Eur. J. Immunol. 1992. 22: 2303-2307

Miriam Palacios, Richard G. Knowles and Salvador Moncada Wellcome Research Laboratories, Kent

Nitric oxide synthasc induction and nonspecific rcsistance

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Enhancers of nonspecific immunity induce nitric oxide synthase: induction does not correlate with toxicity or adjuvancy A rangc of bacterial products and rclatcd synthetic compounds. either alonc or in combination. enhance nonspecific resistance to infcction and tumors. Thcse compounds. which vary in their othcr properties such as pyrogenicity. toxicity and adjuvancy. wcrc used to assess the hypothesis that nonspecific resistancc is mediated by induction of the 1.-argininc: nitric oxidc (NO) pathway. The results obtaincd show that agents which stimulatc nonspecific immunity. such 21s endotoxin. muramyl dipeptide (MDP) and combinations of monophosphoryl lipid A (MPL) with eithcr trehalosc dimycolatc (TDM) or MDP cause a substantial induction of Ca'+-independcnt NO synthasc in murinc lung. In contrast. agents which do not stimulate nonspccific resistance. such as either MPL or TDM alone or thrconyl MDP (ThrMDP). do not inducc NO synthase. This differencc i n the ability of MDP and ThrMDP to induce NO synthase in the lung in vivo was also manifest in pcritoneal macrophages irz virw as wcll as being evident i n the > 100-fold greater potency of MDP in inducing the enzymc in vitro in lung slices. In contrast to the good correlation between induction of NO synthasc and induction of nonspccific resistance. no corrclation was observed with eithcr the toxic cffects of thesc agents or thcir adjuvancy.

1 Introduction Endotoxin (lipopolysaccharide. LPS) from the cell wall of gram-ncgative bactcria has many effects when injected into animals including elevation of body temperaturc. induction of shock. enhancement of nonspccific resistance to pathogenic infections. inhibition of tumor growth. and adjuvant effccts. increasing specific immunity (reviewed in [ 1-71). Monophosphoryl lipid A (MPL) is a nontoxic fraction derived from the LPS of gram-negative bactcria [ 8 ] .which retains some of the bcncficial effccts o f thc LPS in enhancing specific and nonspecific immunity in synergy with other immunostimulatory agcnts such as trehalosc dimycolate (TDM) [X. 91. Thc cell wall of gram-positive bactcria does not contain LPS. but mycobacteria do contain peptidoglycans which haw a similar range of properties. MDP (N-acetylmuramyl-~~-alanyl-~>-isoglutamine. muramy1 dipeptide) has been shown to be the minimal structure which retains these properties. including pyrogenicity. adjuvancy, enhancement of nonspccific resistance. and inhibition of tumor growth [ 10-171. Howcver, of thc range of analogues of MDP which have bccn synthesizcd and tcsted. somc are good adjuvants despite failing to stimulatc nonspecific resistance or exhibit pyrogenicity [ 11, 121. Such compounds are useful tools for determining which of the many effects of endotoxin and relatcd compounds may represent the molecular mechanism(s) underlying their ability to enhancc nonspecific resistancc. [ I 106373 Correspondence: Richard G. Knowlcs. Biochemical Scicnccs. Wcllcome Research Laboratories. Langlcy Court. Beckenham. Kent. BR3 3BS. GB Abbreviations: MDP: Muramyl dipcptidc. N-iicetylmuramylr-alanyl-D-isoglutaminc ThrMDP: Threonyl murrimyl dipcptide. N-acetylmuramyl-~.-threonyl-~~-isoglutamineMPL: Monophosphoryl lipid A TDM: Trchalose dimycolatc 0 VCtI Vcrlagsgcsellsch;ift mhH. D-6Y-IO Wcinhcim. 1992

Induction of Ca'+-indcpendent nitric oxidc (NO) synthase (provisionally EC' 1.14.13.39) by endotoxin in a widc range of tissues in l s i i w results in a sustained production of NO lasting many hours 113-IS]. Induction of this enzyme in macrophages and othcr cells could be suggcsted to bc the basis of nonspescific resistancc [ 161. NO production by this cnzyme has bccn shown t o be an important mechanism by which macrophages can kill or inhibit the growth of many pathogenic organisms it1 igitro (rcvicwed in [ 171) and in at least onc instance in viiw [ 1x1. Induction of this form of NO synthasc in hepatocytcs 113. 191has also becn implicated a s a mechanism by which thcse cells kill malarial parasites [20. 21 ].We have thereforc cxamined thc abilities of LPS. MPL. TDM. MDP and one MDP analogue which docs not enhancc non-spccific resistance. N-acetylmuramy1-I.thrconyl-n-isoglutamine (ThrMDP) [ 11. 121 to induce NO synthasc in i i v o and iii r*irro. in order t o understand the relationship between this induction and the diffcrent beneficial and pathological effccts of LPS and other bacterial products.

2 Materials and methods 2.1 Materials and animals The MDP used was batch ZOHO9351. from Sigma, (St. Louis. MO). ThrMDP. batch no. 1.7I-138/01. was from Syntex Kesearch (Palo A l ~ oCA). . MPL.TDM and refincd standard endotoxin from Srrlinoriell~niinriesora (LPS) were from RIB1 lmmunochcm (obtaincd from Universal Biologicals, London. GB). Brewers thioglycollate was from Difco (E. Molcsey. GB). lA-[U-"C]argininc and thymidinc was obtained from Amersham International (Aylesbury, GB). Other rcagcnts were from Sigma or BDH (Poole. GB). Thc mice used wcrc 30-75-g male BALBk. and thc rats were 200-250-g male Wistars, both from Charles River (Margate. GB) and fed ad libitum.

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Eur. J. Immunol. 1992. 22: 2303-2307

M. Palacios, R. G. Knowles and S. Moncada

100 yg/ml streptomycin. 5774 cells (lo6) in 1 ml medium were incubated in 4.5 cm2wells in 12-well plates for 24 h in the presence of different compounds before removal of medium and extraction for NO synthase assay.

2.2 NO synthase induction

Studies of NO synthase induction were predominantly carried out using the lung, since substantial induction has been observed in this tissue. For these studies of NO synthase induction in vivo, mice were dosed intraperitoneally with compounds in sterile PBS, in a volume of 10 2.3 NO synthase extraction and assay ml/kg body weight, 6 h before removal of the lungs. The doses of the different agents studied as potential NO NO synthase was extracted at 0 "C-4 "C in a homogenizasynthase inducers (up to 10 mglkg body weight) were in the tion buffer (0.32 M sucrose, 50 mM Tris-HC1, 1mM EDTA, range used previously in studies on enhancement of 1 mM dithiothreitol, 100 yg/ml phenylmethylsulphonyl nonspecific resistance [S-121. The lungs were removed fluoride, 10 yglml leupeptin, 10 yg/ml soybean trypsin under pentobarbitone anesthesia (60 mg/kg) following inhibitor, and 2 yg/ml aprotinin, pH 7.0 at 20°C) by 2-3 min perfusion of saline through the portal vein, with homogenization of the lung tissue for 10 s with an Ystral exsanguination via the inferior vena cava, in order to homogenizer before centrifugation at 10000 x g for 20 min remove blood from the tissue.The lung was freeze-clamped at 4 "C. The supernatant obtained from tissues following in vivo induction was used for NO synthase assays as dein liquid nitrogen and stored at - 70°C until assayed. scribed previously [13], measuring the formation of [14C]ciinduction of NO synthase in lung in vitro was studied using trulline from [U-l4c] L-arginine (20 yM, 0.6 yCi/ml) over rat lung slices incubated in Eagle's MEM containing 10% 5 min in the presence of 1mM unlabeled L-citrulline and FCS and 0.1 yg/ml polymyxin B. Following excision and 50 mM L-valine (to inhibit arginase, [13]). Assays were perfusion with sterile PBS via the pulmonary artery, lungs validated by determining that the [14C]citrulline formation were sliced in two directions at 90" at a setting of 1 mm in a was inhibited by 1 mM NGmonomethyl-L-arginine. Because McIlwain tissue chopper, before dispersal in medium and of the absence of large amounts of hemoglobin in lung slices distribution among 20 (4.5 cm2) welldrat lung, 2 ml/well. incubated in vitro and in the extract of 5774 cells, we were Following a 6-h incubation with compounds the slices were able to use the spectrophotometric NO synthase assay [23] removed into 1.5-ml microfuge tubes, frozen in liquid N2 which measures the oxidation of oxyhemoglobin by NO and stored at - 70 "C until assayed. [241. Other studies were carried out using macrophages. For this, thioglycollate-elicited peritoneal macrophages were ob- The Ca2+dependence of the measured activity was assessed tained from mice as described [22],being harvested 4 days by inclusion of 1 mM EGTA to chelate Ca2+. Activity after the injection of thioglycollate. In studies on induction measured was related to the protein concentration deterof NO synthase in these macrophages in vivo, compounds mined by the bicinchinonic acid assay [25] using bovine were injected as described above, 6 h before harvesting the serum albumin as standard, and expressed as pmol citrulmacrophages. In all studies the preparations were macro- line formed/min per mg extract protein. phage-enriched by adherence for 1 h followed by vigorous washing with PBS before use. For studies of induction in v i m the adherent cells (approximately 106 cells in 1 ml 3 Results RPMi 1640 containing 10% FCS and 50 yg/ml gentamycin, in 4.5 cm2 wells in 12-well plates) were incubated for a 3.1 Induction of NO synthase and toxicity in vivo further 6 h in the presence of compounds before removal of Mouse lung was found to contain Ca2+-dependent NO medium and extraction for assay of NO synthase. synthase constitutively (2.0 k 0.64 pmol/min per mg proJ774 monocytic leukemia cells were grown in RPMI 1640 tein, n = 5). There was no significant effect of any of the medium containing 10% FCS, 100 U/ml penicillin G and different inducers used in this study on the Ca2+-dependent Table 1. Induction of &*+-independent NO synthase in vivo by inducers of nonspecific resistancea)

Treat men L group

NO synthasc (pmollmin per mg protein)

Lethargy ( -

Control (sali tic) TDM ( 4 mg/kg) MPL. ( 4 mglkg) TDM + MPL ( 4 : 4 mglkg) MDP(4 mg/kg) MDP t MPL (4 :4 mg/kg) LPS (4 tnglka) LPS + MDP + TDM ( 4 : 4 :4 mglkg)

*

Piloerection

Diarrhea

on on

+) 017

013

*

017 013 013

on

313 313 6th

016

016

314 23

IN 113

014 013

*

015 015

015 015

515 515

013 013 0th 1I4 113 515

0.20 f 0.31 0.34 f 0.50 0.10 f 0.84 3.96 0.67 2.53 f 0.36 4.78 +. 1.43 5.01 +. 0.40 6.06 0.24

717

Loss o f righting reflex

013

515

013 016 014

013 515 515

The observations were carried out over 6 h, and NO synthase activity was determined at 6 h following injection of the agents shown. The activities are expressed as mean f SEM.

Nitric oxide synthase induction and nonspecific resistance

Eur. J. Immunol. 1992. 22: 2303-2307

NO synthase. In contrast, lungs from control mice contained only a low and variable activity of Ca2+-independent NO synthase which was, however, greatly increased following LPS administration (Table 1).

Neither MPL nor TDM alone had any significant effect on the activity of Ca2+-independent NO synthase, but when they were administered together (4 mg/kg of each), a large, highly significant induction of the Ca2+-independent NO synthase was observed which did not differ from the induction caused by LPS (Table 1). MDP at the same dosage also caused a 10-fold increase in Ca2+-independent NO synthase activity.The combination of MPL and MDP at the same doses (4 mg/kg) also had a synergistic effect on the induction of NO synthase.

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Thioglycollate-elicited peritoneal macrophages contained only Ca2+-independent NO synthase, and this activity increased 4-fold following administration of LPS of mice (Fig. 2). Similarly, MDP in vivo induced a 2.5-fold increase in the Ca2+-independent NO synthase activity in these macrophages. However, as in the lung, ThrMDP administration had no significant effect (Fig. 2).

3.2 Induction of NO synthase in vitro Rat lung slices maintained in tissue culture for 6 h contained only Ca2+-independent NO synthase activity. The

i

Lethargy, piloerection, diarrhea and loss of the righting reflex were studied in these animals as gross measures of the pathological changes observed in response to endotoxin and some other bacterial products. Interestingly, the correlation between toxicity and NO synthase induction was poor: although MDP MPL, LPS alone and LPS + MDP + TDM all caused a similar induction of NO synthase, the three groups exhibited dramatic differences in pathological signs, from mild lethargy only (MDP MPL) to extreme lethargy, piloerection, diarrhea and loss of the righting reflex (LPS + MDP TDM). Moreover,TDM + MPL caused a substantial induction of NO synthase but no signs of toxicity (Table 1).

+

+

+

A dose-response curve was obtained for NO synthase induction in mouse lung 6 h after intraperitoneal administration of different doses of MDP (Fig. 1).MDP caused a dose-dependent induction of Ca2+-independent NO synthase with a half-maximal effect achieved at 4.5 mg/kg and a maximal induction similar to that caused by LPS or by LPS + MDP + TDM (Table 1).In contrast, there was no significant change in the enzyme activity following treatment of mice with ThrMDP at up to 10 mg/kg (Fig. 1).

--

---

Control

MDP THRMDP LPS (10) (1 0) (4) DOSE( mgkg body weight)

Figure 2. Induction of NO synthase in mouse peritoneal macrophages in vivo by muramyl peptides. The data are NO synthase activities in thioglycollate-elicited peritoneal macrophages taken 6 h after intraperitoneal injection of MDRThrMDP or LPS at the doses shown, expressed as mean ? SEM (n = 3).

ThryDPQ

c -

0

0.01

0.1

1.o

10

PEPTIDE CONCENTRATION (pg/rnl)

Figure 1. Induction of Ca2+-independent NO synthase in mouse lung in vivo by two muramyl peptides. The data are the NO synthase activities, in the absence of Ca2+, from lungs taken 6 h after intraperitoneal injection of MDP or ThrMDP, expressed as mean k SEM (n = 4).

Figure 3. Induction of NO synthase in rat lung in vitro by muramyl peptides. The data are NO synthase activities in lung slices following 6 h exposure to MDP or ThrMDP at the concentration shown, as mean ? SD from one experiment, representative of three similar experiments.

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M. Palacios, R. G. Knowles and S. Moncada

Eur. J. Immunol. 1992. 22: 2303-2307

Table2. Induction of NO synthase in 5774 murine monocytic leukemia cells and thioglycollate-elicited murine peritoneal macrophages1)

It is clear that all agents or combinations of agents which induce nonspecific resistance also induce Ca2+-independent NO synthase, whereas those which fail to induce nonspecific resistance also fail to induce NO synthase. In particular, substitution of the alanyl residue in MDP by a threonyl residue resulted in the loss of its ability to induce NO synthase in the lung in vivo or in vitro and in macrophages in vivo, in parallel with the reported loss of its ability to induce nonspecific resistance, while retaining its adjuvant effects [ll, 121. Similarly, whereas the LPS derivative MPL and another bacterial product TDM both fail to induce either NO synthase or nonspecific resistance alone, administration of both together causes a synergistic induction of both NO synthase and nonspecific resistance. These findings support the hypothesis that induction of the L-arginine: NO pathway is an important effector mechanism in nonspecific immunity.

NO synthase activity (pmoUniin per mg protein) 5774 cells Peritoneal macrophages

Control

LPS

(10 pg/ml) (20 pglrnl)

MDP ThrMDP (20 pg/rnl)

o* 0 53 -t 15* 0k 0 0t 0

11.1 k 1.78 34.3 3 . w 11.5 t 1 5 9 12.3 1.06

* *

a) NO synthase activity (pmol/min per mg protein) from the cytosol of monocytcs/macrophages, after 24 and 6 h of incubation with different inducers (see Sect. 2.2). The values shown are the means k SEM. n = 3. Significant difference from control: *, p

Enhancers of nonspecific immunity induce nitric oxide synthase: induction does not correlate with toxicity or adjuvancy.

A range of bacterial products and related synthetic compounds, either alone or in combination, enhance nonspecific resistance to infection and tumors...
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