolic
pathway is involved in L-Arg enhancement of phagocytosis, the findings reported here suggest that the NOS
pathway alone may be sufficient to account for the
effect.
-
-
ARGININE AND NEUTROPHIL PHAGOCYTOSIS
endogenous modulator of leukocyte adhesion. Proc. Natl. Acad. Sci.
U.S.A., 88:4651-4655.
Lokeshwar, V.B., and Bourguignon, L.Y.W. (1992)Tyrosine phosphatase activity of lymphoma CD45 (gp180) is regulated by a direct
interaction with the cytoskeleton. J . Biol. Chem., 267:21551-21557.
Lopez Farre, A, Riesco, A, Moliz, M., Egido, J., Casado, S., Hernando,
L., and Caramelo, C. (1991) Inhibition by L-arginine of the endothelin-mediated increase in cytosolic calcium in human neutrophils.
Biochem. Biophys. Res. Commun., 178:884-891.
Madden, H.P., Breslin, R.J., Wasserkrug, H.L., Efron, G., and Barbul,
A. (1988)Stimulation of T cell immunity by arginine enhances survival in peritonitis. J. Surg. Res., 44.558-663.
McCall, T.B., Boughton-Smith, N.K., Palmer, R.M.J., Whittle, B.J.R.,
and Moncada, S. (1989) Synthesis of nitric oxide fiom L-arginine
by neutrophils. Biochem. J., 261:293-296.
McCall, T.B., Feelisch, M., Palmer, R.M.J., and Moncada, S. (1991)
Identification of N-iminoethyl-L-ornithineas a n irreversible inhibitor of nitric oxide synthase in phagocytic cells. Br. J. Pharmacol.,
102:234-238.
Mehta, J.L., Lawson, D.L., Nicolini, F A , Cain, D.A., Mehta, P., and
Schreier, H. (1990) Evidence for generation of a large amount of
nitric oxide-like vascular smooth muscle relaxant by cholesterolrich neutrophils. Biochem. Biophys. Res. Commun., 173:438-442.
Mulligan, M.S., Hevel, J.M., Marletta, M.A., and Ward, P.A. (1991)
Tissue injury caused by deposition of immune complexes is L-arginine dependent. Proc. Natl. Acad. Sci. U S A , 88.6338-6342.
Obara, S., Mishima, K., Yamada, K., Taniguchi, M., and Shimoyama,
M. (1989)DNA-regulated arginine-specific mono(ADP-ribosy1)ation
and de-ADP-ribosylation of endogenous acceptor proteins in human
neutrophils. Biochem. Biophys. Res. Commun., 163:452-457.
Reynolds, J.V., Thom, AK, Zhang, S.M., Ziegler, M.M., Naji, A., and
Daly, J.M. (1988) Arginine, protein malnutrition and cancer. J.
SWg. Res., 45.513-522.
Reynolds, J.V., Daly, J.M., Shou, J., Sigal, R.K., Ziegler, M.M., and
Naji, A. (1990)Immunologic effects of arginine supplementation in
tumor-bearing and non-tumor-bearing hosts. Ann. Surg., 211:202210.
Rosenlund, B.L. (1993)Effects of insulin on free amino acids in plasma
and the role of amino acid metabolism in the etiology of diabetic
microangiopathy. Biochem. Med. Metab. Biol., 49:375-391.
Salvemini, D., De Nucci, G., Gryglewski, R.J., and Vane, J.R. (1989)
33
Human neutrophils and mononuclear cells inhibit platelet aggregation by releasing a nitric oxide-like factor. Proc. Natl. Acad. Sci.
U.S.A., 86r6328-6332.
Schmidt, H.H.H.W., Seifert, R., and Bohme, E. (1989)Formation and
release of nitric oxide from human neutrophils and HL-60 cells by
a chemotactic peptide, platelet activating factor and leukotriene Bq.
FEBS Lett., 244:357-360.
Sigal, R.K., Shou, J., and Daly, J.M. (1992)Parenteral arginine infusion in humans: Nutrient substrate or pharmacologic agent? JPEN
16:423-428.
Tachibana, K., Mukai, K., Hiraoka, I., Moriguchi, S., Takama, S.,
and Kishino, Y. (1985) Evaluation of the effect of arginine-enriched
amino acid solution on tumor growth. JPEN, 9:428-434.
Takeda, Y., Tominaga, T., Tei, N., Kitamura, M., Taga, S., Murase,
J., Taguchi, T., and Miwatani, T. (1975) Inhibitoly effect of L-arginine on growth of rat mammary tumors induced by 7,12-dimethylbenz(a)anthracene. Cancer Res., 352390-2393.
Takema, M., Inaba, K., Uno, K, Kakihara, K.-I., Tawara, K., and
Muramatsu, S. (1991)Effect of L-arginine on the retention of macrophage tumoricidal activity. J. Immunol., 146:1928- 1933.
Terashima, M., Mishima, K., Yamada, K., Torichiya, M., Wakutani,
T., and Shimoyama, M. (1992) ADP-ribosylation of actins by arginine-specific ADP-ribosyltransferase purified from chicken heterophils. Eur. J . Biochem., 204:305-311.
Vente, J.P., Von Meyenfeldt, M.F., Van Eijk, H.M.H.,
Van Berlo,
C.L.H.,Gouma, D.J., Van der Linden, C.J., and Soeters, P.B. (1989)
Plasma-amino acid profiles in sepsis and stress. Ann. Surg., 2095762.
Ward, P.A., and Mulligan, M.S. (1991) New insights into mechanism
Klin. Woof oxyradical and neutrophil mediated lung- injury.
.
chenschr., 69:1009-1011.
Wright. C.D.. Miilsch. A.. Busse, R.. and Osswald. H. (1989) Generation of nitric oxide by human neutrophils. Biochem. Biophys. Res.
Commun., 160:813-819.
Yamaguchi, T., Shimahara, Y., Takada, Y. (1993)Plasma amino acid
profile in relation to arterial ketone body ratio in surgical patients.
Circ. Shock, 40:258-263.
Ye, S.-L., Istfan, N.W., Driseoll, D.F., and Bistrian, B.R. (1992)Tumor
and host response to arginine and branched chain amino acid-enriched total parenteral nutrition. A study involving Walker 256
carcinosarcoma-bearing rats. Cancer, 69:261-270.