Induction of nitric oxide synthase is a necessary precondition for expression of tumor necrosis...

Vol. 184, No. 3, 1992

May 15, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1364-1371

INDUCTION OF NITRIC OXIDE SYNTHASE IS A NECESSARY PRECONDITION FOR EXPRESSION OF TUMOR NECROSIS FACTOR-INDEPENDENT

TUMORICIDAL ACTIVITY BY ACTIVATED MACROPHAGES

Robert Kel!er I Stefano Bassetti I Ruth Keist 1

A l e x a n d e r M u l s c h ~, a n d S t e f a n K l a u s e r 3

1 Immunobiology Research Group, Institute for Immunology and Virology, University of Zurich, Gloriastr. 30,

8028 Zurich, Switzerland

Department of Applied Physiology, University of Freiburg, Freiburg, Germany

3 Department of Biochemistry, University of Zurich,

8057 Zurich, Switzerland

Received March 27, 1992

Various bacteria and bacterial products induce in pure, lymphocyte-free bone marrow-derived mononuc!ear phagocytes (BMM~) the generation of tumor necrosis factor, nitric oxide (NO) synthase, NO and nitrite (NO2) , the flow O f L-arginine to citru!line, and tumoricidal activity. The flow of L-arginine to citrulline and formation of NO/NO 2 on the one hand and expres-

I 1 1 I sion of tumoricidal activity were-not a~ways c~ose~y re~ated; however, these parameters were suppressed in a dose-dependent manner bv the flavoprotein inhibitor, diphenyleneiodonium (DPI)

- • " 1 ~ 1 " " and the L-argzn!ne analogue, N -monomethy~-L-argznzne (NMMA). The findings support the concept of a central role of the NO synthase pathway in the generation of tumor necrosis factor- independent tumoricidal activity by activated macrophages but the exact conditions which enable the transfer of the !ytic principle from the effector to the target cell remain to be elucidated. ~ 1992 Academic Press, Inc.

Activated macrophages represent major effectors in defense

against bacterial infection and neoplasia. Recent evidence

suggested that nitric oxide (NO) derived from L-arginine may be

Abbreviations: BbLM~, bone marrow-derived mononuc!ear phagocytes; DPI, diphenyleneiodonium; EPR, electron paramagnetic resonance; IMDM, Iscove's modified Dulbecc~ medium; LPS,- lipopo!ysaccheride; LTA, lipoteichoic acid; NM~, N -monomethyl-L-arginine; NO, nitric oxide; NO 2, nitrite; NU, neutralizing units; PGMC, peptidog!ycan from M. catarrhalis; TNF~, tumor necrosis factor.

0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of.reproduction in any form reserved. 1364

Vol. 184, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a major mediator of tumor cell killing [i] and of microbiostatic

effects of macrophages against intracellular pathogens such as

Leishmania, Toxoplasma, tubercle bacilli, and the pathogenic

fungus, Cryptococcus neoformans [2-6]. Although formation of NO

and nitrite (NO2) and expression of tumor necrosis factor

(TNF~)-independent tumoricidal activity were both strongly depen-

dent on L-arginine, these parameters did not always coincide [7-

9]. To uncover these discrepancies, the ability of bacteria and

bacterial products to enhance NO synthase activity and to induce

the flow of L-arginine to L-citrulline, the secretion of NO2 and

tumoricidal activity in macrophages was compared in the present

study.

Materials

MATERIAL AND METHODS

Enterococcus faecalis and Moraxella catarrhalis organisms were §electEd, grown, and heat-killed as previously described [7]. Peptidog!ycan from M. catarrhalis was isolated as described (MS in preparation). Lipo-~eichoic acid (LTA) from E. faeca!is and lipopolysaccharide (LPS) from E__:~$oli 0128:Bl2-~ere from Sigma Chemical Co., St. Louis, MO. N~'monomethy!-L-arginine (N~A) was purchased from Bachem, Bubendorf, Switzerland, diphenyleneiodonium chloride from Fluka, Buchs, Switzerland. Conventional sheep anti-mouse TNF~ (neutralizing activity

I00'000 NU/ml), obtained as previously described [i0], neutra- lized the action of rat TNF~ to a similar extent as that of murine TNF~.

Methods

Bone marrow-derived mononuclear phagocytes (BMM~). Bone marrow cells frol male D--~r----~w-~e cultured in medium (IMDM) conditioned with supernatant from strain L929 cells as described [7]. On day 6, BMM~ remaining adherent after repeated washing were cultured for the time interval indicated in medium supplemented with one of the agents under test before their functional activities were measured.

Tumor ~ecrosis factor secretion. Supernatants harvested from 5x10 ~ BMM@fmI~ various time intervals were checked for their cytolytic activity in a 3 h MTT tetrazolium cytotoxicity test, utilizing the TNF~-sensitive WEHI-164/13 cells as targets [i0].

NO synthase activity was determined by measuring the L- arg~-~ine-dependent activation of purified soluble guanylyl cyclase by BMM~ cytosol (10 ~g protein/ml), prepared by centrifugation

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(i00'000 x g for 60 min) of the soluble fraction from homoge- nized BMM~ [11,12].

11 6 i L-citru~ine formation. 10 BMM~/m~ were incubate~4at 37°C in medium supplemented with 2% FCS, 1 ~Ci L-[guanidino- C]-arginine and the agent under test. An aliquot of the acified supernatant was mixed with 5-sulfosalicylic acid, kept for 60 min at 4°C before centrifugation and separation of amino acids on a Biotronik LC 6001 amino acid analyzer [9].

NO[ determination, i00 B! cell-free supernatants from 106 BMM~7~l or sodium nitrite standard solutions were mixed with 50 ~l Griess reagent and incubated for 10 min at room temperature before NO2 concentration was determined by measuring absorbance at 550 nm in a microplate reader [7].

Assay of TNF~-independent tumoricida! activity. Resting day 6 BMM~'-Q-~e--[ncubated for 24 h in medium with an agent to be tested. The medium was then replaced by new medium supplemented with the same agent and TNF~-resistan~4P-815 murine mastocytoma cells that had been prelabeled with [ C]-thymidine (initial effector/target cell ratio i:i and 2.5:1; [I0]). After 36 h interaction, radioactivity in cell-free supernatants was measured and the percentage of specific thymidine release was calculated.

RESULTS

In B~M~ derived from the same pool, M. catarrhalis (MC)

organisms, MC peptidoglycan, LTA from E. faecalis and LPS from

E. coli enhanced NO synthase activity and induced the flow of L-

arginine to citrulline and the production of NO2; in contrast,

E. faecalis organisms affected these parameters only little.

Induction of the NO synthase pathway was not always followed by

the generation of tumoricida! activity (LPS, Table i); on the

Table 1. Ability of bacteria and bacterial products to trigger in BMM~ within 24 h NO synthase activity,

the generation of L-citrulline and nitrite from L-arginine, and/or tumoricidal activity

Pretreatment of BMM~ NO synthase activity %radioactivity associated Nitrite production Tumoricidal activit~

24h (nmol cGMP/mg after 24 h with (~M/IO 6 BMM~/24 h) (% cl4-thymidine GC/min) L-arginine L-eitrulline release from P-g15

mas~ocytoma)

Medium only (control) 60 (~3) 92 (!2) 0 (zO) 0 0 (~I)

M. eatarrhalis 5 pg/ml 375 (~38) 61 (!3) 36 (~2) 121 (~8) 55 (~8)

PG M. catarrh. 10 ~g/ml ~09 (~16) 65 (!3) 23 (~2) 91 (~6) 89 (~4)

E. faecalis I0 pg/ml 57 (~20) 86 (!6) 7 (!3) ii (!2) 45 (~7)

LTA E. faeealis I0 ~g/ml 659 (~15) 54 (~3) 38 (~3) 124 (~8) 55 (!6)

LPS E. cell 0128:B12 I0 ng/ml 475 (Z27) 38 (!5) 52 (~4) 132 (~7) 0 (~3)

These values (+ SD) are derived from a single, typical experiment performed in triplicate.

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VOI. 184, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

other hand, tumoricidal activity could also be expressed when NO

synthase activity was barely enhanced (E. faecalis); there was

thus not necessarily a direct quantitative correlation between

production of NO/N02 and expression of tumoricidal activity.

The analysis of the kinetics of the macrophage response to

the microbial agents showed that peak secretion of TNF~ was

reached within 2 h while NO synthase activity required approxi-

mately four hours for maximum expression. The other macrophage

activities were not demonstrable before 8 to 16 h interaction

and reached their maximum mostly after 24 h (not shown).

To verify that secretion of NO2 and expression of tumoricidal

activity were both dependent on NO synthase, the effects of the

NO synthase inhibitor DPI [13] and of NMMA on the macrophage

parameters were determined. Although peptidoglycan from M.

catarrhalis (PGMC) and LTA differed considerably in their abi-

lity to trigger the secretion of nitrite and/or tumoricidal

activity, both functions were suppressed in a concentration-

dependent manner by DPI (Fig. I) and NMMA (not shown; 7).

To elucidate whether induction of TNF~ secretion and NO

synthase activity were in any way related, the effects of anti-

TNF~ and of N~IA and DPI on the macrophage response were investi-

gated. Anti-TNF~ markedly suppressed the secretion of TNF~

without affecting the NO2 secretion or TNF~-independent tumori-

cidal activity. On the other hand, DPI abolished both the secre-

tion of NO2 and tumoricidal activity expressed against P-815

mastocytoma cells but did no diminish the secretion of TNF~

(Fig. 2).

DISCUSSION

Earlier work has indicated that secretion of NO/NO 2 and

expression of tumoricidal activity by activated macrophages may

be directly correlated; in particular, both functions were sup-

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100

-~ 9o ~

~ ao 6 ~ o ~

~,-~ 60'

~ > ,

~ 40- " x ~

=L'~ i ~"~, 30-

b~ 2O

N ~

o C 0.5 1.0 2.5

#M DPI

Fig. i. Concentration-dependent suppression by DPI of nitrite secretion and tumoricidal activity against P-815 mastocytoma cells induced in BMM~ ( [] , control) by PGMC (5 ,Ug/m!; O) and LTA from E. faecalis (5 ~g/ml;Z~). Values (+ SD) are derived from five-" ' exper iments , each per formed in t ~ ' i p l i c a t e .

pressed by arginase and analogues of L-arginine and this suppres-

sion was reversed by L-arginine [1,7,9,10,14]. NO is derived

from L-arginine by oxidation of one of the two chemically equi-

I oo

~ • ® 8 0 -

r n . ~ .

~ 6o.

o ~ 4 0 -

~ .

~ $ 2o.

o alocabcabc ,~acabcabc abcabcabc

TNF~x Nitrite tumoricidal secretion secret ion activity

Fig. 2. S~ion of TNF~ and NO[ and tumoricidal activity against P-815 mastocytoma cells induce~ in BMM~ by PGMC and LTA from E. faeca- !is, are differently affected by anti-TNF~ and DPI. Valu-~s--~--~) are means from 6 to 9 experiments, each performed in triplicate. a) resting BMM~; b) PGMC 5 ~g/ml; c) LTA 5~g/ml.

control; ~ anti-TNF~ i000 NU/well; ~ DPI 2.5 ~M.

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valent guanido nitrogens, a process catalyzed by NO synthase

[15,16]. Its inducible, ca!modulin-independent form present in

macrophages is expressed in response to eytokines, such as

interferon y, and bacterial !ipopo!ysaccharide. Although produc-

tion of NO/NO2 and expression of tumoricidal activity are strong-

ly dependent on L-arginine, these two functions did not always

coincide [7,8,9]. To elucidate the significance of the NO syn-

thase pathway in these processes, we have now measured the

activity of the enzyme in response to various microbial agents

in relation to the flow of L-arginine to citru!!ine, the forma-

tion of NO/NO2, and expression of TNF~-independent tumoricidal

activity.

The microbial agents differed considerably in their ability

to trigger in resting macrophages the NO synthase pathway and

expression of tumoricidal activity (for example E. faecalis

organisms vs LPS; Table i). On the other hand, formation of NO2

and tumoricida! activity triggered in macrophages by microbial

agents were similarly susceptible to suppression by two diffe-

rent classes of NO synthase inhibitors, the flavoprotein inhibi-

tor, DPI (Fig. i), and the L-arginine analogue, NMMA [7], under-

lining the central role of the NO synthase pathway in these

processes. However, measurements of NO by EPR [9] and of NO

synthase activity did not provide a plausible explanation why

low NO could be associated with considerable cytotoxicity (E.

faecalis, S. epidermidis; 7) while high level NO was not

consistently accompanied by target cell damage (LPS; 8). Proba-

bly nascent, free NO is too labile to assure the transfer of the

lytic activity from the effector to the target cell. The occa-

sional quantitative divergence might therefore be explained by

the thesis that NO can be metabolized via different ways and/or

stored in stabilized form in complexes from which it can be

1369


released as required. In addition to recent evidence for complex

formation of nitric oxide with iron [9,17,18] and/or with thiols

[19,20], and for a protein-bound storage of these complexes

[21], further work is required to strengthen this concept.

Considering previous reports that macrophage-derived TNF~

increased in fibroblast-like L-929 cells the formation of

nitrite [ii] and that TNF~ was able to activate soluble guany-

late cyc!ase in glomeru!ar mesangial cells via an L-arginine-

dependent mechanism [22], the question arose whether expression

of these two macrophage activities was in any way related. The

findings that anti-TNF~ suppressed TNF~ activity without affec-

ting NO2 secretion nor tumoricidal activity against P-815 masto-

cells while NMMA and DPI abolished NO2 secretion and cytoma

TNF~-independent tumoricidal activity without impairing the

release of TNF~ (Fig. 2), are evidence against the existence of

a close relationship between these parameters.

Acknowledgments: The work of the Immunobiology Research Group was supported by the Swiss National Science Foundation (grant 31-25717.88) and the Roche Research Foundation. We thank Prof. R.M. Zinkernage! for sheep anti-mouse TNF~, Mr. John Gustafson for the isolation of peptidoglycan from M. catarrhalis, and Mrs. E. Huf and Miss D. Herbring for technica~T--assistance.

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[13] Stuehr, D.J., Fasehun, O.A., Kwon, N.S., Gross, S.S., Gonza!ez, J.A., Levi, R. and Nathan, C.F. (1991) FASEB J. 5: 98-103.

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