Antioxidants attenuate endotoxin-induced activation of alveolar macrophages

Antioxidants attenuate endotoxin-induced activation of alveolar macrophages Cynthia Mendez, MD, Iris Garcia, BS, and Ronald V. Maier, MD, FAGS, Seattle, Wash.

Background. Endotoxin (lipopolysaccharide [LPS]) stimulation of tissue-fixed macrophages induces the generation of toxic oxidants. However, recent studies also implicate redox changes in both the signal transduction pathways for cytokine genes and the generation of physiologicaUy active arachidonic acid metabolites. Because cytokines and arachidonic acid metabolites initiate and perpetuate deleterious sfstemic inflammatory responses, we tested whether macrophage activation could be modulated by antioxidants. Methods. Rabbit alveolar macrophages were obtained by bronchoalveolar lavage, isolated, treated with the antioxidants vitamin E or N-acetylcysteine (NAG), and stimulated with an optimal dose of LPS (10 ng/ml). Assays were performed for tumor necrosis factor (TNF), procoagulant activity, and prostagtandin E2. Total cellular RNA was extracted for Northern blot analysis of TNF messenger RNA. Results. Exposure of the macrophage to the antioxidants vitamin E and NAG inhibited TNF production,, accumulation of TNF messenger RNA, procoagulant activity expression, and prostaglandin E2 production. Conclusions. Macrophage signal transduction of LPS is dependent on the generation of reactive oxygen intermediates that can be blocked both at the level of the lipid membrane (vitamin E) and at the in#acellular level (NAG). This suggests a potential therapeutic role for antioxidants in disease states such as adult respiratory distress syndrome and multiple organ failure syndrome, which are characteffzed by excessive macrophage activation. (SURGERY1995;118:412-20.) From the Depmlment of Surgery, University of Washington, Seattle, Wash.

DESPITE ADVANCESIN THE CAREof the critically ill patient, adult respiratory distress syndrome (ARDS) and multiple organ failure syndrome continue to be significant causes o f morbidity and death. O n e o f the key features in the origin o f these disorders is an overexuberant systemic acuvation of the i m m u n o i n f l a m m a t o r y system. The tissue-fixed m a c r o p h a g e is a key c o m p o n e n t in the svstemic inflammatory response a n d produces many mediators thai initiate, perpetnate, and m o d u l a t e this response, t Thus modification of the activated macrop h a g e response represents a p o t e n t i a l p o t e n t therapeutic avenue for treatment o f the critically ill surgical patient. In critically ill patients with sepsis and ARDS. antioxidants are d e p l e t e d a n d reactive oxygen intermediates are elevated in both the lungs a n d circulating leukocytes. 2'3 Also, treatment with exogenous antioxidants has b e e n found to decrease mortality in Supported bv National Institutes of Health grants GM-07037and GM45873. Presented at the Fiflv-sixthAnnual Meeting of the Sociew of University Surgeons, Denver, Colo.. Feb. 9-11. 1995, Repnnt requests: Cynthia Mendez. MD. Depamnent of SurgelT, Universityof Washington, Mail Stop RF-25, 1959N.E. Pacific.Seattle, WA 98195. Copyright 9 1995 by Mosby-YearBook, Inc. 0039-6060/95/$3.00 + 0 11/6/65011 412

SURGERY

animal models o f sepsis. 4'5 In addition to their well-known toxic extracellular effects, reactive oxygen intermediates have recently b e e n postulated to be involved in intracellular signaling pathways. Antioxidants have b e e n shown to decrease endotoxin-stimulated cytokine p r o d u c t i o n in whole blood. 6' 7 Redox changes have been showu to be involved in the activation of certain inflammatory cells, including the endothelial cell, lymphocyte, a n d monocyte, s-t2 Several specific intracellular signaling mechanisms have been f o u n d to be influenced by r e d o x changes including calcium shifts, tyrosine kinase activation, a n d the nuclear transcription factor NF-kB. 13' 14 Because o f the recently described potential role of r e d o x changes in cellular a n d intracellular activation, we hypothesized that antioxidants could m o d u l a t e the endotoxin-induced excessive activation o f the macrophage. The effects of two well-defined antioxidants, lipid soluble vitamin E and cell p e r m e a b l e N-acetylcysteine (NAG), on lipopolysaccharide (LPS)-stimulated alveolar m a c r o p h a g e activation were investigated. As markers of activation we assayed several central mediators implicated in the pathophysiology o f multiple organ failure syndrome a n d ARDS: t u m o r necrosis factor (TNF), prostaglandin E 2 (PGE2), and p r o c o a g u l a n t activity (PEA).

Surgery Volume 118, Number 2

Mendez, Garcia, Maier

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Animals. Male New Zealand white rabbits (1.5 to 2 kg) were purchased from R and R Rabbittry (Stanwood, Wash.). Rabbits were housed at the University of Washington vivarium and received food and water ad libitum. Animals were used within 5 days of delivery. Animal use protocols were approved by the University of Washington Animal Care Committee and met the standards of the National Institutes of Health for animal care and use.

Material. Escherichia coli 01 ll:B LPS, vitamin E in the form of ct-tocopherol' succinate, and N-acetylcysteine (NAC) were purchased from Sigma Chemical Company (St. Louis, Mo.). Rabbit TNF and glyceraldehyde 3-phosphate dehydrogenase (GADPH) complementary DNAs were gifts from T. Shirai (Asahi Chemical Co., Tokyo, Japan) and A. Clowes (University of Washington, Seatde, Wash:), respectively. [32p] deoxycytidine triphosphate was purchased from Amersham (Arlington Heights, Ill.). Phosphate-buffered saline solution was purchased from Gibco (Grand Island, N.Y.). RPMI-1640 was purchased from Biowhittaker (Walkersville, Md.). Macrophage isolation and culture. Alveolar macrophages were obtained in the following manner. Rabbits were killed with an overdose of pentobarbital, the trachea was isolated by using sterile technique, and in situ bronchoalveolar lavage was performed by using six 40 ml aliquots of normal saline solution at 4 ~ C. The bronchoalveolar lavage fluid was then centrifuged, and the cellular pellets were resuspended at 1 x 106 cells/ml in RPMI-1640 with 50 p g / m l gentamicin. Previous studies have shown that this method of isolation yields

a population of cells that is greater than 95% macrophages) 5 Preparations with more than 5% neutrophils were excluded from study. For functional assays including TNF, PCA, and PGE2 production, 1 x 106 cells (1 ml) were plated into each well o f a 12-well tissue culture plate (Costar, Cambridge, Mass.). For RNA extraction 1.0 x 107 cells were plated in 100 m m plates ( C o m i n g Inc., Coming, N.Y.). Macrophage viability was assessed at 24 hours via trypan blue exclusion. Experimental design. Culture wells containing 1 x 106 cells were stimulated with a previously determined optimal dose of LPS (10 n g / m l ) after a 4~hour exposure to normal medium without antioxidants or medium with the antioxidant vitamin E (50 to 100 p g / m l ) or NAC (0.1 to 10 m m o l / L ) . Before stimulation with LPS some cells were washed with RPMI-1640 to remove the antioxidant from the external medium. The cells were incubated at 37 ~ C in 5% CO2 for 18 hours. Supernatants were then collected and stored at -70 ~ C until TNF and PGE 2 assays were performed. The remaining macrophage monolayers were frozen in phosphate-buffered saline solution at -70 ~ C for subsequent PCA assay. TNF assay. TNF was measured with the biologic cytotoxicity assay of Flick and Gifford. 16 Transformed mouse fibrobiasts (NCTC clone L929; American Type Culture Collection, Rockville, Md.) were pretreated with actinomycin D (Sigma Chemical Company) at 1 p g / m l for 15 minutes in RPMI-1640 supplemented with 5% horse serum (Hyclone Laboratories, Logan, Utah) and added at 5 x 104 cells/well to serial dilutions of the supematants collected from the stimulated macro-

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Fig. 2. Effect of NAC on TNF (units/ml) production by macrophages in response to LPS. Exposure to NAC inhibited TNF production (closed bars), and this inhibition persisted after washing NAC from tbe medium before LPS stimulation (hatched bars). #p <_0.01 compared with LPS alone (n = 6). phages. T h e plates were then incubated in 5% CO2 at 37 ~ C for 24 hours. T h e cells were then fixed a n d stained in 0.1~ crystal violet in 20% methanol. Dried monolayers were solubilized in 0.1 m m o l / L sodium citrate in 50% methanol, a n d the absorbance was read at 550 nm with a kinetic microplate reader (Molecular Devices, Menlo Park, Calif.). O n e umt o f TNF activity was defined as that activity that p r o d u c e d 50% cytolysis of the L929 monolayer. A linear regression was p e r f o r m e d to d e t e r m i n e the p o i n t between serial dilutions where the 50% cytolysis e n d p o i n t occurred. The E. coli 0111:B4 LPS was tested against the L929 cell line a n d showed no direct cytotoxicity. Vitamin E and NAC were tested and f o u n d not to interfere with the assay. PC& assay. Alveolar m a c r o p h a g e PCA was determ i n e d by a modified two-stage amidolytic assay with the c h r o m o g e n i c substrate $2222 (Kabi. Franklin. Ohio). 17 Frozen m a c r o p h a g e monolayers were thawed at r o o m temperature, sonicated, a n d iced. Each 100 pl sample containing the lysate o f 105 cells was run in duplicate in a 96-well plate (Costar). Standards were p r e p a r e d from rabbit brain thromboplastin with calcium ( 11.6 m m o l / L). PC& o f the reconstituted thromboplastin was assigned a value o f 100.000 units/100 pl. Dilutions of the standard solution were p r e p a r e d to give a range from 100 to 25.000 units PCA/100 pl. The reaction mixture (100 pl) containing 400 pg $2222 a n d 2 units Proplex-T (Baxter Healthcare. G a r d e n Grove. Calif.) in phosphate-buffered saline solution was a d d e d to each sample or standard. Absorbance at 405 n m was read for 15 minutes on the kinetic microplate reader. Reaction rates, expressed as m e a n optical density p e r minute. were plotted with a log-log curve fitting routine (SOFT-

max; Molecular Devices) with typical correlation coefficients from 0.98 to 0.99. PGEz assay. PGE 2 levels were d e t e r m i n e d with an enzyme-linked immunoassay kit (Perspective Diagnostics, Cambridge, Mass.). Supernatants from stimulated macr o p h a g e (100 pl) or standard solutions with a known a m o u n t of PGE 2 (100 pl) were a d d e d in duplicate to 96-well plates coated with a goat anti-rabbit antibody. A rabbit polycolonal antibody to PGE2 was a d d e d to each well, a n d the plate was kept at 4 ~ C overnight. Alkaline p h o s p h a t a s e - c o n j u g a t e d PGE,2 (100 pl), which competes with the PGE2 in the samples for anti-PGE2 antibody b i n d i n g sites, was a d d e d to each well, a n d the plate was placed at 4 ~ C for 3 hours. T h e plate was then washed, para-nitrophenyl p h o s p h a t e substrate (300 pl) was a d d e d to each well, a n d the plate was incubated at 37 ~ C for I hour. Stop solution was then a d d e d , a n d the absorbance was read at 405 n m on the kinetic microplate reader. T h e a m o u n t o f color present was inversely p r o p o r t i o n a l to the concentration of PGE2 in the sample. A 4-parameter logistic standard curve was g e n e r a t e d (SOFTmax) a n d used to d e t e r m i n e the concentration of PGE 2 in each sample. R N A isolation a n d N o r t h e r n blot analysis. After 3 hours o f stimulation with LPS, total cellular RNA was isolated from alveolar m a c r o p h a g e s (15 x 106 cells) by guanidium t h i o c y a n a t e - p h e n o l c h l o r o f o r m extraction as described.18 Fifteen m i c r o g r a m samples of RNA were electrophoresed in d e n a t u r i n g 1% fornlaldehyde-agarose gels a n d transferred to nylon m e m b r a n e s (Nytran; Schleicher a n d Schuell, Keene, N.H.). T h e m e m b r a n e s were cross-linked by ultraviolet light a n d subjected to N o r t h e r n blot analysis via hybridization with [32p]

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deoxycytidine triphosphate labeled TNF or GADPH c o m p l e m e n t a r y DNA by r a n d o m priming. T h e membranes were then subjected to autoradiography at -70 ~ C. M e m b r a n e s also u n d e r w e n t p h o s p h o r i m a g i n g to quantitate the a m o u n t o f radioactivity that correlates directly with the a m o u n t of RNA present. Statlstieal analysis. Values are expressed as m e a n _+ SEM. Data were analyzed by ANOVA with post hoc testing by Fisher's p r o t e c t e d least significant difference. Significance is defined as a p value of less than 0.05.

RESULTS Cytotoxicity o f antioxidants. Neither vitamin E n o r NAC at the dosages used in the study were f o u n d to be cytotoxic to the m a c r o p h a g e s after a 24-hour exposure as assessed by m o r p h o l o g i c analysis a n d trypan blue exclusion. T N F p r o d u c t i o n . LPS (10 n g / m l ) - s t i m u l a t e d TNF p r o d u c t i o n by m a c r o p h a g e s was inhibited when the cells were exposed to lipid soluble vitamin E at concentrations of 50 p g / m l to 1 m g / m l (Fig. 1). This inhibition was statistically significant (p < 0.01, vitamin E ->50 p g / m l ) when c o m p a r e d with TNF p r o d u c t i o n in response to LPS alone (Fig, 1). The inhibition of TNF p r o d u c t i o n by vitamin E persisted, albeit to a lesser degree, after the cells were washed and vitamin E was removed from the external m e d i u m before LPS stimulation (p < 0.05, vitamin E ->50 p g / m l ) . Exposure of the m a c r o p h a g e s to the cell p e r m e a b l e antioxidant NAC was also f o u n d to inhibit LPS-induced TNF p r o d u c t i o n (Fig. 2) (p < 0.01, NAC ->1 m m o l / L ) . T h e inhibitory effects o f NAC were overall less complete a n d r e m a i n e d essentially u n c h a n g e d after this antioxidant was removed from the extracellular m e d i u m before stimulation by LPS. N o r t h e r n blot analysis was p e r f o r m e d to d e t e r m i n e whether the effect on m a c r o p h a g e TNF p r o d u c t i o n by the antioxidants vitamin E and NAC was due to inhibition of gene activation a n d TNF messenger RNA (mRNA) synthesis. T h e r e was a low level of constitutive TNF m R N A p r o d u c t i o n that was significantly augm e n t e d by t r e a t m e n t of the macrophages with LPS (10 n g / m l ) . This increase in TNF mRNA was abrogated by t r e a t m e n t with both vitamin E (100 p g / m l ) a n d NAC (1 a n d 10 m m o l / L ) (Fig. 3). Although to a lesser degree, the inhibition of TNF m R N A p r o d u c t i o n was also seen when the antioxidants were r e m o v e d from the m e d i u m , a n d correlated well with the effects n o t e d on functional e n d - p r o d u c t TNF production. T h e m a t c h e d mRNA levels of the h o u s e k e e p i n g gene GADPH were used to normalize the TNF m R N A levels a m o n g the various experimental conditions. 19 P h o s p h o r i m a g i n g of the N o r t h e r n blot m e m b r a n e to quantitate the mRNA response revealed a 93% reduction in LPS-induced TNF mRNA by vitamin E (100 pg)

Fig. 3. Northern blot analysis for TNF mRNA in macrophages. Exposure to vitamin E and NAC led to decreased levels of TNF mRNA, and this effect persisted after washing anfioxidants from the medium.

when present in the external m e d i u m at the time of LPS exposure and a 58% reduction in TNF mRNA after removal of extracellular Vitamin E, when c o m p a r e d with I 2 S stimulation alone (Fig. 4). T r e a t m e n t of the macrophages with NAC (10 r e t o o l / L ) led to a 61% reduction o f TNF mRNA when c o m p a r e d with LPS alone. Removing NAC from the m e d i u m before LPS exposure showed a persisten t 58% reduction in TNF mRNA. PC_&. Alveolar m a c r o p h a g e levels of PCA were increased 10 times above baseline by exposure of the m a c r o p h a g e s to LPS (10 n g / m l ) . T h e i 2 S - i n d u c e d increase in PC& was inhibited by a 4-hour incubation of the m a c r o p h a g e s with vitamin E (Fig. 5). T h e inhibition of LPS-stimulated PCA by vitamin E reached statistical significance at all concentrations tested (50 to 1000 p g / m l ) (p < 0.01). T h e inhibitory effects of vitamin E on PC& persisted after this antioxidant was removed from the m e d i u m before LPS stimulation. Exposure of tile m a c r o p h a g e s to NAC alone resulted in a small increase in PC& over baseline. However, NAC t r e a t m e n t for 4 hours led to a d o s e - d e p e n d e n t inhibition o f LPS-induced PC& that was also seen after washing the cells before LPS stimulation (Fig. 6). The inhibition of PC& reached statistical significance at 10 m m o l / L concentration (p < 0.05). P G E 2 p r o d u c t i o n . Production of the arachidonic acid metabolite PGE2, a major m o d u l a t o r of the iminune response, was also m e a s u r e d as a m a r k e r for

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Fig. 4. Phosphorimaging of Northern membranes, normalized for loading, from Fig. 3. Exposure of macrophages to vitamin E led to 93% reduction in TNF mRNA levels. Exposure to NAC (10 mmol/L) led to 61% reduction in TNF mRNA. Removing either antioxidant from the medium before LPS stimulation was associated with 58% reduction in TNF mRNA levels compared with LPS stimulation alone. 12"

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Fig. 5. Effect of vitamin E on PCA production by macrophages in response to LPS. Data are expressed as fold increase over nonstimulated control. Exposure to vitamin E inhibited PCA expression (closedbars), and this inhibition persisted after washing vitamin E from the medium before LPS stimulation (hatched bars). *p -< 0.05, #p -< 0.01 compared with LPS alone (n = 6). arachidonic acid metabolism. LPS stimulation of the macrophages led to a 20-fold increase in PGE2 levels compared with baseline. Similar to TNF and PC& both vitamin E a n d NAC exposure led to a marked decrease in LPS-stimulated PGE2 production (Figs. 7 a n d 8).

DISCUSSION Endotoxin (LPS)-induced activation of the macrophage has b e e n partially characterized a n d appears to

involve the interaction of LPS with LPS-binding protein in serum and subsequent b i n d i n g of this complex to the cell surface receptor CD 14. 20 However, the intracellular pathways for LPS activation are less well understood. Activation of macrophage inflammatory genes are believed to involve both tyrosine kinase activation and phosphorylation a n d translocation of the transcription factor NF-kB. 21' 22 V i t a m i n E (c~-tocopherol) is a lipid soluble hydro-

Surgery Volume 118, Number 2

Mendez, Garcia, Maier

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phobic antioxidant that becomes intercalated in cellular lipid bilayer membranes. Vitamin E is known to protect cells u n d e r g o i n g lipid peroxidation by interrupting the p r o p a g a t i o n of free oxygen radicals in the membrane. % O n the basis of the same reaction involved in the inhibitio n of lipid peroxidation, vitamin E facilitates the conversion of superoxide to hydrogen p e r o x i d e for subsequent potential detoxification to water (Fig. 9). In addition, a n d potentially most important, vitamin E

functions as a direct scavenger of reactive oxygen intermediates. T h e cell p e r m e a b l e NAC is also a well-known antioxidant with multiple functions. NAC's antioxidant activity is in part due to its ability to donate cysteine groups a n d thus act as substrate to increase the intracellular levels of the e n d o g e n o u s intracellular antioxidant glutathione. 14 Gluthathione is used as an electron recipient by the enzyme glutathione peroxidase to detoxify hy-

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Fig. 9. Intracellular pathways for reactive oxygen intermediates and proposed sites of action for antioxidante vitamin E and NAC. Vitamin E that is embedded in the lipid membrane can facilitate conversion of superoxide (O2-) to hydrogen peroxide (H202). NAC increases intracellular glutathione (GSH) levels, thus increasing the cell's ability to detoxify H2O 2 by its conversion to H20 via the enzyme glutathione peroxidase. In addition, both vitamin E and NAC can function as direct scavengers of reactive oxygen intermediates (OH). AA, Arachidonic acid; GSSG, oxidized glutathione; SOD, superoxide dismutasel

d r o g e n p e r o x i d e and form innocuous water (Fig. 9). Cysteine groups from NAC are also used by other r e d o x sensitive enzymes with oxidized sulfhydryl groups. Furthermore, NAC itself can also function as a direct reactive oxygen intermediate scavenger (Fig. 9) similar to vitamin E. T h e r e are several mechanisms by which the antioxidante studied could have suppressed the LPS-induced p r o d u c t i o n of m a c r o p h a g e inflammatory mediators. The fact that TNF m R N A is suppressed by vitamin E and NAC suggests a pretranscriptional ( m e m b r a n e interaction or signal transduction) level or transcriptional level

of regulation by reactive oxygen intermediates. Antioxidants have been previously shown to affect the activation of various tyrosine kinases a n d NF-kB mobilization in lymphocytes. 8' 10, 13, ]4 However, antioxidants have also b e e n shown to inhibit endothelial cell E-selectin expression in an NF-kB i n d e p e n d e n t manner. ]] As m e n t i o n e d previously both tyrosine phosphorylation and NF-kB mobilization have a role in m a c r o p h a g e activation, but it is uncertain which o f these mechanisms is most i m p o r t a n t in the control of m a c r o p h a g e inflammatory m e d i a t o r production. F u r t h e r m o r e , antioxidants may well block a yet unidentified enzyme with an

Surgery Volume 118, Number 2 active site (possibly c o n t a i n i n g a cysteine g r o u p ) t h a t is m o r e p r o f i c i e n t w h e n i n t h e o x i d i z e d state. T h e effect o f t h e a n t i o x i d a n t s s t u d i e d o n P C A exp r e s s i o n m a y also b e a c c o u n t e d f o r b y i n h i b i t i o n o f t h e a c t i v a t i o n a n d m o b i l i z a t i o n o f NF-kB. It is k n o w n t h a t NF-kB is i n v o l v e d i n t h e t r a n s c r i p t i o n a l a c t i v a t i o n o f tiss u e f a c t o r o r PCA. I n a d d i t i o n , t h e i n h i b i t i o n o f PGE2 p r o d u C t i o n , as s h o w n by t h e s e a n t i o x i d a n t s , h a s b e e n s h o w n to d e c r e a s e P C A p r o d u c t i o n via d e c r e a s e d levels o f i n t r a c e l l u l a r cyclic a d e n o s i n e m o n o p h o s p h a t e . 24 T h e i n h i b i t o r y effects o f v i t a m i n E a n d N A C o n PGE2 p r o d u c t i o n is m o r e p r e d i c t a b l e b e c a u s e t h e g e n e r a t i o n o f a r a c h i d o n i c acid m e t a b o l i t e s is d e p e n d e n t o n a series o f o x i d a t i v e r e a c t i o n s . V i t a m i n E h a s also b e e n s h o w n to i n h i b i t t h e l i b e r a t i o n o f l a b e l e d a r a c h i d o n i c acids f r o m the cellular lipid membrane suggesting an inhibition of p h o s p h o l i p a s e A 2. 25 I n t e r e s t i n g l y , t h e i n h i b i t i o n o f m a c r o p h a g e a c t i v a t i o n by a n t i o x i d a n t s i n o u r m o d e l o v e r r i d e s t h e p o t e n t i a l s t i m u l a t o r y effects t h a t d e c r e a s i n g PGE2, a w e l l - k n o w n i n h i b i t o r o f m a c r o p h a g e activat i o n , h a s i n this system. T h e fact t h a t t h e i n h i b i t i o n o f m a c r o ~ ) h a g e a c t i v a t i o n m e a s u r e d i n this s t u d y persists a f t e r w a s h i n g t h e cells b e f o r e s t i m u l a t i o n w i t h LPS s u p p o r t s t h e c o n c e p t o f a predominant membrane linked or intracellular mecha n i s m . T h e fact t h a t v i t a m i n E is s o m e w h a t less effective as a n i n h i b i t o r o f a c t i v a t i o n w h e n w a s h e d away suggests t h e p r e d o m i n a n t site o f v i t a m i n E's e f f e c t is a t t h e level of the lipid membrane. A portion of the vitamin E that is loosely a d h e r e n t to t h e cell surface, w i t h o u t b e i n g e m b e d d e d i n t h e cell m e m b r a n e , m a y p a r t i c i p a t e b y n e u t r a l i z i n g e x t r a c e l l u l a r reactive o x y g e n i n t e r m e d i a t e s that function in a paracrine or autocrine fashion. The lack o f a s i g n i f i c a n t d i f f e r e n c e i n t h e d e g r e e o f i n h i b i t i o n b y N A C a f t e r r e m o v i n g it f r o m t h e m e d i u m provides additional support for a completely intracellular l o c a t i o n as t h e site o f a c t i o n f o r this a n t i o x i d a n t . T h e overall lesser effect o f N A C also s u p p o r t s t h e c o n c e p t t h a t r e d o x b a s e d r e a c t i o n s a t t h e level o f t h e cell m e m b r a n e d u r i n g i n i t i a t i o n o f Cell signal t r a n s d u c t i o n is critical to m a c r o p h a g e activation. I n c o n c l u s i o n , it a p p e a r s t h a t t h e LPS s t i m u l a t o r y p a t h w a y i n t h e m a c r o p h a g e possesses a signal t r a n s d u c t i o n m e c h a n i s m t h a t is sensitive to r e d o x c h a n g e s . T h e e x a c t p o i n t i n t h e s i g n a l i n g p a t h w a y p o s s e s s i n g this s e n , sitivity is c u r r e n t l y u n s p e c i f i e d . H o w e v e r , its e x i s t e n c e suggests a p o s s i b l e r o l e f o r a n t i o x i d a n t s i n disease states s u c h as A R D S a n d m u l t i p l e o r g a n f a i l u r e t h a t a r e c h a r a c t e r i z e d by excessive m a c r o p h a g e activation. REFERENCES

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3. Pacht ER, Timerman AP, Lykens MG, et al. Deficiency of alveolar fluid glutathione in patients with sepsis and adult respiratory syndrome. Chest 1991;100:1397-403. 4. Brackett DJ, Lerner MR, Wilson MF. Dimethyl sulfoxide antagonizes hypotensive, metabolic, and pathologic responses induced by endotoxin. Circ Shock 1991;33:15663. 5. Peck MD, Alexander JW. Survival in septic guinea pigs is Influenced by vitamin E, but not by vitamin C in enteral diets. JPEN 1990;15:433-6. 6. DeForge LE, Fantone JC, Kenney JS, Remick DG. Oxygen radical scavengers selectively inhibit interleukin 8 production in human whole blood. J Cfin Invest 1992;90:2123-9. 7. DeForge LE, Preston AM, Takeuchi E, et al. Regulation of interleukin 8 gene expression by oxidant stress. J Biol Chem 1993;268:25568-76. 8. Schreck R, Rieber P, Baeurele PA. Reactive oxygen intermediates as apparently widely used messengers of the NF-kB transcription factor and H1V-1. EMBOJ 1991;10:2247-58. 9. Marui N, Offerman MK, Suerlick R, et al. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-seusitive mechanism in human vascular endothelial cells. J Clin Invest 1993;92:1866-74. 10. Kelly KA, Hill MR, Youkhana K, et al. Dimethyl sulfoxide modulates NF-kB and cytokine activation in lipopolysaccharidetreated murine macrophages. Infect Immun 1994;62:3122-8. 11. Faruqi R, de la Motte C, DiCorleto PE. alpha-Tocopherol inhibits agonist-induced monocytic cell adhesion to cultured human endothelial cells. J Clin Invest 1994;94:592~600. 12. Ziegler-Heitbrock HWL, Sternsdorf T, Liese J, et al. Pyrrolidine dithiocarbamate inhibits NF-kB mobilization and TNF production in human monocytes. J Immunol 1993;151:6986-93. 13. Schreck R, Meier B, Mannel DN, et al. Dithiocarbamates as potent inhibitors of nuclear factor kB activation in intact cells.J Exp Med 1992; 175:1181-941 14. Staal FJ, Anderson MT, Staal GE, et al. Redox regulation of signal transduction: tyrosine phosphorylation and calcium influx. Proc Natl Acad Sci 1994;91:3619-22. 15. Maier RV, Hahnel GB, Pohhnan TH. Endotoxin requirements for alveolar macrophage stimulation. J Trauma 1990; 30:$4957. 16. Flick DA, Gifford GE. Comparison of in vitro cell cytotoxic assays for tumor necrosis factor. J Immunol Methods 1984; 68:167-75. 17. Surprenant YM, Zuckerman SH. A novel microtiter plate assayfor the quantitation ofprocoagulant activity on adherent monocytes, macrophage, and endothelial cells. Thromb Res 1989;53:339-46. 18. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal Biochem 1987;162:156-9. 19. TsoJY, Sun XH, Kao TH, et al. Isolation and characterization of rat and human glyceraldehyde 3-phosphate dehydrogenase cDNA's: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 1985;13:2485-502. 20. Tobias PS, Soldau K, Kline L, et al. Cross-linking of lipopolysaccharide (LPS) to CD-14 on THP-1 cells mediated by LPS-binding protein. J Immunol 1993;150:3011-21. 21. Vincenti MP, Burrell TA, Taffet SM. Regulation of NF-kB activity in murine macrophages: effect of bacterial lipopolysaccharide and phorbol ester. J Cell Physiol 1992;150:204-13. 22. Weinstein SL, Gold MR, DeFranco AL. Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages. Proc Natl Acad Sci 1991;88:4148-52. 23. Packer L. Interactions among antioxidants in health and disease: vitamin E and its redox cycle. Proc Soc Exp Biol Med 1992; 200:271~6. 24. Williams JG, Garcia I, Maier RV. Prostaglandin E2 mediates lipopolysaccharide-induced macrophage procoagulant activity

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DISCUSSION Dr. Ori D. Rotstein (Toronto, Canada). You show that the antioxidants alone, in the absence of I,PS, are able to induce some TNF a n d procoaga~lant activity expression, and I wonder how you might put this into the whole scheme of how antioxidants might be working in the cell. My second question is related to methodologic issues a n d presentation of data. There was a clear reduction in the level of the TNF mRNA in the antioxidant-u-eated groups, and you stated during your talk that there was n o change in the total RNA loading. However, in the original blots there seemed to be some reduction in the GAPDH levels in the treated cells. Will you tell us about the relative levels of TNF mRNA after normalization for GAPDH? You suggested that reactive oxygen species were neutralized by the antioxidants a n d that is how they might be working. In response to LPS, macrophages actually d o n ' t produce very m u c h in the way of reactive oxygen species. There is some suggestion that, in fact, the alteration in the redox state of the cell overall may be more important than its ability to alter the a m o u n t of reactive oxygen species. Have you done these kinds of studies in cells fi-om patients with chronic granulomatosis disease where they might alter the redox state of the cell without actually needing to inhibit reactive oxygen species?

Surgery August 1995 Dr. Mendez. In response to your first question regarding how tbe antioxidants alone would increase PC&, it is intriguing. Preliminary data by o t h e r investigators suggest that altet~ adon in redox state enhances gene expression for PCA. Regarding phosphorimaging, we did normalize for GADPH. The inhibitory effects of vitamin E produced 93% reduction in the mRNA levels when it was in the medium a n d about 60% when it was removed. NAC inhibited about 60% in both washed and unwashed cells. In regard to the redox state of the cell, NAC functions (in addition to scavenging free oxygen radicals) to increase the cellular level of glutathione. We are currently measuring the redox state of our cells to define how that relates to LPS activation. Dr. H. H a n k Sims (Providence, R.I.). Have you run the critical control showing that the level of antioxidants you used prevents oxidant production in the alveolar macrophages in response to phorbol esters? Second, do you have any insight into which reactive oxygen species are involved here? Does this involve induction of the Klebanoff pathway, which could be proved with azide, or more distal products of the oxidative burst, which could be proved with taurine? Dr. Mendez. We have not actually measured the production of warious reactive oxygen species and the effect of the antioxidants. As far as exactly which reactive oxygen intermediate is involved, I c a n ' t really hypothesize just yet. It could be just the general redox state of the celL Antioxidants could affect the enzymatic abilities of the various enzymes involved in signal transduction.