[CANCER RESEARCH 47, 5590-5594, November 1, 1987]
Synthesis of Nitrite and Nitrate in Murine Macrophage Cell Lines1
Dennis J. Stuchr and Michael A. Marietta3
Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02140
ABSTRACT
Synthesis of nitrite (NCV) and nitrate (NOD was studied in the
macrophage cell lines RAW 264.7, WEHI-3, PU5-1.8, J774A.1, and
P388D1 and compared to the synthesis by thioglycolate-elicited perito
neal macrophages from ('31 I/I le and C3H/HeJ mice. Treatment with
Escherichia coli lipopolysaccharide (LPS) induced NO, /NO, synthesis
by all the cell lines except P388D1, which remained unresponsive at the
highest LPS concentration (SO nK/nil). Recombinant murine -y-interferon
induced NOr/NOr synthesis in only two cell lines (PU5-1.8 and RAW
264.7), although it activated synthesis by ( 31 I/I Io and C3H/HeJ mac
rophages. Dual signal treatments consisting of lymphokines or •y-interferon plus LPS stimulated NO> /NO> synthesis by all five cell lines and
each line showed enhanced synthesis as compared to that induced by any
single stimulus. Heat-killed Bacillus Calmette-Guerin and purified mycobacterial protein derivative stimulated NO, /NO, synthesis in three
of five cell lines, while dextran sulfate, zymosan, and the synthetic
adjuvant muramyl dipeptide were ineffective. Nitrite represented 50-75%
of the total NO. /NO, produced in all cases. The kinetics of LPSinduced NO..7NO, synthesis in J774A.1 and C3H/He macrophages
were identical; a 6-h lag phase was followed by a 24- to 48-h period in
which NO, and NO, were in a ratio of approximately 3:2 at all time
points.
served (9). Precursor-product studies with macrophage cultures
clearly show the precursor to NO2~ and NO3~ is i arginine
(10). Furthermore, the NO2~/NO3~ nitrogens are exclusively
derived from the terminal guanido-nitrogens of arginine (10).
The whole animal experiments reporting ammonia as the pre
cursor (6, 7) can now be interpreted as ammonia incorporation
into glutamine or directly into carbamoyl phosphate which is
required for the biosynthesis of arginine. Saul and Archer (7)
did not see incorporation from [l5N]glutamate but the difficul
ties of competing pathways and dilution were probably respon
sible for this negative result, as glutamate via transamination
produces aspartate that in turn provides a nitrogen for arginine
biosynthesis. It is entirely possible that the basal and immuno
stimulated NO2~/NO3~ synthesis are both macrophage proc
esses where the basal reaction is due to resident tissue macro
phages.
Further experiments have shown that stimulated macro
phages also synthesize yV-nitrosamines when secondary amines
are added to the cell culture medium (11). The amines must be
present while the macrophages are actively engaged in NO2~/
NOr synthesis and simply adding NO2~ to nonstimulated cells
does not yield nitrosamines (11). Thus this process may be a
previously unrecognized contributing factor in nitrosamine carcinogenesis. Additionally, the NO2~/NO3~ synthesized may be
INTRODUCTION
The association between exposure to high levels of nitrate
and gastric cancer (1-3), and the potential for the endogenous
formation of A'-nitrosamines by reaction of nitrite derived from
nitrate by bacterial reduction with endogenous or exogenous
amines, has led to studies on the sources of nitrate exposure as
well as on the chemistry of the nitrosation reaction. Exposure
to nitrate is, at least in part, environmental. However, recent
studies show that mammals maintained on a low nitrate diet
excrete more nitrate than they ingest (4, 5) and when rats were
treated with Escherichia coli LPS4 a 10-fold increase in urinary
nitrate resulted (6). These results show that there is a basal
level of N( ) f synthesis and that the synthesis can be enhanced
by stimulation of the immune system. The relationship of the
basal synthesis to that in immunostimulated animals is un
known. Studies by Wagner et al. (6) and Saul and Archer (7)
showed that ammonia could serve as a precursor to nitrate in
the basal synthesis. In addition, Wagner et al. (6) showed that
ammonia was a precursor in the LPS-induced synthesis of
nitrate.
Our previous work established that primary murine macro
phage cultures, when stimulated with E. coli LPS and lympho
kines such as IFN-7 synthesize both NO2~ and NO3~ (8, 9). A
lag time, dependent on the stimulant used has also been obReceived 4/6/87; revised 7/17/87; accepted 7/24/87
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by USPHS Grant CA26731 awarded by the Department of Health
and Human Services, National Cancer Institute.
2 Present address: Department of Medicine, Cornell University Medical Cen
ter, New York, NY 10021.
'To whom requests for reprints should be addressed, at The University of
Michigan, College of Pharmacy, 428 Church St., Ann Arbor, MI 48109.
4 The abbreviations used are: LPS, lipopolysaccharide; SMEM, supplemented
minimal Eagle's medium; BCG, Bacillus Calmette-Guerin; hk BCG, heat-killed
Bacillus Calmette-Guerin; IFN--y, r-interferon; DS, dextran sulfate; PPD, purified
protein derivative; Ik, lymphokines; MDP, yV-acetylmuramyl-L-alanyl-o-isoglu-
involved in effector functions of activated macrophages or may
represent by-products of a pathway crucial to the activation of
these cells for bactericidal and tumoricidal activity. NO2~ is
toxic to bacteria (12), and the time course of NO2~/NO3"
production during BCG infection in mice closely parallels the
acquisition of increased nonspecific bacterial resistance (8).
Two recent reports by Hibbs et al. (13, 14) suggest a role for
NO2~/NO3~ synthesis in mediating toxicity to neoplastic cells
during coculture with activated macrophages.
Many questions remain regarding NO2~/NO;T synthesis and
the role of this synthesis in the tumoricidal activity of macro
phages. Studies addressing the various aspects of macrophage
biology have been aided by the availability of well-characterized
macrophage tumor cell lines (15). We report here our studies
of NO2~/NO;f synthesis by several macrophage cell lines. A
comparison with results obtained by using primary murine
macrophages shows that macrophage cell lines are useful
models to further study the biochemistry of NO2"/NO3~ syn
thesis.
MATERIALS AND METHODS
Cell Culture Media. Powdered Eagle's minimal essential medium
without phenol red (Flow Laboratories) was supplemented with sodium
pyruvate (110 mg/liter); glucose (3.5 g/liter); glutamine (584 mg/liter);
penicillin (50 units/ml); streptomycin (50 Mg/ml); 15 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid; and 10% HyClone low endotoxin defined calf serum (HyClone Laboratories, Logan, UT), final pH
7.3-7.4 (SMEM). RPMI 1640 could not be used due to its high NCV
content (from calcium nitrate). All supplements were cell culture grade
and were obtained from Sigma Chemical Co, St. Louis, MO, unless
otherwise noted. SMEM was prepared with distilled deionized water
which was endotoxin-free by the Limulus amebocyte assay (detection
limit, 0.025 ng/ml; Sigma) and was stored or transferred in either
nonpyrogenic plastic labware or heat-treated endotoxin-free glassware.
5590
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MACROPHAGE
SYNTHESIS
OF NITRITE
AND NITRATE
LPS and BCG. E. coli LPS (serotype, 0.127: B8; Sigma) was diluted
in saline to a concentration of 250 «ig/mland stored at -10°C until
use. BCG (American Type Culture Collection 19274) was grown in
On bos medium (16). Twelve- to 14-day cultures were sedimented,
resuspended in 95% saline-5% dimethyl sulfoxide, and stored frozen at
-80°C. To prepare hk BCG, frozen BCG were thawed, centrifuged,
resuspended at 1.0 x 10" per ml in Hanks' buffered saline solution, and
incubated in a water bath at 60°t'for 1.5 h.
-v-Interferon. Murine recombinant IFN-7 was a generous gift of
Genentech, Inc. (San Francisco, CA). This preparation was determined
by Genentech to have a specific activity of 1.97 x 107units/ml (antiviral
units) and a concentration of 1.5 mg/ml. A stock solution of 1 x IO4
units/ml in culture medium was stored at 4*C and diluted as needed.
Other Activating Agents. DS (M, 500,000) and zymosan A were
obtained from Sigma; M DP was from Behring Diagnostics; and PPD
was from Connaught Laboratories (Toronto, Ontario, Canada). Stock
solutions (25x) of these compounds were prepared in SMEM (PPD)
or saline and sterile filtered (0.2 urn] (PPD), or autoclaved (zymosan
and DS) and stored at 4*C prior to use.
Lymphokines. Spleen supernatants containing LK were prepared
from the spleens of C3H/He or C3H/HeJ mice that had been infected
with BCG 9 to 12 days previously (9). LK solutions contained signifi
cant levels of nitrite and nitrate (approximately 100-200 /¿M)
and were
dialyzed (Spectrapor dialysis tubing, cutoff M, 6000-8000) against 10
to 20 volumes of serum-free SMEM. Dialysis brought nitrite/nitrate
levels down to less than 20 pM. LK solutions were sterilized by filtration
(0.2 firn; Waters Associates) and stored in aliquots of 1 to 4 ml at
-80'C.
100-
50
LPS (/ig/ml)
Fig. I. NO2~/NOs~ synthesis by macrophage cell lines as a function of LPS
concentration. RAW 264.7 (A), WEHI-3 (•),PU5-1.8 (D), and P388DI (A).
Macrophages ( 1 x 10*)were cultured for 72 h in 1 ml medium containing various
amounts of LPS. Values are the final levels of M >. plus N( >< in the medium at
72 h and represent the mean for three cultures ±SD. The background concentra
tion of NO2~/NO3" in the medium ranged between 10 and 15 x 10"* M.
Source and Culture of Primary Macrophages and Macrophage Cell
Lines. Thioglycolate-elicited peritoneal macrophages were obtained
from C3H/He and C3H/HeJ mice and cultured as described previously
(8). The macrophage cell lines J774A.1, P388D1, RAW 264.7, PU51.8, and WEHI-3 were obtained from the American Type Culture
Collection. The cell lines were certified to be free of Mycoplasma at the
time of purchase. Macrophage cell lines were grown on 100-mm plastic
tissue culture dishes in high glucose Dulbecco's modified Eagle's me
dium (M. A. Bioproducts) supplemented with 10% calf serum and 4
IHMglutamine. Culture medium for PU5-1.8 and P388D1 cell lines
was also supplemented with nonessential amino acids (Gibco). Cells
were passaged every 3-6 days by diluting a suspension of the cells 1:10
in fresh medium. For experiments, the cells were removed from the
culture dishes by vigorous pipeting, and were centrifuged and resus
pended in SMEM to a concentration of 1 x 10" cells/ml. Cells were
plated at 1 ml/well in 24-well culture plates (Costar), allowed to adhere
for 2 h, and thereafter the medium was replaced with fresh SMEM
containing the various agents.
Sample Collection and Measurement of Nitrite and Nitrate. In all
cases, the supernatants were collected after the appropriate incubation
period and stored at -10°C until analysis. Nitrate and nitrite were
measured by an automated procedure (17). Briefly, samples were passed
through a column containing copper-plated cadmium filings, thereby
reducing sample NO.i to M):~. The sample then undergoes reaction
with the G r¡essreagent (1% sulfanilamide/0.1% naphthylethylenediamine dihydrochloride/4.3% H.,PO4) to form a chromophore absorbing
at 543 nm. NO2" was measured independently of NO3~ by bypassing
the cadmium column.
Assay of Cell Viability. Cell viabilities in several viewing fields were
estimated by using trypan blue dye exclusion (0.4% in saline).
RESULTS
LPS-induced NO2~/NO3~ Synthesis. Fig. 1 shows NO2"/
NO3~ production by the macrophage cell lines PU5-1.8, WEHI3, RAW 264.7, and P388D1 following 72 h of culture with
different concentrations of LPS. LPS induced NO2"/NO3"
synthesis by the cell lines PU5-1.8, WEHI-3, and RAW 264.7,
but not by P388D1. The dose-response curve for the three
responding cell lines shows synthesis was stimulated by 1 ng/
ml LPS and began to plateau at >1 ¿ig/ml.In contrast, the
Fig. 2. Kinetics of LPS-induced NOr/NOr
synthesis by C3H/He macro
phages and the J774A. 1 cell line. A, Oliilimacrophages (l.0 x IO6cells) were
preincubated for 4 h at 37*C prior to addition of medium containing LPS (10
Mg/ml) at the time 0. Points, average level of NO2~ (O) and NOr (A) ±SD in
the culture supernatant at each time point. The No; 'NO, concentration in the
control cultures (•)is presented as the sum of the NO. and NO3~ amount at
each time point. B, J774A.1 cells (1.0 x 10*/well) were preincubated for 4 h at
37'C prior to addition of medium containing LPS (25 »jg/ml)at time 0. Points,
mean level of NO2" (O) and NO3~ (A) present in four culture supernatants ±SD.
The data for controls (•:cultures not receiving LPS) represent the background
levels of NOj" and NO3" in the supernatants at each time point.
P388D1 cell line did not synthesize NCV/NCV even at LPS
concentrations of 50 Mg/ml. None of the cell lines produced
NOr/NOr
spontaneously (not shown). The higher doses of
LPS (1 and 50 Mg/ml) were toxic, viabilities at 72 h ranged
from 10% viable for RAW 264.7, 30-50% for WEHI-3, and
40-70% for PU5-1.8 compared to 85-95% for control cultures.
P388D1 remained 95% viable regardless of the LPS concentra
tion. Nitrite represented approximately 50-75% of the total
NO2~/NO3~ produced in all cases (not shown).
Kinetics of NO2~/NO3~ Synthesis. Fig. 2 depicts the kinetics
of NO2~ and NO3~ synthesis in response to LPS for thioglycolate-elicited macrophages from C3H/He mice (Fig. 2A) and the
J774A.1 macrophage cell line (Fig. 2B). In both cases, there
was a lag phase of about 6 h followed by a near linear increase
in NO2~ and NO3~ for 48 h, after which synthesis decreased
and plateaued between 48 and 72 h. Nitrite represented ap
proximately 60% of the total NO2~ plus NO3~ present at all
time points for both the J774A.1 cell line and C3H/He mac
rophages. As shown in Fig. 2, LPS-treated C3H/He macro
phages routinely produced more NO2~/NOr per cell than
identically treated J774A.1 macrophages.
IFN--,-stimulated NO2~/NO3~ Synthesis. NOr/NOr
synthe-
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MACROPHAGE
SYNTHESIS
sis by the five macrophage cell lines following a 72-h culture
with different concentrations of IFN-7 is shown in Fig. 3. Only
the PU5-1.8 and RAW 264.7 cell lines generated NOr/NOr
in response to IFN-7. PU5-1.8 and RAW 264.7 produced
NO2-/NOr at IFN-7 concentrations ranging from 10 to 10,000
units/ml. An IFN-7 concentration of 100 units/ml appeared
optimal for PU5-1.8, while RAW 264.7 response was maximal
at the lowest concentration tested (10 units/ml). The cell lines
WEHI-3, J774A. 1, and P388D1 did not synthesize N<V/NO3~
at the highest IFN-7 concentration tested (10,000 units/ml).
Nitrite represented approximately 60% of the total NO2~/NO3~
produced in all cases (not shown).
Enhancement of NO2~/NO3~ Synthesis by IFN-7 or LK. Table
1 lists NO2~/NO3~ production by the five macrophage cell lines
in response to dual activation stimuli (IFN-7 or LK plus LPS)
and that induced by LK treatment alone. Concurrent treatments
with either IFN-7 or LK plus LPS stimulated NO2~/NO3"
production by all five macrophage cell lines. Each cell line
showed enhanced NO2~/NO3~ synthesis in response to dual
signal treatments over that induced by LPS, IFN-7, or LK
alone. The RAW 264.7 cell line produced the greatest amounts
of NOr/NO3~ when cultured with IFN-7 plus LPS, while the
OF NITRITE
AND NITRATE
cell line P388D1, which did not respond to either IFN-7 or
LPS alone, synthesized the least. LK-treated J774A.1 and
P388D1 macrophages produced NO2~/NO3~, although both
were unresponsive to IFN-7 (Fig- 3). NO2 represented approx
imately 50 to 75% of the total NOr/NOr
synthesized in all
cases (Table 1).
NO2~/NO3~ Synthesis by Macrophage Cell Lines Treated with
Heat-killed BCG, PPD, MDP, Zymosan, and DS. Studies have
shown that hk BCG as well as the mycobacterial components
PPD and MDP can activate macrophage cell lines (18, 19).
Also, yeast particles (zymosan) and polyanionic compounds
(DS) have been used effectively as activating agents (19). Table
2 shows NOr/NOr
production by the five macrophage cell
lines following treatment with these agents. Heat-killed BCG
and PPD stimulated NOr/N(V
synthesis by the PU5-1.8,
RAW 264.7, and WEHI-3 cell lines, but not by J774A.1 or
P388D1. PPD also induced synthesis by the three lines that
responded to hk BCG. In contrast, MDP did not stimulate
NO2~/NO3~ synthesis by any of the cell lines except RAW
264.7, where it caused a small amount of NO2~/NO3~ produc
tion at the highest concentration tested (100 Mg/ml). Zymosan
in general was not a stimulus of NO2"/NO3~ synthesis, although
RAW 264.7 cells treated with zymosan (250 Mg/ml) generated
a small amount of NCV/NOr.
DS (10-500 /ig/ml) did not
induce synthesis by any of the cell lines (not shown). In separate
experiments, identical concentrations of MDP, zymosan, and
DS were incapable of triggering NO2~/NO3~ synthesis by either
100-
C3H/He or C3H/HeJ macrophages (not shown).
Comparison between Macrophage Cell Lines and Murine Per
itoneal Macrophages. Table 3 summarizes the results obtained
with the murine macrophage cell lines in terms of their NO2~/
NO3~ production, and compares them to results obtained using
thioglycolate-elicited C3H/He and C3H/HeJ primary mouse
macrophages. Multisignal activation treatments (IFN-7 or LK
plus LPS) induced NO2"/NO3~ synthesis by both primary mac
O
IO
IOO
IOOO I »IO4
r-IFN (U/ml)
Fig. 3. NO2"/NO3" synthesis by macrophage cell lines as a function of IFN--x
concentration. RAW 264.7 (A), WEHI-3 (•),PUS-1.8 (D), J774A.1 (O), and
P388D1 (A). Macrophages (1 x IO6)cultured for 72 h in 1 ml medium containing
various amounts of IFN-% Points, final levels of NOr plus NOr in the medium
at 72 h and represent the mean for three cultures ±SD. The background
concentration of NO2" plus NO3" in the medium ranged between 10 and 15 x
10-* M.
Table 1 NOi~/NO,~ synthesis by macrophage cell lines treated with lymphokines,
lymphokines plus LPS or lymphokines plus IFN-y
Macrophages (I x Ml'' nil) were cultured for 72 h in medium containing LK
(1:3), LK plus LPS (1 xg/ml), or IFN-y (500 units/ml) plus LPS (1 Mg/ml).
NOz'/NOs" values are the mean of triplicate cultures ±SD. Numbers in paren
theses, NOr levels (nmol/10* cells) at 72 h.
cells)PU5-1.8RAW
NOr + NO3- (nmol/10«
LPS154
+
±6
10)261
(95 ±
264WEHI-3J774P388D1LK
±10
(130
±7)144
±2
2)104
(87 ±
±5
(69
15)63
±
4)•
ND, not determined.
±4
(34 ±3)IFN-y
rophages and the cell lines. LPS alone was also an effective
stimulus for both primary and transformed macrophages, with
the exception of the P388D1 cell line and the LPS-insensitive
C3H/HeJ macrophages. From here the responses of the cell
lines diverge from that of primary C3H/He and C3H/HeJ
macrophages, with only three of five cell lines producing NO2~/
Table 2 NOi~/NOi~ production by macrophage cell lines treated with hk BCG,
PPD, MDP, and zymosan
Macrophages were cultured at I x lOVml for 72 h with the various stimulants.
Concentration units are bacteria per ml for hk BCG and u^ ml for PPD, MDP,
and zymosan. Values are the mean for triplicate cultures ±SD. In some cases,
nmol NOr produced is also reported (shown below the NO2~ plus NO3~ value).
cells)hk
26468
)PPD
BCG (ml '
±7
4)208
(50 ±
±17
(113±8)104
(5±D14
±5
(57
4)29±
±3
ND33
±6
(19 ±4)LK106
kg/ml)MDP
±9
(595)9±2
±
±7
(75
4)75±
±527
±714
149±
LPS77
+
±16
ND°106
NOr + NO," (nmol/ 10"
Gig/ml)Zymosan
±248
111±
±427
±42±596 ±53±921
1Oil0±63±43±81Â
±
±637
±551
110±
±526
±490
1019±90±70±
±
1049
±
±73±36±
±556
1634
±
±817±60±81
10±20±29±61
12±1NT3±20±81
±615±26±41
(/¿g/ml)10'10*10»100so1010010125010010PU5-1.847
±211
±35±8RAW
±63±2
±2WEHI-330
±6J7740±20±30±20±10±30Â
* NT, not tested.
±4
(22 ±
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MACROPHAGE SYNTHESIS OF NITRITE AND NITRATE
Table 3 Summary ofNO2~/NO}~productionby macrophagecell linesand murineperitonealmacrophagesin responseto the variousactivatingtreatments
Macrophage cell lines
Activating treatments"
PU5-1.8
+*IFN-y
LPS
+LK
+IFN--X
+LK + LPS
+hk + LPS
+PPD
BCG
+MDPZymosan
Murine peritoneal macrophages
RAW 264
WEHI-3
J774
++
+
+
-
-
++
++
++
++
NT±
NT±
_—
+
+
+
+
±
+
+
—
-
±
+
+
—
-
++
-
P388D1
C3H/He
C3H/HeJ
_
_
_
—
—
—
—_+++++NT—-" Highest doses: LPS, 50 Mg/ml; IFN-f, IO4 units/ml; LK, 1:3 dilution, hk BCG, 107/ml; PPD, 100 ng/ml; MDP, 100 ^g/ml; DS, 500 Mg/ml; zymosan, 250 (ig/
-DS+
* +, >20 nmol NO2~/NO3~ produced per 10s cells; ±,10-20 nmol/106 cells; -, <10 nmol at the highest dose tested; NT, not tested.
NOr in response to hk BCG, and only two of five in response
to IFN-T; although both of these treatments caused NO2~/
NOi synthesis in primary macrophages. Agents that did not
stimulate NOr/NO3"
synthesis in either C3H/He or C3H/
HeJ macrophages (MDP, zymosan, and DS) also caused little
or no synthesis by the macrophage cell lines.
DISCUSSION
This report establishes the use of murine macrophage cell
lines as models to study macrophage synthesis of NO2"/NO3~.
Several macrophage cell lines established by different means
(15) were induced to synthesize NO2~/NO3~ by cytokines and
bacterial stimulants that were found effective with primary
murine macrophages (8, 9). These results show NO2~/NO3~
synthesis to be a general property of murine macrophages
following activation.
LPS-induced NO2~/NO3~ synthesis by the macrophage cell
lines was identical to that observed by using primary C3H/He
macrophages in every respect, including the dose-response,
kinetics, and NO2~/NO:T ratio. Indeed, the products NO2~ and
NO3" were present in an approximate 3/2 ratio irrespective of
the inducing agent, cell line, or incubation time. These results
are identical to those obtained using activated primary mouse
macrophages (20). Furthermore, we have found that NO2~ or
NO, added to unstimulated macrophage cultures are stable.
The addition of either aniónto activated macrophages does not
perturb this ratio (9). In other words the 3/2 ratio plus either
added anión makes up the total NO2~/NO3~ found at the end
of the experiment (9) with no equilibration of the added anión.
This implies a common mechanism of synthesis and suggests
that NO3~ does not arise via oxidation of preformed NO2~, but
rather is formed concurrently along with NO2~ in a specific
ratio.
LPS did not induce NO2~/NO3" synthesis by the P388D1
cell line, consistent with reports showing the P388D1 macro
phages are resistant to the cytostatic and cytotoxic effects of
LPS (18). Although the P388D1 response to LPS was phenotypically similar to that of LPS-resistant primary C3H/HeJ
macrophages (21), it probably reflects a distinct defect. For
example, LPS treatment induces interleukin 1 production by
P388D1 but not C3H/HeJ macrophages (22). Also, the
P388D1 cell line was established from the DBA/2 murine
strain, which is LPS responsive (23, 24). P388D1 macrophages
did not synthesize NO2~/NO3~ in response to any of the single
stimuli. This suggests that they are generally unable to be
activated by single signals for NO2~/NO3~ synthesis.
IFN-7 is capable of stimulating NO2~/NO3~ synthesis in
primary C3H/He and C3H/HeJ
macrophages (9), but induced
NO2 /NO3 synthesis in only two of five cell lines. This differ
ence may be related to the report that macrophage cell lines
express fewer receptors for IFN-7 than primary macrophages
(25). However, IFN-7 has been shown to increase the expres
sion of la antigen on WEHI-3 and P388D1 macrophages (26,
27), indicating that IFN-7 may still induce responses other than
NO2"/NO3~ synthesis in the cell lines. Interestingly, concurrent
treatment with IFN-7 plus LPS caused NO2"/NO3~ synthesis
by all five cell lines, including P388D1, which was unresponsive
to either signal alone. This synergy between IFN-7 and LPS is
similar to that reported for induction of cytolytic capability in
primary macrophages (28, 29) as well as for NO2~/NO3~ syn
thesis by primary C3H/HeJ macrophages (9). LK treatment
also induced NO2~/NO3~ synthesis in two IFN-7-unresponsive
macrophage lines (WEHI-3 and J774A.1), supporting reports
that additional activating factors are present along with IFN-7
in LK preparations (30, 31).
Heat-killed BCG as well as the mycobacterial protein deriv
ative PPD stimulated NO2"/NO3" synthesis in three of five cell
lines. Interestingly, WEHI-3 was among the responsive
while two lines thought to represent more functionally
macrophages (J774A.1 and P388D1) were not. The
capacity of hk BCG and PPD to stimulate NO2"/NO3"
group,
mature
similar
synthe
sis suggests that PPD may be the stimulatory principle in hk
BCG. Surprisingly, the synthetic mycobacterial peptide frag
ment MDP was unable to induce NO2~/NO3~ synthesis in any
of the primary or transformed macrophages, although MDP is
known to augment antibody-independent tumorilytic capacity
of several macrophage cell lines (32), as well as prime macro
phages for enhanced Superoxide production (33). Zymosan and
dextran sulfate did not induce NO2"/NO3~ synthesis, although
these agents are reported to cause cytostasis in macrophage cell
lines (18), and trigger secretion of colony-stimulating factors,
prostaglandin E, and a-interferon (34-36).
While a functional role for NO2"/NO3~ synthesis in the
biology of activated macrophages has not yet been established,
the process has direct bearing on nitrosamine-induced carcinogenesis. Nitrosamines can be endogenously synthesized by the
acid-catalyzed chemical reaction of secondary amines with
NO2~-derived nitrosating agents (37). Because of the catalysis
by acid, endogenous formation of ./V-nitroso compounds has
always been considered to occur in the stomach; however, the
lysosomes of macrophages are also acidic (38). Therefore, macrophage-derived NO2~ could participate in the endogenous
formation of JV-nitrosamines by reaction with secondary amines
in the stomach or possibly through a similar reaction within
macrophage lysosomes. In fact, we have found immunostimulated macrophages in culture carry out ,/V-nitrosation reactions
on a variety of secondary amines added to the culture medium
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MACROPHAGE
SYNTHESIS
OF NITRITE
(11). Interestingly, /V-nitrosation did not occur in cultures of
unstimulated macrophages that had NO2~ and the amine added,
indicating that jV-nitrosation was a property of cells actively
engaged in NO2~/NO3~ synthesis. This implies that the simple
acid-catalyzed reaction of the amine and NO2~ is not the source
of macrophage-synthesized nitrosamines but that they are in
stead derived from the reaction of the amine with a precursor
to NO2~. In either case this process, if active in vivo, could
potentially generate carcinogenic ./V-nitroso compounds during
immunostimulation and may, therefore, contribute to the carcinogenesis associated with chronic inflammatory conditions
(39).
The finding that macrophage cell lines can be activated for
NO2"/NOj~ synthesis has provided a convenient system to
investigate the mechanisms of biosynthesis of these molecules,
as well as providing a source of pure, homogeneous macro
phages for protein purification studies. Additionally, the asso
ciation between macrophage NO2"/NO3~ synthesis and nitrosamine formation indicates that macrophage cell lines will be
valuable for investigating the role of endogenous NO2"/NO3~
synthesis in nitrosamine carcinogenesis.
ACKNOWLEDGMENTS
We are grateful to Dr. Radha lyengar for helpful discussions and
comments on the manuscript.
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Synthesis of Nitrite and Nitrate in Murine Macrophage Cell Lines
Dennis J. Stuehr and Michael A. Marletta
Cancer Res 1987;47:5590-5594.
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