Receptor-Mediated Activation of Murine Peritoneal Macrophages by Antithrombin III Acts as a Costimulatory Signal for Nitric Oxide Synthesis

CELLULAR IMMUNOLOGY ARTICLE NO.

188, 33– 40 (1998)

CI981337

Receptor-Mediated Activation of Murine Peritoneal Macrophages by Antithrombin III Acts as a Costimulatory Signal for Nitric Oxide Synthesis Jae-Yong Kwak,* Sang-Youel Park,*,† Myung Kwan Han,‡ Hye-Soo Lee,§ Myung-Hee Sohn,¶ Uh-Hyun Kim,‡ John R. McGregor,\ Wolfram E. Samlowski\,1 and Chang-Yeol Yim* *Department of Internal Medicine, ‡Department of Biochemistry, ¶Department of Nuclear Medicine, §Department of Clinical Pathology, Chonbuk National University Medical School, Chonju, Chonbuk 560-182, Korea; †Department of Veterinary Medicine, Chonbuk National University Veterinary School, Chonju, Chonbuk 560-182, Korea; and \Cancer Immunotherapy Program, Huntsman Cancer Institute and Department of Internal Medicine (Hematology/Oncology), University of Utah Medical School, Salt Lake City, Utah 84132 Received February 20, 1998; accepted June 11, 1998

cascades (1). Antithrombin III (ATIII)2 is one of the major inhibitors of activated clotting proteases, including thrombin and factors IXa, Xa, XIa, and XIIa. This a2-glycoprotein has a molecular weight of approximately 58,000 (2, 3) and is found in the plasma and extracellular fluid in high concentrations (150 –350 mg/L) (4). The mechanism by which ATIII inactivates clotting proteins is thought to be due to the interaction of a critical L-arginine residue in ATIII with the active serine center of the clotting protein (5). The rate of ATIII–thrombin interactions is markedly enhanced by the binding of heparin to an e-lysine residue in ATIII (3, 6). This interaction appears to result in a conformational change which exposes the enzyme-binding Larginine and therefore enhances the potency of ATIII (7, 8), without altering the stoichiometry of the reaction (3, 6). In vivo, heparin-like substances on endothelial cells result in the selective activation of antithrombin at the blood– endothelial interface, protecting these surfaces against clot formation (9, 10). Once ATIII– thrombin complexes are formed, these complexes are released from the endothelium into the circulation and cleared via the liver, where they are degraded (11). A specific cellular mechanism for uptake of serine protease inhibitor (SERPIN)– enzyme complexes via SERPIN–enzyme complex receptors has been identified (12). Little is yet known about the potential for protease inhibitors to act as signaling molecules for other biologic processes. Since ATIII is a major protease inhibitor in the coagulation system, we designed experiments to evaluate whether ATIII could influence NO

We evaluated the effect of antithrombin III (ATIII), a serine protease inhibitor (SERPIN), on induction of nitric oxide (NO) synthesis in murine peritoneal macrophages. Incubation of macrophages with ATIII plus interferon-g (IFN-g) but not ATIII alone induced nitrite accumulation (a metabolite of NO) in a dose-dependent manner. Expression of the inducible nitric oxide synthase isoform was confirmed by Western blot. NO synthesis was inhibited by NG-monomethyl-Larginine, by complexing ATIII with thrombin or by rabbit anti-human ATIII antiserum. Addition of polymyxin B to macrophage cultures failed to inhibit ATIII/IFN-g-induced NO synthesis, excluding lipopolysaccharide contamination. 125I-ATIII bound to macrophages in a dose-dependent, specific, and saturable manner, with a Km of ;7.1 nM. Our results demonstrate that ATIII, but not ATIII/thrombin complex, acts to costimulate macrophage activation and NO synthesis via a novel receptor mediated mechanism, which may indicate a role for SERPINs in macrophage activation. © 1998 Academic Press

INTRODUCTION Complex and apparently overlapping systems of protease inhibitors have evolved to protect host tissues from damage by enzymes, such as those released by macrophages and neutrophils as a part of immune responses during infection and inflammation, as well as during activation of the complement and clotting

2 Abbreviations used: ATIII, antithrombin III; NO, nitric oxide; IFN-g, interferon-g; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; TNF, tumor necrosis factor-a; SERPIN, serine protease inhibitor; PEM, peritoneal exudate macrophages; MLA, NG-monomethyl-L-arginine; SEC, serine protease inhibitor– enzyme complex; A2M-R, a2-macroglobulin receptor.

1

To whom correspondence and reprint requests should be addressed at 4c 416 Division of Hematology/Oncology, University of Utah, 50 N. Medical Drive, Salt Lake City, UT 84132. Fax: 801-5855469. E-mail: [email protected]. 33

0008-8749/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

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synthesis by macrophages. We report here that native ATIII, but not ATIII complexed to thrombin, can act as a costimulatory signal with IFN-g to induce NO synthesis by macrophages through a novel receptor-mediated pathway which appears independent of lipopolysaccharide (LPS)-induced cell activation. MATERIALS AND METHODS Animals. Specific pathogen-free BALB/c mice (6 – 8 weeks of age) were obtained from Harlan SpragueDawley (Indianapolis, IN) and housed at the Chonbuk National University Hospital Animal Care Facility or the University of Utah Vivarium. Mice were maintained under guidelines established by the Chonbuk National University Hospital and University of Utah Animal Care Committees, which also approved experimental protocols. Mice were age and sex matched at the onset of each experiment. All experiments described in this paper were performed at least twice with highly concordant results. Macrophages. Macrophages were obtained from mice injected with 2 ml of sterile 3% thioglycollate (Difco Laboratories, Detroit, MI). Four days after injection, peritoneal exudate cells were harvested by peritoneal lavage with 6 ml of ice-cold phosphate-buffered saline (PBS)/mouse. These cells were washed twice and then suspended at 2 3 106 cells/ml in RPMI 1640 medium supplemented with 5% fetal calf serum (Hyclone Laboratories, Inc., Logan, UT), 100 units/ml penicillin G (Sigma Chemical Co., St. Louis, MO), 50 mg/ml streptomycin (Sigma), and 2 mM glutamine (Sigma) (working medium). After a 2-h incubation in 75cm2 culture flasks (a final volume of 20 ml/flask) (Becton Dickinson Labware, Oxnard, CA) at 37°C in a humidified 95% air/5% CO2 atmosphere, nonadherent cells were removed by repeated washing with ice-cold PBS. Adherent cells (peritoneal exudate macrophages, PEM) were harvested using a cell scraper (Baxter S/P brand diSPo cell scraper, Baxter Health Care Corporation, McGraw Park, IL) and resuspended at 106 cells/ml in working medium for further experiments. Macrophage cultures. PEM (106 cells/ml in working medium) were cultured with or without various reagents including recombinant mouse IFN-g (Genzyme, Cambridge, MA), LPS (endotoxin from Escherichia coli serotype 0128:B12, Sigma), NG-monomethyl-L-arginine (MLA, Chem-Biochem Research Inc., Salt Lake City, UT), human or rat ATIII (Green Cross Co., Seoul, Korea, or Sigma), rabbit anti-human ATIII polyclonal IgG antibody (Daeil Biotech Co., Ltd., Seoul, Korea), purified rabbit IgG (Sigma), and polymyxin B (Sigma). ATIII/thrombin complex was made by incubating equimolar concentrations (equivalent to a final concentration of 12.5 IU/ml ATIII) together for 15 min prior to addition to macrophage cultures. All inhibitors were

reconstituted in medium and filter sterilized prior to addition to cell cultures. All reagents and media for tissue culture experiments were tested by Limulus amoebocyte lysate assay (detection limit 10 pg/ml; Whittaker Bioproducts, Walkersville, MD) to exclude LPS contamination. Nitrite measurement. Biologically produced NO is rapidly oxidized to nitrite and nitrate in aqueous solutions (13, 14). Nitrite concentrations in the cell-free culture supernatants, therefore, served as a reflection of NO production and were measured using a previously described colorimetric assay (15). Following a 48-h incubation in 96- or 24-well plates at 37°C in a humidified 95% air/5% CO2 atmosphere, nitrite concentration was measured in the cell-free culture supernatants derived from macrophage cultures. Briefly, 50-ml aliquots of the culture supernatants dispensed into 96-well microtiter plates (flat bottom) (Nunc, Roskilde, Denmark) were incubated with 100 ml of a 1:1 mixture of 1% sulfanilamide (Sigma) in 30% acetic acid and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride (Sigma) in 60% acetic acid at room temperature. After 5 min, absorbance was measured at 570 nm using a microtiter plate reader (MR700, Dynatech Laboratories Inc., Alexandria, VA). Concentrations were determined from a linear standard curve obtained from serial concentrations (6.25–200 mM) of sodium nitrite (Sigma) in working medium. Results of triplicate measurements were presented as means 6 SD. Western blot analysis for inducible nitric oxide synthase (iNOS) enzyme induction. Cells were homogenized in 100 ml of ice-cold lysis buffer (20 mM Hepes, pH 7.2, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin). The homogenates containing 20 mg of protein were separated on a 10% SDS–PAGE and transferred from slab gels to nitrocellulose sheets (Schleicher & Schuell, Dassel, Germany) in a Western blot apparatus (Bio-Rad, Hercules, CA) run at 50 V for 2 h. The nitrocellulose was blocked with 2% bovine serum albumin and then incubated for 4 h with 1 mg/ml antiiNOS antibody (Transduction Laboratories, Lexington, KY). The binding of antibody was detected with antimouse IgG conjugated to an alkaline phosphatase (Sigma). Immunoblots were developed using a phosphate substrate solution (Pierce, Rockford, IL). Assay of TNF secretion. TNF secretion by macrophages was measured by a modification of an enzymelinked immunosorbent assay (ELISA) (16) using a kit (Factor-Test-X mouse TNF ELISA kit, Genzyme Corporation, Cambridge, MA). The kit was a solid-phase ELISA employing the multiple antibody sandwich principle and was sensitive to TNF concentrations in medium above 15 pg/ml. Aliquots (0.1 ml) of the culture supernatants and standards were dispensed into a 96-

ACTIVATION OF NO SYNTHESIS BY ATIII PLUS IFN-g

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FIG. 1. Induction of NO synthesis in murine peritoneal macrophages by ATIII in the presence of IFN-g. Thioglycolate-elicited BALB/c mouse peritoneal macrophages were cultured in 96-well microtiter plates (2 3 105 cells in a final volume of 0.2 ml/well) with medium alone, 5 U/ml IFN-g, 25 ng/ml LPS, or LPS/IFN-g in the presence of increasing concentrations (0 –12.5 IU/ml) of ATIII. Following a 48-h incubation, nitrite concentration was measured in the cell-free culture supernatants as a reflection of NO synthesis. Results of triplicate samples are expressed as means 6 SD (A). As a control for endotoxin contamination of ATIII, parallel wells were cultured with 5 mg/ml polymyxin B. After 48 h, nitrite concentration was measured in the cell-free culture supernatants as a reflection of NO synthesis (B). Results of triplicate samples are shown as means 6 SD.

well microtiter plate (precoated with hamster antimouse TNF monoclonal antibody) and incubated at 37°C for 2 h. After washing the plate four times with a wash buffer, 0.1 ml of saturating concentrations of a horseradish peroxidase-conjugated goat polyclonal anti-mouse TNF antibody was added to each well. Following a 1-h incubation at 37°C, the plate was washed four times again and 0.1 ml of a substrate solution (a 1:1 mixture of 0.01 N hydrogen peroxide in buffered solution with tetramethylbenzidine in methanol) was then added which initiates a peroxidase-catalyzed color change that is subsequently stopped by acidification (0.1 ml of 1 M sulfuric acid). The absorbance measured at 450 nm was used to calculate the concentration of mouse TNF present in samples, based on a standard curve (from 0 to 2240 pg/ml TNF in working medium). Results are presented as means 6 SD of triplicate samples. Iodination of ATIII and binding study. Purified ATIII was iodinated with [125I]NaI (10 mCi/ml, Amersham, UK) using Iodo-beads (Pierce) according to the instruction manual. Radiolabeled ATIII had a specific activity of 3 3 104 cpm/ng protein. Murine macrophages (2 3 105 cells in a final volume of 0.2 ml/well) were incubated in a 96-well microtiter plate in working medium at 37°C for 60 min. After washing with PBS, increasing concentrations of 125I-labeled ATIII (125IATIII) were added to each well and incubated at 0°C

for 50 min. After washes with PBS and 0.2 N NaOH, cell lysates were harvested onto glass fiber filters (Flow Laboratories, McLean, VA). Radioactivity associated with each filter was determined by a gamma counter. RESULTS ATIII induces NO synthesis by PEM in the presence of IFN-g. We first tested whether human ATIII induces NO synthesis by macrophages. BALB/c mouse PEM were cultured in 96-well microtiter plates (at a final volume of 0.2 ml) with medium alone, IFN-g (5 U/ml), LPS (25 ng/ml), or LPS/IFN-g in the presence of increasing concentrations of ATIII (0 –12.5 IU/ml). After a 48-h incubation, nitrite accumulation was measured in the cell-free culture supernatants as a reflection of NO synthesis. Treatment of cells with ATIII alone, over a wide range of concentrations, did not induce significant nitrite production in the absence of IFN-g. In the presence of a fixed concentration of IFN-g (which by itself did not activate nitric oxide synthesis), addition of increasing concentrations of ATIII stimulated nitrite production in a dose-dependent manner (Fig. 1A). Addition of ATIII did not affect the low levels of PEM nitrite synthesis induced by LPS alone (;7 mM) or the near-maximal stimulation induced by LPS/ IFN-g (;70 mM). To verify that this was not an artifact of human ATIII in murine cells, rat ATIII (Sigma) was

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FIG. 2. Relationship of ATIII/IFN-g-induced nitrite synthesis to L-arginine:NO pathway and iNOS expression. Mouse macrophages were cultured with LPS (25 ng/ml)/IFN-g (5 U/ml) or ATIII (12.5 IU/ml)/IFN-g (5 U/ml) in the presence or absence of 0.5 mM MLA. Following a 48-h incubation, nitrite was measured in the cell-free culture supernatants as a reflection of NO synthesis (A). Results of triplicate samples are expressed as means 6 SD. Demonstration of iNOS expression in macrophages treated with ATIII/IFN-g. Macrophages were cultured in 24-well culture plates (2 3 106 cells in a final volume of 2 ml/well) with medium alone, 25 ng/ml LPS, or 12.5 IU/ml ATIII in the presence or absence of 5 U/ml IFNg. At 48 h, the cell free supernatants were collected and nitrite accumulation was measured (B). Results are expressed as the mean 6 SD of triplicate wells. Cells were lysed in electrophoresis buffer. Following SDS–PAGE gel electrophoresis, samples were evaluated for iNOS expression by Western blotting (inset).

also tested. A similar dose–response relationship between rat ATIII and NO synthesis was also observed following costimulation with IFN-g (data not shown). Human ATIII was used for all subsequent experiments. Control experiments revealed that nitrite synthesis induced by ATIII/IFN-g was not inhibited by addition of 5 mg/ml polymyxin B [which is known to bind and inactivate the activity of LPS lipid A (17)] excluding inadvertent endotoxin contamination (Fig. 1B). In contrast, addition of polymyxin B markedly inhibited LPS/ IFN-g-induced nitrite synthesis. As a further control, bacterial endotoxin was undetectable in reagents by Limulus amoebocyte lysate assay. Inhibition of ATIII/IFN-g-induced NO synthesis by MLA. A competitive NO synthase inhibitor, MLA (18), was used to establish that the nitrite detected in cell cultures was derived from the L-arginine:NO pathway. PEM were cultured with ATIII/IFN-g in the presence or absence of MLA (0.5 mM). Cultures incubated with LPS/IFN-g served as controls. Addition of MLA profoundly inhibited nitrite production in both experimental and control cultures (Fig. 2A). Since NO synthesis was not observed at baseline, these results suggested that the NO induced by ATIII/IFN-g was derived from the cytokine-inducible L-arginine:NO pathway.

Demonstration of iNOS induction in macrophages treated with ATIII/IFN-g. The possibility that iNOS expression was induced by ATIII/IFN-g was further confirmed by Western blotting. BALB/c mouse PEM (106 cells/ml) were cultured in 24-well plates with medium alone, ATIII (12.5 IU/ml) or LPS (25 ng/ml) in the presence or absence of IFN-g (5 U/ml). After a 48-h incubation, supernatant was removed and nitrite accumulation was measured. The residual cell pellet was lysed. After electrophoretic separation of cellular proteins on SDS–PAGE gels and transfer to nitrocellulose, blots were stained using an iNOS-specific antiserum. Treatment of cells with medium alone, IFN-g alone, LPS alone, or ATIII alone induced no or very small amounts of nitrite synthesis (,5 mM). In these groups, iNOS protein expression could not be detected. In the presence of IFN-g, both ATIII and LPS induced significant nitrite accumulation (25.0 6 1.7 and 79.1 6 2.1 mM, respectively). These cell populations stained strongly for iNOS enzyme, with a molecular size of 130 kDa (Fig. 2B). These results established that activation of macrophages with ATIII/IFN-g induces iNOS protein expression. Inhibition of macrophage activation by anti-ATIII antiserum. Further evidence that ATIII-induced nitric oxide synthesis acted through an LPS-independent pathway was provided in experiments using rabbit

ACTIVATION OF NO SYNTHESIS BY ATIII PLUS IFN-g

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FIG. 3. Inhibition of ATIII/IFN-g-induced NO synthesis by anti-human ATIII antibody. Mouse macrophages were cultured with IFN-g (5 U/ml)/ATIII (12.5 IU/ml). Serial dilutions of rabbit anti-human ATIII polyclonal IgG antibodies were added (A). Following a 48-h incubation, nitrite was measured in the cell-free culture supernatants as a reflection of NO synthesis. In a second experiment, mouse macrophages were cultured with IFN-g (5 U/ml)/LPS (25 ng/ml) or IFNg (5 U/ml)/ATIII (12.5 IU/ml) and incubated with saturating concentrations of rabbit anti-human ATIII polyclonal IgG antibodies (12.5 mg/ml) or an equivalent concentration of control rabbit IgG (B). Results are expressed as the mean 6 SD of triplicate samples.

anti-human ATIII polyclonal IgG antibodies. This experiment revealed that nitrite synthesis induced by ATIII/IFN-g was almost completely inhibited by addition of increasing concentrations of ATIII antiserum, with saturation above 12.5 mg/ml (Fig. 3A). NO production induced by LPS/IFN-g was not significantly affected (Fig. 3B). Preimmune rabbit IgG did not inhibit either ATIII/IFN-g- or LPS/IFNg-induced nitrite production. These results suggested that ATIII could act as a LPS-independent second signal to induce NO synthesis by murine macrophages. Effect of ATIII on TNF secretion by macrophages. Possible differences in signaling mechanisms during macrophage activation by ATIII and LPS were evaluated. BALB/c mouse PEM were cultured in 96-well microtiter plates with medium alone, LPS alone (25 ng/ml), or ATIII alone (12.5 IU/ml), without added IFN-g. After 48 h, TNF secretion into culture supernatants was measured. Nitrite accumulation was measured as a control for cell activation. LPS incubation of PEM cultures resulted in induction of both TNF secretion (Fig. 4A), as well as nitrite production (Fig. 4B). In contrast, ATIII by itself failed to induce measurable TNF secretion by murine peritoneal macrophages and did not induce appreciable NO synthesis. Formation of ATIII/thrombin complexes inhibits NO production. Formation of serine protease complexes with ATIII are thought increase receptor-mediated clearance of the complex (11). We therefore questioned

whether formation of ATIII complexes with serine proteases would alter iNOS costimulatory activity. Equimolar quantities of ATIII and thrombin (equivalent to 12.5 IU/ml ATIII) were coincubated for 15 min at 25°C. The ability of equivalent concentrations of complexed ATIII or native ATIII to stimulate NO synthesis were then tested. This experiment demonstrated that formation of ATIII/thrombin complexes completely abrogated the ability of ATIII to stimulate NO synthesis (Fig. 5). This finding established that uncomplexed ATIII was responsible for the majority of the costimulatory activity. ATIII binds to macrophages via a cell surface receptor. To establish the nature of the ATIII interaction with macrophages, increasing concentrations of 125I-ATIII were added to PEM. Parallel wells with addition of excess unlabeled ATIII (at a 100 nM concentration) served to assess binding specificity. After a 50-min incubation (on ice), PEM-bound radioactivity was measured. The results of this experiment demonstrated that binding was concentration dependent, saturable, and specific (Fig. 6). Semilogarithmic plots revealed half-maximal ATIII binding (Km) was 7.1 nM. At saturation, 32 fmol of ATIII bound to 106 macrophages, corresponding to 1.8 3 104 molecules bound/cell. DISCUSSION Experiments reported herein established that ATIII is a novel triggering signal which induces iNOS expres-

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FIG. 4. ATIII and LPS have differential effects on TNF secretion by murine macrophages. Peritoneal macrophages were cultured in microtiter wells (2 3 105 cells in a final volume of 0.2 ml/well) with medium alone, 25 ng/ml LPS, or 12.5 IU/ml ATIII (without IFNg). At 48h, TNF (A) and nitrite (B) concentrations were measured in the cell-free culture supernatants. Results are expressed as the mean 6 SD of triplicate wells.

sion by murine peritoneal macrophages. In the presence of IFN-g, ATIII induced high output NO synthesis by peritoneal exudate macrophages in a dose-dependent manner. Neither ATIII nor IFN-g alone were able to activate macrophages and induce significant NO synthesis. Both human and rat ATIII were able to costimulate NO synthesis, suggesting a conserved signal sequence in the ATIII molecule. No additional macrophage NO production was observed when ATIII was added to macrophages stimulated by LPS/IFN-g. These results may indicate that concentrations of IFN-g (5 U/ml) and LPS (25 ng/ml) employed in the present study already resulted in maximum macrophage NO synthesis. Alternatively, it is possible that ATIII costimulation employs the same receptor-binding mechanism or a shared intracellular signaling pathways as LPS, resulting in overlapping activation signals. Our subsequent experiments (discussed below) appeared to suggest differences in LPSand ATIII-induced cell activation, however. Addition of 0.5 mM MLA, a potent competitive inhibitor of NOS enzymes, to cell cultures in the presence of ATIII/IFN-g demonstrated a near complete inhibition of nitrite production, confirming the origin of nitrite from the L-arginine:NO pathway. Further proof that the inducible iNOS isoform was activated by ATIII was obtained from Western blot analysis, which demonstrated that both LPS and ATIII exposure of cells in the presence of IFN-g resulted in marked induction of iNOS enzyme expression.

FIG. 5. Effects of ATIII/thrombin complex formation on NO synthesis. ATIII/thrombin complexes were formed by incubating equimolar concentrations of both ATIII and thrombin at 25°C for 15 min. PEM were cultured with either 12.5 IU uncomplexed ATIII or ATIII/thrombin complexes (containing 12.5 IU ATIII) in the presence of 50 U/ml IFN-g. Control PEM were cultured in medium alone (control) or with IFN-g (50 U/ml) or ATIII (12.5 IU/ml) alone. After 48 h, nitrite accumulation in the cell-free culture supernatants was measured as a reflection of NO synthesis. This experiment was performed twice with similar results (expressed as the mean 6 SD of triplicate samples).

ACTIVATION OF NO SYNTHESIS BY ATIII PLUS IFN-g

FIG. 6. ATIII binding to macrophages. Mouse macrophages (2 3 105 cells in a final volume of 0.2 ml/well) were incubated with increasing concentrations of 125I-labeled ATIII on ice for 50 min. The plate was washed with PBS and 0.2 N NaOH. Cells were harvested onto glass fiber filters and their radioactivity was determined by a gamma counter. Parallel samples incubated with excess unlabeled ATIII served to establish binding specificity.

In order to exclude the possibility that inadvertent endotoxin contamination of ATIII, medium, or reagents contributed to NO synthesis, we performed control experiments in the presence of 5 mg/ml polymyxin B, which is known to bind and inactivate the lipid A portion of LPS (17). Polymyxin B did not inhibit ATIIIinduced NO synthesis. Furthermore, bacterial endotoxin was undetectable in reagents by Limulus amoebocyte lysate assay. We also noted that ATIII, but not LPS, lost activity after a 7-day storage at 4°C (data not shown), which also argues against inadvertent endotoxin contamination (endotoxin is stable under these conditions). Thus we concluded that the interaction of ATIII with cells appeared to be mediated via a mechanism distinct from LPS activation of PEM. Further evidence to support this difference was provided by incubating PEM with anti-human ATIII antibody, which markedly inhibited nitrite synthesis induced by ATIII/IFN-g, but not cell activation induced by LPS/IFN-g. Preimmune rabbit IgG (a control) did not inhibit either ATIII/IFN-g- or LPS/IFN-g-induced nitrite production. Further evidence that ATIII and LPS differ in their capacity to induce cellular activation signals was derived from experiments evaluating TNF induction by the two agonists. LPS is known to activate TNF secretion by macrophages (19). Unlike LPS, ATIII alone did not induce TNF secretion. When taken together, our data suggest that ATIII costimulation of NO synthesis in macrophages is independent of LPS and appears to involve different signaling pathways from those activated by LPS.

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Previous investigators have established that formation of ATIII/thrombin complexes induces clearance via hepatocyte SERPIN– enzyme complex (SEC) receptors (20). The SEC receptor is capable of interacting with a substantial range of protease inhibitor/protease complexes, such as a-1-antitrypsin/elastase, ATIII/thrombin, heparin cofactor II/thrombin, plasminogen activator/plasminogen activator inhibitor, and a-1-anti-chymotrypsin/cathepsin-G complexes (21). We therefore tested whether ATIII/thrombin complexes would have increased activity in activating NO synthesis by murine PEM. To our surprise, while native ATIII showed substantial costimulatory activity, NO synthesis was completely abrogated by formation of ATIII/thrombin complexes. This finding suggested that the effects of the native ATIII molecule were most likely not mediated by the SEC receptor, but rather via a distinct signaling pathway. A further experiment was performed to characterize whether ATIII activated macrophages by interacting with a specific cell surface receptor. Classical binding studies, testing the ability of 125I-ATIII to bind to macrophages were performed. Incubation of macrophages with 125I-ATIII resulted in specific, concentration-dependent binding of ATIII. ATIII binding sites were half-maximally occupied (Km) at a total ATIII concentration of 7.1 nM. Receptor binding appeared to be saturable at approximately 50 nM ATIII. Calculations suggested a Vmax of 32 fmol/106 cells. This experiment clearly established that ATIII interactions with macrophages were receptor mediated. We estimate that approximately 1.8 3 104 ATIII receptors are expressed per murine peritoneal macrophage. We have identified a novel signal that is capable of costimulating macrophage NO synthesis. This receptor-mediated process is activated by ATIII in the presence of IFN-g as a costimulatory signal and is distinct from known LPS-activated signaling pathways. Further work will be needed to characterize the ATIII receptor itself, as well as its physiologic function.

ACKNOWLEDGMENTS This work was supported by a grant (No. 2) from Kye Nam, Kim Jae Jung Memorial Fund, and a grant from the National Institutes of Health (CA67404).

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