Pharmacological Research, Vol. 37, No.

3, 1998

COMPARISON OF NITRIC OXIDE PRODUCTION BY

MONOCYTE r MACROPHAGES IN HEALTHY SUBJECTS AND PATIENTS
WITH ACTIVE PULMONARY TUBERCULOSIS

BAHAR TUNÇTANU , HAMZA OKUR†, C. HALUK ÇALIŞIR‡, HAKAN

˘
ABACIOGLU§, ˙ICLAL ÇAKICI, ˙ILKER KANZIK and
NURETTIN˙ ABACIOGLU ˘

Department of Pharmacology, Faculty of Pharmacy and †Department of Microbiology, Faculty

of Medicine, Gazi Uni¨ ersity, Ankara, ‡Ankara Ataturk
¨ Chest Disease and Chest Surgery
¨
Center, Ankara, and §Department of Microbiology, Faculty of Medicine, Dokuz Eylul
˙
Uni¨ ersity, Izmir, Turkey

Accepted 23 December 1997

The aim of the present study was to determine the NO production by human cultured
macrophages Žm f . and to compare the NO production between healthy subjects and
patients with active pulmonary tuberculosis. The bioassay method was used for assessment
of validation. Lipopolysaccharide Ž125 ng mly1 .-activated m f from healthy and diseased
subjects released a substantial amount of NO. NO synthase inhibitor, N G-nitro-L-arginine
methyl ester, Ž0.1 mmol ly1 . suppressed NO synthesis significantly in m f of healthy
subjects. Nitrite formation measured by the diazotization method in the supernatants taken
from cultured m f of tuberculous patients were significantly lower than the healthy
subjects. The supernatants obtained in both subjects caused relaxations of guinea-pig aorta
reversed by methylene blue Ž10 m mol ly1 .. There was a significant difference between
relaxations of healthy and diseased supernatants. Nitrite formation measured by the
bioassay method in the supernatants taken from cultured m f of tuberculous patients was
significantly higher than the healthy subjects. It was concluded that NO production
appeared to be decreased in tuberculosis. The reason for decreased production of NO in
tuberculosis may be related to the interaction of several cytokines andror eicosanoids by
means of the disease related induction of immune reactions.
Q 1998 The Italian Pharmacological Society

KEY WORDS: nitric oxide, monocytermacrophages, human, pulmonary tuberculosis.

INTRODUCTION products andror lymphokines has yielded negative

results w5]7x. However, there were several studies
reporting that human m f inactivated or activated
Nitric oxide ŽNO. is produced by different cell types, with tumor cells, cytokines andror bacterial products
and it is an important regulator and mediator of released NO w8]10x. Also, some investigators have
several processes including monocytermacrophages noted that human peripheric blood monocytes ex-
Žm f .-mediated cytotoxicity for pathogenic microor- press iNOS mRNA and protein in response to bacte-
ganisms w1, 2x. Rodent m f exposed to certain cy- rial products andror cytokines and that m f in the
tokines in vitro w1, 3x show markedly increased levels lungs of people with clinically active M. tuberculosis
of nitrite and nitrate production w4x. In contrast to infection often express catalytically competent iNOS
rodent m f , in most studies, stimulation of human w11]13x. In addition, NO produced by murine m f is
m f with Mycobacterium tuberculosis, bacterial an important antibacterial factor, although its role
in human tuberculosis remains unclear.
In the present study, we aimed to determine the
U
Corresponding author. NO production by human cultured m f and to com-

1043]6618r98r030219]08r$25.00r0rfr970284 Q1998 The Italian Pharmacological Society

220 Pharmacological Research, Vol. 37, No. 3, 1998

pare the NO production between healthy subjects observed, L-NAME 0.1 mmol ly1 w15, 16x was added
and patients with active pulmonary tuberculosis by to culture media for to confirm the NO-like activity.
using the diazotization method. We also aimed to The culture plates were centrifuged at 1000 g for 5
assess the validation of the bioassay method. min before medium replacement. Viability and num-
ber of cells were ) 90% throughout the period of
culture.
MATERIALS AND METHODS
Determination and measurement of nitrite
Patient selection concentration
A total of six patients with newly diagnosed active Diazotization method The accumulated concen-
¨ Chest
pulmonary tuberculosis hospitalised at Ataturk trations of nitrite which is a stable end product of
Disease and Chest Surgery Center, Ankara were NO in the supernatants were quantified by a mi-
studied. The mean age was 31.8" 4.4 years Žrange croplate assay method according to Green et al. w17x.
21]46, 6 men.. The diagnosis of tuberculosis was Samples Ž100 m l. were incubated with an equal
based on the demonstration of acid fast bacilli in volume of Griess reagent Ž1% sulphanylamider0.1%
sputum and was confirmed by a positive culture for N-1-naphtylethylene diamine dihydrochlorider2.5%
M. tuberculosis. None of the patients had clinical H 3 PO4 . at room temperature for 10 min. The absor-
evidence of the acquired immunodeficiency syn- bance at 550 nm was determined with a microplate
drome and none were on corticosteroids or other reader ŽDiagnostic Pasteur, LP 400. using a refer-
immunosuppresive agents, and antituberculous ther- ence filter of 620 nm. Sodium nitrite diluted in
apy. complete medium was used as nitrite standard. Ni-
trite concentrations in the supernatants were calcu-
Healthy subjects lated on the basis of the standard curve obtained
Five healthy persons with no respiratory symp- with known concentrations of sodium nitrite.
toms were studied as healthy subjects. The mean age Bioassay method Albino guinea-pigs of either
was 25.4" 1.0 years Žrange 23]29, 4 women and 1 sex Ž250]400 g. were killed by a sharp blow on the
men.. head and exsanguinated. The thoracic aorta was
rapidly removed, cleared of adhering periadventitial
Isolation and culture of monocytes
fat and cut into rings of 3 mm width. The rings were
Monocytes were isolated from heparinised venous
denuded of endothelium by gentle rubbing of the
blood using a discontinuous Ficoll gradient w14x.
luminal surface with ends of small forceps w18x and
After Ficoll]Hypaque centrifugation the mononu-
mounted in 10 ml organ baths filled with warmed
clear cells were washed three times by centrifugation
Ž378C., oxygenated Ž95% O 2r5% CO 2 . Krebs]
in RPMI 1640 medium containing penicillin Ž50 units
Henseleit solution ŽpH 7.4.. The changes in isomet-
mly1 ., streptomycin Ž50 m g mly1 . and L-glutamine
Ž300 m g mly1 . and finally resuspended Ž5 = 10 5 cells ric tension were measured by a Nihon Kohden force
displacement transducer ŽModel TB-611 T. and
mly1 . in RPMI 1640 medium, 20% heat-inactivated
recorded on a Nihon Kohden polygraph ŽModel
FCS and autologous serum. Samples Ž200 m l. of
RM-6000.. The tissues were equilibrated under rest-
monocyte suspension Žadjusted to 10 5 cellsrwell.
ing tension of 1.5 g for 1.5 h and the Krebs]Henseleit
were plated in U-bottom 96-well culture plates and
solution was changed every 15 min during equilibra-
incubated for 1]1.5 h at 378C in 5% CO 2r95% air
tion period. Rings were preconstricted with nore-
humidified atmosphere. The non-adherent cells were
pinephrine Ž10 m mol ly1 . prior to the experiments.
removed by washing with RPMI 1640. Monocytes
The absence of endothelium was confirmed by the
obtained in this manner were 90% pure as de-
lack of relaxation in response to 100 m mol ly1
termined by Giemsa staining and cell viability Žmea-
acetylcholine. At the end of each experiment, nitrog-
sured by Trypan blue exclusion. was more than 95%.
lycerin Ž10 m mol ly1 . was added for confirming and
RPMI 1640 medium and 10% FCS were added to
indicating the relaxing ability of the tissue.
the wells and cultured at 378C for periods of 3]4
Sodium nitrite diluted in Krebs]Henseleit solu-
days to allow cells to differentiate. During the cul-
tion was used as nitrite standard. A cumulative
ture period, the medium was changed every 48 h.
concentration-response curve to the relaxant effects
When the monocytes differentiated to macrophages
of sodium nitrite was constructed. Responses to
morphologically, 200 m l of the mixture of LPS Ž125
sodium nitrite and supernatants were calculated as
ng mly1 . and 10% FCS in RPMI 1640 medium Žtotal
the percentages of the preconstricted tissues. The
200 m l. were added to the culture every 48 h. At
amount of nitrite in the cell-free supernatants were
selected time-points during 2]18 days, cell-free su-
calculated by probit-regression analysis.
pernatants Ž100 m l. from identically treated wells
were pooled and replaced with fresh medium and Drugs
then immediately measured by the diazotization Ficoll]Hypaque ŽHistopaque-1077., RPMI 1640
method. For the bioassay studies, the remaining medium, lipopolysaccharide ŽLPS. Ž E. coli 0.11:B4.,
portion was frozen at y208C. When a difference in G
L-glutamine, penicillin-streptomycine solution, N -
nitrite levels in the absence or presence of LPS was nitro-L-arginine methyl ester ŽL-NAME., nore-
Pharmacological Research, Vol. 37, No. 3, 1998 221

pinephrine bitartrate ŽArterenol., acetylcholine hy- RESULTS

drochloride, methylene blue and superoxide dismu-
tase ŽSOD. were obtained from Sigma Chemical Co. Nitrite concentrations measured by the diazo-
ŽSt. Louis, MO, USA.. Sodium nitrite, sulphany- tization method
lamide, N-1-naphtylethylene diamine dihydrochlo- Nitrite levels in the medium were 2.15" 0.29 m mol
ride and H 3 PO4 were obtained from Merck ly1 Ž30 samples.. When the m f obtained from
ŽGermany.. Fetal calf serum ŽFCS. and nitroglycerin healthy subjects were added to the medium, nitrite
ŽPerlinganit . were purchased from Seromed concentrations significantly increased Ž3.09" 0.07
ŽGermany. and Schwarz-Pharma ŽGermany., respec- m mol ly1 , 17 samples, two donors.. Activation of the
tively. The composition of the Krebs]Henseleit so- cells with LPS Ž125 ng mly1 . significantly augmented
lution was Žmmol ly1 .: NaCl, 118.2; KCl, 4.6; CaCl 2 , nitrite concentrations in the medium Ž7.00" 0.86
2.6; MgSO4 , 1.5; NaHCO3 , 25.0; glucose, 11.1. m mol ly1 , 37 samples, five donors.. NO production
in the absence or presence of LPS was significantly
Statistical analysis suppressed by the NOS inhibitor, L-NAME Ž0.1 mmol
m f culture supernatant samples from healthy ly1 . Ž1.35" 0.10 m mol ly1 , 15 samples, five donors..
subjects and patients with active pulmonary tubercu- Nitrite levels were 2.34 "0.11 m mol ly1 Ž46 samples,
losis collected at selected time-points were studied six donors. in the m f culture supernatants obtained
using both methods. Each measurement in the dia- from patients with active pulmonary tuberculosis
zotization method was performed in triplicate. Val- and significantly higher than the medium. Activation
ues are expressed as means " SEM. Statistical com- of the cells with LPS Ž125 ng mly1 . did not change
parisons were made by Kruskal]Wallis non-para- the nitrite concentrations Ž2.17" 0.07 m mol ly1 , 46
metric ANOVA followed by Dunn’s multiple com- samples, six donors.. NO production in the absence
parisons test and Student’s unpaired t-test where or presence of LPS was not affected by L-NAME,
appropriate. A P value of - 0.05 was considered respectively Ž2.20" 0.08 m mol ly1 , 12 samples, six
statistically significant. donors.. Nitrite production in the m f supernatants

Fig. 1. Nitrite concentrations measured by the diazotization method in the medium, m f culture supernatants obtained
from healthy subjects and patients with active pulmonary tuberculosis. m f were activated with LPS Ž125 ng mly1 . for 2]18
days. L-NAME Ž0.1 mmol ly1 . was added to the medium if a difference in nitrite levels was observed in the absence or
presence of LPS. Statistical comparison was made by Kruskal]Wallis non-parametric ANOVA test followed by Dunn’s
multiple comparisons test. Statistically significant difference from wmedium; q healthy subjects m f wqx; ' healthy subjects
m f wqx q LPS Ž P - 0.05..
222 Pharmacological Research, Vol. 37, No. 3, 1998

Fig. 3. Relaxation responses caused by the medium, m f

Fig. 2. A representative record showing the effect of culture supernatants obtained from healthy subjects and
supernatant collected from culture medium on nore- patients with active pulmonary tuberculosis and contrac-
pinephrine ŽNE, 10 m mol ly1 . preconstricted aorta ring. tion responses addition with methylene blue Ž10 m mol
ŽA. Supernatant m mol ly1 ; ŽB. methylene blue Ž10 m mol ly1 l. on norepinephrine ŽNE, 10 m mol ly1 . preconstricted
ly1 .; ŽC. nitroglycerin Ž10 m mol ly1 .; ŽW. wash. aorta ring. m f were activated with LPS Ž125 ng mly1 . for
2]18 days. Statistical comparison was made by Student’s
t-test. Statistically significant difference from wmedium;
q
healthy subjects m f wqx q LPS Ž P- 0.05.. B, Methy-
in the absence or presence of LPS obtained from lene blue.
tuberculous patients Ž2.34" 0.11 m mol ly1 or 2.17"
0.07 m mol ly1 , respectively, 46 samples, six donors.
was significantly lower than the healthy subjects responses were reversed to constriction as 4.63"
Ž3.09" 0.07 m mol ly1 Ž17 samples, two donors. or 1.70% for medium, 6.53" 1.60% for healthy sub-
7.00" 0.86 m mol ly1 Ž37 samples, five donors., re- jects and 7.77" 1.30% for tuberculous patients by
spectively. ŽFig. 1.. the addition of methylene blue Ž10 m mol ly1 . w20x.
Vasorelaxation responses for healthy subjects and
patients were significantly higher than the medium.
Relaxation responses induced by the addition There was a significant difference by means of relax-
of supernatants ation responses with the culture supernatants
NO synthessis in activated human m f was de- between healthy subjects and tuberculous patients
monstrated using a bioassay method based on the ŽFig. 3.. SOD Ž160 units mly1 . w20x did not change
NO-dependent relaxation of an endothelium-de- the relaxation response in the experiments Ždata not
nuded norepinephrine Ž10 m mol ly1 .-constricted ar- shown..
terial ring w19, 20x. The supernatants Ž1 ml. collected
from the medium and the m f culture obtained from
healthy subjects and patients with active pulmonary Nitrite concentrations measured by bioassay
tuberculosis were added into the organ bath. Addi- method
tion of the supernatants caused vasorelaxation ŽFig. Nitrite concentrations in the medium, the m f
2.. Vasorelaxations caused by the medium, the m f culture obtained from healthy subjects and patients
culture obtained from healthy subjects and patients with tuberculosis were 1.40" 0.20 m mol ly1 Žseven
with tuberculosis were 4.20" 0.6% Ž7 samples., 5.50 samples., 2.00" 0.50 m mol ly1 Ž17 samples, five
" 1.2% Ž17 samples, five donors. and 11.20" 1.70% donors. and 5.20" 1.40 m mol ly1 Ž38 samples, six
Ž38 samples, six donors., respectively. The relaxation donors., respectively. The calculated nitrite concen-
Pharmacological Research, Vol. 37, No. 3, 1998 223

w23x showed that infection with virulent M. tubercu-

losis did not induce the formation of NO by human
monocyte-derived m f and this process was not af-
fected by LPS or cytokines. In our study, decreased
nitrite levels in tuberculosis may be attributed to the
interaction among cytokines, NO and prostaglandins.
Human m f produce ‘deactivators’ such as PGE 2 ,
IL-4, IL-10 and TGFb when an appropriate stimulus
applied w24x. It has been shown that infection with
M. tuberculosis induced a significant activation of
phospholipase A 2 activity of m f w23x. These deacti-
vators have been shown to downregulate a number
of functional responses in both murine w25, 26x and
human m f w27, 28x and to inhibit NO production in
murine m f also w29x. It has been reported that LPS
causes the induction of iNOS and COX-2 in rodent
m f w30x. The formation of COX metabolites has no
effects on NOS activity whereas large amounts of
NO inhibit the degree of COX-2 activity and induc-
tion w30x. IL-10 is of particular interest as its produc-
tion in response to chronic infection by intracellular
pathogens, such as Mycobacteria, has been impli-
cated in the suppression of the host immune re-
sponse in humans w31x. The interplay between induc-
tion and ‘deactivation’ of antimicrobial response by
m f will also be affected by the presence of IFNg ,
Fig. 4. Calculated nitrite levels according to the relax- released as a response to LPS. The reason of the
ations of the medium, m f culture supernatants obtained
from healthy subjects and patients with active pulmonary affection is that IFNg is a potent activator of m f
tuberculosis. m f were activated LPS Ž125 ng mly1 . for and inducer of NO release and it also downregulates
2]18 days. Statistical comparison was made by Student’s other m f responses including production of IL-10
t-test. Statistically significant difference from wmedium; by human m f w32, 33x. Since IL-10 inhibits NO
q
healthy subjects m f wqx q LPS Ž P - 0.05.. production, the presence of IFNg will clearly modu-
late the outcome of the m f response to LPS, partic-
ularly with respect to sustaining antimicrobial activ-
trations were significantly different between healthy ity w34x. It has been shown that patients with active
subjects and tuberculous patients ŽFig. 4.. pulmonary tuberculosis have an increased secretion
of prostaglandins, IL-1, IL-6, IL-10, IL-12, TGFb ,
TNF, IFNg w35]40x. In ¨ i¨ o activated T cells in the
DISCUSSION tuberculous patients may result in faster detection
of cytokine production in ¨ itro. Th1 cells secrete
Although some investigators have been unable to IL-2 and IFNg stimulating NO production, whereas
show NO production by human m f w5]7x, others Th2 cells secrete IL-4, IL-10 and TGFb inhibiting
have noted induction of only low to modest levels of NO production w40x. In tuberculosis, Th1 type re-
NO production w8]10, 21x. In this study, NO levels sponses are diminished during development of tu-
were moderately measured by the diazotization berculosis, but Th2 type responses are either unaf-
method in the LPS-activated human m f culture and fected or enhanced w41x. Defective proliferative re-
NO synthase inhibitor, L-NAME significantly su- sponsiveness andror deficient IL-2 and IFNg secret-
pressed the NO production. Although NO has been ing capacity to Mycobacteria have previously been
directly implicated in the stasis or killing of My- reported in patients with severe tuberculosis w36, 42x.
cobacteria w21x, the role of NO and other nitrogen In a recent study w43x, Dlugovitzky et al. showed that
oxides in infected humans remains a matter of con- IFNg and IL-2 levels were low and IL-4 and IL-10
siderable debate. It has been shown that NO pro- levels were high in tuberculosis patients showing
duction does not increase in human m f activated or advanced pulmonary disease. Therefore, inadequate
infected in ¨ itro with BCG or M. tuberculosis andror IFNg production could result in the failure of m f
LPS w12, 22x. In our work, we also did not measure activation and promote disease progression and so
an increase in NO production in tuberculosis. On we concluded if there was a reduction of NO pro-
the contrary, nitrite levels measured by the diazoti- duction in tuberculosis, prostaglandins would in-
zation method in the patients were significantly lower crease in the medium and suppress the immune
than those of the healthy subjects. Dumarey et al. response to mycobacteria.
224 Pharmacological Research, Vol. 37, No. 3, 1998

Supernatants obtained from healthy subjects and ACKNOWLEDGEMENTS

tuberculous patients caused relaxations on the
guinea pig aorta and the vasorelaxations were re-
versed by the addition of methylene blue, an inhibi- This study was supported by the Research Founda-
tor of guanylyl cyclase in vascular smooth muscle tion of Gazi University ŽProject Code No: EF-
w44x, suggesting that the monocytermacrophage-de- 02r93-5..
rived vasorelaxant was NO Žor its stable product
nitrite .. In contrast to the diazotization method,
supernatants from tuberculous patients induced sig- REFERENCES
nificantly higher relaxations and calculated nitrite
levels than those induced by supernatants from
healthy subjects. A possible explanation of this dif- 1. Stuehr DJ, Marletta MA. Induction of nitriternitrate
synthesis in murine macrophages by BCG infection,
ference may be the involvement of non-nitrite va- lymphokines or interferon-g . J Immunol 1987; 139(2):
sorelaxant substances such as prostaglandins which 518]25.
can not be measured by the diazotization method. It 2. Hibbs J, Jr, Taintor RR, Vavrin Z, Rachlin EM.
has been shown that exposure of monocytes to bac- Nitric oxide: a cytotoxic activated macrophage effec-
tor molecule. Biochem Biophys Res Commun 1988
terial LPS increases the prostaglandin content of 157: 87]94.
supernatants w35x. On the other hand, possible rea- 3. Ding AH, Nathan CF, Stuehr DJ. Release of reactive
sons of low vasorelaxation responses and diminished nitrogen intermediates and reactive oxygen interme-
nitrite levels from healthy subjects estimated in diate from mouse peritoneal macrophages compar-
ison of activating cytokines and evidence for inde-
bioassay than the diazotization method might de-
pendent production. J Immunol 1988; 141(7):
pend on: Ž1. the non-selective measurement manner 2407]12.
of the later procedure, Ž2. the interference between 4. Marletta MA, Yoon PS, Iyengar R, Leaf CD, Wish-
the medium and Griess reagent, Ž3. the influence of nok JS. Macrophage oxidation of L-arginine to nitrite
the conversion of nitrite to nitrate and Ž4. the mask- and nitrate: nitric oxide is an intermediate. Biochem-
istry 1988; 27: 8706]11.
ing effect of contractile factors on vasorelaxations. 5. Cameron ML, Granger DL, Weinberg JB, Kozumbo
Tracey et al. w45x showed that the diazotization reac- WJ, Koren HS. Human alveolar and peritoneal
tion did not detect NO in a selective manner. This macrophages mediate fungistasis independently of
L-arginine oxidation to nitrite or nitrate. Am Re¨
method has been shown to be 10]100-fold less sensi-
Respir Dis 1990; 142: 1313]19.
tive than the bioassay for NO. Also it has been 6. Murray HW, Teitelbaum RF. L-Arginine-dependent
established that an interference occurs between the reactive nitrogen intermediates and the antimicrobial
medium and Griess reagent. Such a medium includ- effect of activated human mononuclear phagocytes. J
ing various salts like KCl, CaŽNO 3 . 2 and MgSO4 in Infect Dis 1992; 165: 513]17.
7. Schneemann M, Schoedon G, Hofer S, Blau N,
concentrations less than 10y4 mmol ly1 , causes an Guerero L, Schaffner A. Nitric oxide synthase is not
error of approximately "0.2% w46x. Therefore, the a constituent of the antimicrobial armature of human
differences among the methods may depend on the mononuclear phagocytes. J Infect Dis 1993; 167:
presence of these salts ŽKCl 5.4= 10y3 mmol ly1 ; 1358]63.
8. Martin JHJ, Edwards SW. Changes in mechanisms of
CaNO 3 ? 4H 2 O 5.8= 10y4 mmol ly1 and MgSO4 4.1 monocytermacrophage-mediated cytotoxicity during
= 10y4 mmol ly1 . in the RPMI 1640 medium. An- culture. J Immunol 1993; 150: 3478]486.
other possibility of low nitrite level estimations in 9. Mautino G, Paul-Eugene N, Chanez P, Vignola AM,
terms of percentage relaxations in bioassay other Kolb JP, Bousquet J, Dugas B. Heterogenous sponta-
neous and interleukin-4-induced nitric oxide produc-
than those obtained by the diazotization method
tion by human monocytes. J Leukoc Biol 1994; 56:
may depend on the conversion of nitrite to nitrate 15]20.
when supernatants were kept frozen at y208C. 10. Zembela M, Siedlar M, Marcinkiewicz J, Pryjma J.
However, the nitrite concentrations that we mea- Human monocytes are stimulated for nitric oxide
sured were found unchanged or slightly increased release in vitro by some tumor cells but not by
cytokines and lipopolysaccharide. Eur J Immunol
Ždata not shown., so we can exclude this possibility. 1994; 24: 435]9.
Finally, the relaxation responses induced with the 11. Reiling N, Ulmer AJ, Duchrow M, Ernst M, Flad
supernatants might be masked by contractile factors H-D, Hauschildt S. Nitric oxide synthase: mRNA
Žsuch as PGF2 a , TxA 2 , leukotrienes . released from expression of different isoforms in human mono-
cytesrmacrophages. Eur J Immunol 1994; 24: 1941]4.
activated m f w24x. 12. Weinberg JB, Misukonis MA, Shami PJ, Mason SN,
It was concluded that NO production appeared to Sauls DL, Dittman WA, Wood ER, Smith GK, Mc-
be decreased in tuberculosis. The reason of de- Donald B, Bachus KE, Haney AF, Granger DL.
creased production of NO in tuberculosis may be Human mononuclear phagocyte inducible nitric ox-
ide synthase ŽiNOS.: analysis of iNOS mRNA, iNOS
related with the interaction of several cytokines protein, biopterin, and nitric oxide production by
andror eicosanoids by means of the disease related blood monocytes and peritoneal macrophages. Blood
induction of immune reactions. 1995; 86(3): 1184]95.
Pharmacological Research, Vol. 37, No. 3, 1998 225

13. Nicholson S, Bonecini-Almedia MG, Lapa e Silva JR, inhibits parasite killing and nitrogen oxide produc-
Nathan C, Xie Q-W, Mumford R, Weidner JR, tion by IFN-g-activated macrophages. J Immunol
Calaycay J, Geng J, Boechat N, Linhares C, Rom W, 1992; 148: 1792]7.
Ho JL. Inducible nitric oxide syntheses in pulmonary 30. Swierkosz TA, Mitchell JA, Warner TD, Botting RM,
alveolar macrophages from patients with tuberculo- Vane JR. Co-induction of nitric oxide synthase and
sis. J Exp Med 1996; 183: 2293]302. cyclo-oxygenase: interactions between nitric oxide
¨
14. Boyum A. Isolation of mononuclear cells and granu- and prostanoids. Br. J. Pharmacol 1995; 114: 1335]42.
locytes from human blood. Isolation of mononuclear 31. Sieling PA, Abrams JS, Yamamura M. Immunosup-
cells by one centrifugation, and of granulocytes by pressive roles for IL-10 and IL-4 in human infection.
combining centrifugation and sedimentation at 1 g. In vitro modulation of T cell responses in leprosy. J
Scand J Clin Lab In¨ est 1968; 21(Suppl. 97): 77]89. Immunol 1993; 150: 5501]6.
15. Belenky SN, Robbins RA, Rubinstein I. Nitric oxide 32. Schreiber S, Perkins SL, Teitelbaum SL, Chappel J,
synthase inhibitors attenuate human monocyte Stah PD, Blum JS. Regulation of mouse bone mar-
chemotaxis in vitro. J Leukoc Biol 1987; 53: 498]503. row macrophage mannose receptor expression and
16. Salvemini D, De Nucci G, Gryglewski RJ, Vane JR. activation by prostaglandin E and IFN-g . J Immunol
Human neutrophils and mononuclear cells inhibit 1993; 151: 4973]8.
platelet aggregation by releasing a nitric oxide-like 33. Chomarat P, Rissoan M-C, Banchereau J, Miosec P.
factor. Proc Natl Acad Sci USA 1989; 86: 6328]32. Interferon gamma inhibits interleukin 10 production
17. Green LC, Wagner DA, Glogowski J, Skipper PL, by monocytes. J Exp Med 1993; 177: 523]8.
Wishnok JS, Tannenbaum SR. Analysis of nitrate, 34. Roach TIA, Barton CH, Chatterjee D, Liew FY,
nitrite, and w 15 Nxnitrate in biological fluids. Anal Blackwell JM. Opposing effects of interferon-on
Biochem 1982; 126: 131]8. iNOS and interleukin-10 expression in lipopolysac-
18. Furchgott RF, Zawadzki JV. The obligatory role of charide- and mycobacterial lipoarabinomannan-
endothelial cells in the relaxation of arterial smooth stimulated macrophages. Immunology 1995; 85:
muscle by acetylcholine. Nature 1980; 288: 373]6. 106]13.
19. Stuehr DJ, Gross SS, Sakuma I, Levi R, Nathan CF. 35. Ellner JJ, Spagnuolo PJ. Suppression of antigen and
Activated murine macrophages secrete a metabolite mitogen induced human T lymphocyte DNA synthe-
of arginine with the bioactivity of endothelium-de- sis by bacterial lipopolysaccharide: mediation by
rived relaxing factor and the chemical reactivity of monocyte activation and production of prosta-
nitric oxide. J Exp Med 1989; 169: 1011]20. glandins. J Immunol 1979; 123: 2689]95.
20. Stuehr DJ, Kwon NS, Gross SS, Thiel BA, Levi R, 36. Onwubalili JK, Scott GM, Robinson JA. Deficient
Nathan CF. Synthesis of nitrogen oxides from L- immune interferon production in tuberculosis. Clin
arginine by macrophage cytosol: requirement for in- Exp Immunol 1985; 59: 405]13.
ducible and constitutive components. Biochem Bio- 37. Fujiwara H, Kleinhenz ME, Wallis RS, Ellner JJ.
phys Res Commun 1989; 161(2): 420]6. Increased interleukin-1 production and monocyte
21. Denis M. Killing of Mycobacterium tuberculosis suppressor cell activity associated with human tuber-
within human monocytes activation by cytokines and culosis. Am Re¨ Respir Dis 1986; 133: 73]7.
calcitriol. Clin Exp Immunol 1991; 84: 200]6. 38. Toossi Z, Gogate P, Shiratsuchi H, Young T, Ellner
22. Keller R, Keist R, Joller P, Groscurth P. Mononu- JJ. Enhanced production of TGF-b by blood mono-
clear phagocytes from human bone marrow progeni- cytes from patients with active tuberculosis and pres-
tor cells; morphology, surface phenotype, and func- ence of TGF-b in tuberculous granulomatous lung
tional properties of resting and activated cells. Clin lessions. J Immunol 1995; 154: 465]73.
Exp Immunol 1993; 91: 176]82. 39. Cadranel J, Philippe C, Philippe B, Milleron B, Fou-
23. Dumarey CH, Labrousse V, Rastogi N, Vargaftig queray B, Mayaud C, Baud L. Increased expression
BB, Bachelet M. Selective Mycobacterium avium-in- and occupancy for tumour necrosis factor on blood
duced production of nitric oxide by human mono- monocytes from tuberculosis patients. Clin Exp Im-
cyte-derived macrophages. J Leukoc Biol 1994; 56(1): munol 1993; 94: 51]6.
36]40. 40. Surcel H-M, Troye-Blomberg M, Paulie S, Andersson
24. Nathan CF. Secretory products of macrophages. J G, Moreno C, Pasvoli G, Ivanyi J. Th1rTh2 profiles
Clin In¨ est 1987; 79: 319]26. in tuberculosis, based on the proliferation and cy-
25. Bogdan C, Vodovotz Y, Nathan C. Macrophage de- tokine response of blood lymphocytes to mycobacte-
activation by interleukin 10. J Exp Med 1991; 174: rial antigens. Immunology 1994; 81: 171]6.
1549]55. 41. Fenton MJ, Vermeulen MW. Immunopathology of
26. Edwards CK, Hedegaard HB, Zlotnik A, Gangad- tuberculosis: roles of macrophages and monocytes.
haram PR, Johnston RB, Pabst MJ. Chronic infec- Infect Immunol 1996; 64(3): 683]90.
tion due to Mycobacterium intercellulare in mice: 42. Toossi Z, Kleinhenz ME, Ellner JJ. Defective inter-
association with macrophage release of prostaglandin leukin-2 production and responsiveness in human
E 2 and reversal by injection of indomethacin, mu- pulmonary tuberculosis. J Exp Med 1986; 163:
ramyl dipeptide, or interferon-g . J Immunol 1986; 1162]72.
136: 1820]7. 43. Dlugovitzky D, Torres-Morales A, Rateni L, Farroni
27. De Waal Malefyt R, Abrams J, Bennett B, Figdor MA, Largacha C, Molteni O, Bottasso O. Circulating
CG, De Vries JE. Interleukin-10 ŽIL-10. inhibits profile of Th1 and Th2 cytokines in tuberculosis
cytokine synthesis by human monocytes: an autoreg- patients with different degrees of pulmonary involve-
ulatory role of IL-10 produced by monocytes. J Exp ment. FEMS Immunol Med Microbiol 1997; 18(3):
Med 1991; 174: 1209]20. 203]7.
28. Silva JS, Twardzik DR, Reed SG. Regulation of 44. Martin W, Villani GM, Jothianandan D, Furchgott
Trypanosoma cruzi infections in vitro and in vivo by RF. Selective blockadge of endothelium-dependent
transforming growth factor b ŽTGF-b .. J Exp Med and glyceryl trinitrate-induced relaxation by hemo-
1991; 174: 539]45. globin and by methylene blue in the rabbit aorta. J
29. Gazzinelli RT, Oswald IP, James SL, Sher A. IL-10 Pharmacol Exp Ther 1985; 232: 708]14.
226 Pharmacological Research, Vol. 37, No. 3, 1998

45. Tracey WR, Linden J, Peach MJ, Johns RA. Com- 46. Karayannis MI, Piperaki EA, Maniadaki MM. Ki-
parison of spectrophotometric and biological assays netic determination of nitrite based on the Griess
for nitric oxide ŽNO. and endothelium-derived relax- reaction and stopped flow technique. Anal Lett 1968;
ing factor ŽEDRF.: nonspecificity of the diazotisation 19(1 &2): 13]23.
reaction for NO and failure to detect EDRF. J
Pharmacol Exp Ther 1990; 252(3): 922]8.