INFECTION AND IMMUNITY, June 1988, p. 1593-16010019-9567/88/061593-09$02.00/0Copyright C) 1988, American Society for Microbiology

Endotoxin-Associated Protein: Interleukin-l-Like Activity on SerumAmyloid A Synthesis and T-Lymphocyte Activation

MARGARET A. JOHNS,' JEAN D. SIPE,' L. B. MELTON,2 T. B. STROM,2 AND W. R. McCABE1*

Maxwell Finland Laboratory,* Thorndike Memorial Laboratory, and Division of Infectious Diseases and Arthritis, BostonCity Hospital, Boston University School of Medicine, Boston, Massachusetts 02118,1 and Renal Division, Department of

Medicine, Beth Israel Hospital, Boston, Massachusetts 022152

Received 2 November 1987/Accepted 1 March 1988

Bacterial endotoxins or lipopolysaccharides (LPS) elicit a variety of biologic activities in intact animals andvarious in vitro systems. LPS from most gram-negative bacteria have appeared to have similar biologicactivities regardless of the species of origin or method of preparation of the LPS. More recent studies havesuggested differences in the effects of protein-rich as opposed to protein-free LPS in inducing mitogenesis oflymphocytes from endotoxin-resistant C3H/HeJ mice. These studies examine other activities of endotoxin-associated protein (EAP), purified to less than 0.007% contamination with LPS, and demonstrate that thismaterial has activity mimicking some of the effects of interleukin-1 (IL-1). EAP proved to be as potent as LPSin eliciting rises in concentrations of serum amyloid A (SAA) and was active in both endotoxin-sensitive (CF1)and endotoxin-resistant (C3H/HeJ) mice. In contrast to LPS, which mediates its SAA-inducing activity byrelease of an inducer (IL-1) from LPS-stimulated macrophages, EAP appeared to act directly to induce SAAproduction, in that incubation with macrophages failed to increase its activity. EAP also exhibited IL-i-likeactivity in the lymphocyte-activating factor assay when both CF1 and C3H/HeJ thymocytes and macrophageswere tested. The lymphocyte-activating factor activity of EAP was not blocked by addition of polymyxin B. Inaddition, EAP exerted stimulatory activity on resting human T lymphocytes, costimulated with Sepharose-bound anti-CD3 monoclonal antibody 64.1, comparable to that observed with purified human monocyte IL-1.These studies indicate that proteins from procaryotic cells may act as cytokines for some eucaryotic cells.

Endotoxin or lipopolysaccharide (LPS), the major compo-nent of the cell wall of gram-negative bacilli, is composed oflipid A attached through a basal core portion to a terminalpolymeric oligosaccharide structure (14). LPS has beenreported to induce an extensive and broad variety of effectson the immunoregulatory, humoral mediator, and hemato-poietic systems in humans and numerous animal species (20,21). Differing types of LPS containing variable amounts ofprotein, depending on the method of LPS isolation, havebeen used in studies delineating the biologic activities ofLPS. In general, studies over the past 10 to 15 years haveindicated that the lipid A portion of LPS is responsible formost of the biologic activities induced by endotoxin (re-viewed in reference 25), but even the lipid A portion of LPSmay contain significant amounts of protein (22).

Certain strains, C3H/HeJ (HeJ) and C57BL/1OScCR, ofinbred mice have been shown to be genetically resistant tothe lethal effects of LPS (4, 30). Subsequent studies byMorrison et al. (19) and Sultzer and Goodman (32) demon-strated that, although protein-free preparations of LPS in-duced mitogenesis of splenic lymphocytes from most strainsof mice, no mitogenic response was observed with spleniclymphocytes from HeJ mice. In contrast, protein-rich LPSpreparations and partially purified protein extracts of LPSwere shown to induce mitogenesis of splenic lymphocytesfrom HeJ mice (19, 29, 32). Several other biologic activities,distinct from those elicited by lipid A, have been shown to beinduced by these endotoxin-related proteins (6, 10, 11, 18,24). Similarly, LPS induces elevations in concentrations ofthe acute-phase reactant serum amyloid A (SAA), when

* Corresponding author.

injected into most strains of mice, whereas LPS-nonre-sponder HeJ mice have been found to be extremely resistantto SAA elevation by protein-free LPS but do exhibit a

significant increase in SAA levels after injection of protein-containing LPS (36).The recognition that the C3H/HeJ strain of mice is refrac-

tory to many of the biologic effects of lipid A (6, 10, 11,18, 19, 24, 29-32), due to a mutation at the Ips locus on

chromosome 4 (37), has provided a tool for examination ofthe biologic activity of cellular components other than LPSof gram-negative bacilli. Proteins may be associated with or

contaminate various endotoxin preparations. In previousstudies, proteins coextracted with LPS have been termedlipid A-associated protein and endotoxin protein, but in thiscommunication they are called endotoxin-associated protein(EAP) for distinction from other moieties. These LPS-associated proteins, extracted by various methods, havebeen shown to exhibit similar polyacrylamide gel electropho-retic patterns and to react with antiserum to outer membraneproteins (OMPs) (9). Use of LPS-refractory HeJ mice hasallowed demonstration of mitogenic stimulation and polyclo-nal activation of splenocytes from this mouse strain by EAP(19, 29, 32). EAP has also been shown to deliver a stimula-tory signal to macrophages, distinct from that of lipid A,which results in differentiation of macrophages into killercells (6). Macrophages also play a central role in mediatingmany of the effects of LPS as a result of the production of a

central mediator, interleukin 1 (IL-1) (36). IL-1 production isan extremely sensitive indicator of the effects of LPS, andIL-1 has been shown to induce SAA synthesis by hepa-tocytes (16, 26, 28, 35). The present study examines theIL-1-like activity of EAP in inducing SAA synthesis by a

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mechanism distinct from the initiation of SAA synthesis byprotein-free LPS, using the lymphocyte-activating factor(LAF) assay in endotoxin-sensitive (CF1) and endotoxin-resistant (C3H/HeJ) mice. We also examine the stimulatoryactivity of EAP on resting human T lymphocytes in theresting T-cell costimulation assay. These findings demon-strate that some proteins from procaryotic cells may act ascytokines for various eucaryotic cells.

MATERIALS AND METHODS

Mice. Male and female C3H/HeJ (HeJ) mice, aged 4 to 10weeks, were obtained from Jackson Laboratories, Bar Har-bor, Maine, and female CF1 mice, 6 to 8 weeks old, wereobtained from Charles River Laboratories, Wilmington,Mass. The mice were fed and watered ad libitum.

Reagents. RPMI 1640 medium containing 25 mM HEPES(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) andglutamine (RPMI), Hanks balanced salt solution (HBSS),and heat inactivated fetal calf serum (FCS) were obtainedfrom M. A. Bioproducts, Walkersville, Md. Gentamicin andpolymyxin B were obtained from Sigma Chemical Co., St.Louis, Mo., and penicillin and streptomycin were obtainedfrom Burroughs-Wellcome, Research Triangle Park, N.C.Tritiated thymidine (6.7 Ci/mmol) was obtained from NewEngland Nuclear Corp., Boston, Mass. Flexible polyvinylchloride microtiter plates were obtained from Cooke Engi-neering Co., Alexandria, Va., and Linbro tissue cultureplates were from Flow Laboratories, McLean, Va. Phyto-hemagglutinin (PHA) was obtained from Burroughs-Well-come.LPS. Endotoxin was extracted from Salmonella minne-

sota S218 by the method of Westphal et al. (38), employingextraction with hot aqueous phenol. These preparationscontained less than 3% (usually less than 1%) protein asdetermined by the method of Lowry et al. (13).EAP. Protein-rich endotoxin was extracted from S. min-

nesota S218 by the method of Boivin et al., utilizing coldtrichloroacetic acid (3). EAP was extracted from this Boivinendotoxin by the hot-phenol extraction of Westphal andco-workers as modified by Goodman and Sultzer (11, 32).This preparation, as noted by others (9, 19), is heteroge-neous, consisting primarily of OMPs. The EAP preparationsused in these studies were composed primarily of threemajor protein bands of 40,300, 37,600, and 18,600 Mr, similarto observations of others (9, 10), when analyzed by poly-acrylamide gel electrophoresis on a 10 to 18% sodiumdodecyl sulfate gradient gel. No bands indicative of LPSwere detectable by silver staining of the polyacrylamide gels.Analysis of the EAP preparations for contaminating LPSrevealed less than 0.007% by weight, as determined bymouse lethality assays using actinomycin D-sensitized CF1mice, and less than 0.002% by weight by the Limulusamoebocyte lysate assay (performed by courtesy of RonBerzosky, M.A. Bioproducts).

Preparations of EAP were insoluble in a variety of di-luents. Material used for studies was prepared by wettingwith a small amount of ethanol (0.05 ml of 95% ethanol in0.95 ml of distilled water or RPMI 1640 or phosphate-buffered saline, etc.), made up to a 1-mg/ml solution, andsonicated into a fine suspension with a 5-s burst from aBranson sonifier.

Heat, trypsin, and pronase treatment of EAP. EAP wastreated with trypsin and pronase to evaluate the effects ofdigestion with these proteolytic enzymes on SAA-inducing

activity. EAP (1 mg/ml) was suspended in 0.01 M phosphatebuffer (pH 7.5), and 35 ,ug of trypsin (2x recrystallized;Sigma) or pronase (grade B; Calbiochem, La Jolla, Calif.)was added per ml. These mixtures were incubated at 37°Cfor 24 h, and enzyme activity was terminated by heating at90°C for 45 min. SAA activity was assayed as describedbelow after injection of 1.0 pg of enzyme-treated EAP intoC3H/HeJ mice. An additional sample was heated to 100°Cfor 30 min and tested similarly.Mouse peritoneal exudate cells. CF1 or HeJ mice were

injected intraperitoneally with 3 ml of 3% sterile thioglyco-late. Peritoneal exudate cells were harvested 5 or 6 days laterby peritoneal lavage with RPMI medium, washed three timesin HBSS with 15% FCS, and then suspended in RPMI with20% FCS. The cells were allowed to adhere to glass slidesfor 3 h in RPMI containing 20% FCS. After 3 h at 37°C in 5%C02, the nonadherent cells were removed by washing withHBSS, and the cover slips were covered with RPMI con-taining 2% FCS, 100 ,ug of streptomycin per ml, and 100 U ofpenicillin per ml, with and without various concentrations ofEAP or LPS, for 40 h at 37°C in 5% CO2.Human macrophages. Human peripheral blood mononu-

clear cells were harvested from heparinized whole blood byFicoll-Hypaque (Ficoll, Type FP; Pharmacia, Inc., Piscat-away, N.J.) gradient centrifugation. The cells were washedthree times in HBSS with 15% homologous human serumand then suspended in RPMI with 20% homologous humanserum to approximately 106 cells per ml. Samples of 0.25 mlof the cell suspension were laid on sterile glass cover slips(22 by 22 mm) and incubated for 3 h at 37°C in 5% CO2. Thecover slips were washed with HBSS to remove nonadherentcells and were then covered with RPMI with 2% homologoushuman serum, 100 mg of streptomycin per ml, and 100 U ofpenicillin per ml and incubated for 5 days at 37°C in 5% CO2with one change of medium after 2 or 3 days. At day 5, theadherent cells had the cytologic appearance of differentiatedmacrophages. The medium was replaced with the samemedium containing various concentrations of strain S218LPS or EAP and incubated for 24 h. All experiments utilizedidentical cover slip monolayers for treated cells and con-trols. Cells in several areas of the control monolayers werecounted, and the total number of macrophages per culturewas estimated.SAA determination. SAA concentration in serum samples

was measured by solid-phase radioimmunoassay. This assaywas based on competitive binding between SAA in serumsamples and a standard amount of 125I-labeled amyloid Afibril protein (0.5 ng, 10 pCi/pug) (27). Flexible microtiterplates containing rabbit anti-amyloid A antibodies bound tothe wells were used as the solid phase. Groups of four micewere injected intraperitoneally with various doses of EAP,LPS, or macrophage supernatants and bled 18 h later fromthe retroorbital plexus. Serum samples were incubated at60°C for 1 h before testing to expose antigenic sites forbinding. Samples of 200 p.l, diluted in 2% casein barbitalbuffer (25 mM, pH 8.6), were added to individual wells,followed by 20 ,ul of labeled standard. The plates were thenincubated at 4°C for 16 h, washed in water, cut into individ-ual wells, and counted in a gamma scintillation spectrome-ter. The results expressed were the averages of triplicatedeterminations for individual mice, and the arithmetic meanand standard deviation for each group of four mice weredetermined.LAF assay. The LAF assay was carried out utilizing

modifications of the technique described by Gery et al. (7, 8).(i) Thymocyte preparation. Six- to 8-week-old mice were

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sacrificed by CO2 asphyxiation, and their thymus glandswere removed surgically. The thymuses were placed in a60-mm petri dish containing RPMI 1640 with 10% FCS, 0.05mM 2-mercaptoethanol, and penicillin-streptomycin (100 Uand 100 ,ug/ml). Thymocytes were gently teased out of thecapsules by using sterile forceps and were then transferredto a sterile 60-gauge screen fitted into the top of a 50-mlcentrifuge tube containing cold RPMI with additives. Thecells were pressed through the screen into the tube by usingthe plunger of a 5-ml syringe. The thymocytes were thencounted, washed once (870 x g, 10 min), and suspendedin cold RPMI with additives to 1.5 x 107 cells per ml. Try-pan blue exclusion was used to assess the viability of thethymocytes.

(ii) Assay preparation. Linbro 96-well tissue culture plateswere used for the LAF assay. Samples (macrophage super-natants or EAP) were diluted 1:5, 1:50, and 1:500 in RPMI.A purified human IL-1 standard, obtained as a 1:100 dilutionfrom Charles Dinarello (Tufts University School of Medi-cine), was used as a positive control. Portions (50 ,ul) of eachsample, standard, or medium control (RPMI) were added towells in triplicate, followed by 50 ,ul of PHA (0.2 ,ug per well)and 100 plI of thymocyte suspension. The plates were in-cubated at 37°C in 5% CO2 for 3 days; then 0.5 ,uCi of[3H]thymidine in RPMI was added, and the plates werereincubated for 18 h further. Cells were harvested ontoglass-fiber filters (23-995 grade 934 AH; M.A. Bioproducts)by using a Mini Mash Cell Harvester (M.A. Bioproducts).The filter paper wells were punched out and placed infilmware tubes (Nalgene, Rochester, N.Y.). A 3-ml volumeof Betafluor scintillation fluid (National Diagnostics) wasplaced in each tube, and the tubes were sealed by heat,placed in scintillation vials, and counted in a Tracor AnalyticCounter, model Delta 300.

Resting T-cell costimulation assay. (i) Cell preparation.Plateletphoresis by-products from the Dana-Farber CancerInstitute were used as a source of mononuclear cells. Periph-eral blood mononuclear cells were isolated by Ficoll-Hy-paque (Pharmacia) density centrifugation. These cells werewashed three times in RPMI 1640 medium containing 5 mMHEPES buffer and 10% heat-inactivated pooled normalhuman AB serum (Biobee, Inc., Boston, Mass.) (completemedium). Cells were then suspended in 200 ml of completemedium at a density of approximately 3 x 106/ml andcultured overnight in 650-ml plastic culture flasks (Costar,Cambridge, Mass.) in a 5% CO2 incubator at 37°C. Nonad-herent cells were removed by gently decanting the culturemedium and saved for further accessory-cell depletion asdescribed below.

(ii) Accessory-cell depletion. A combination of adherenceand lytic procedures were used to complete accessory-celldepletion. These procedures have been previously described(39, 40). Briefly, 2.0 x 106 plastic-nonadherent cells wereapplied to acid-washed nylon-wool columns (1.2 g of nylonwool per 20-ml syringe) and incubated at 37°C for 1 h. Cellswere then eluted from the columns with 20 ml of completemedium at a flow rate of -0.1 ml/min. Accessory-cell lysiswas effected by incubating the eluted cells for 1 h in 5 mML-leucine methyl ester (39) dissolved in RPMI 1640. Afterwashing three times with complete medium, cells weretreated with saturating quantities of monoclonal antibodies4F2 and LB3.1 (anti-DR) for 45 min, after which rabbitcomplement (Pel-Freez) was added with constant rocking for1 h at 37°C. Recovered cells were washed three times andsuspended in complete medium at approximately 2.0 x 106cells per ml in 650-ml plastic culture flasks. These were

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FIG. 1. Effect of LPS on SAA inducer production by humanperipheral blood monocytes. Each point represents the mean SAAconcentration for a group of four C3H/HeJ mice 18 h after theintraperitoneal injection of 0.5 ml of cell-free supernatant from strainS218 LPS plus differentiated human blood mononuclear cells (0) oran equivalent amount of strain S218 LPS alone (0). Small quantitiesof LPS incubated with human macrophages induced substantialSAA response; 250 jig of LPS alone was required to stimulate SAAproduction.

incubated overnight at 37°C, and nonadherent cells (T cells)were removed the next day by gentle decanting. Cells werethen cultured as described below.

(iii) Cell culture. Cultures consisting of 5.0 x 104 T cellswere incubated in 96-well flat-bottomed plates (Costar) incomplete medium. EAP was dissolved in complete medium,filtered twice through 0.2-,um-pore-size disposable filters(Gelman, Ann Arbor, Mich.), and added to the cultures atthe indicated concentrations. Sepharose-bound anti-CD3monoclonal antibody 64.1 (Sepharose-64.1) was prepared aspreviously described (33) and added to the cultures at agel-to-medium ratio of 1:280. Purified monocyte IL-1 wasgenerously supplied by Charles Dinarello. The final volumeof each well was 0.1 ml. Cells were cultured for 72 h in a 5%CO2 incubator at 37°C and approximately 94% humidity.During the last 4 h, [3H]thymidine (New England NuclearCorp.) was added at 1 puCi per well. At the end of 72 h, thecells were harvested and [3H]thymidine incorporation wasmeasured with a Beckman LS2800 scintillation counter.

RESULTS

Effect of LPS and of LPS-stimulated human macrophagesupernatants on SAA induction. LPS-induced SAA synthesishas been shown to require intermediate production of SAAinducer (IL-1) by macrophages (28). HeJ mice do not mountan acute-phase SAA response to LPS, except in very largedoses, because their macrophages do not respond to protein-free LPS and fail to produce the SAA inducer. In vitrostimulation of SAA-inducer production by LPS has beendemonstrated in numerous studies employing mouse andhuman macrophages (26, 28; reviewed in reference 35), andSAA inducer produced after LPS stimulation of macro-phages from LPS-responding species has been shown toinduce SAA production in LPS-resistant HeJ mice. Nearlyall of the previous studies of LPS effects on SAA productionhave employed protein-depleted LPS from Escherichia coliK235. Initial studies indicated that the minimal and maximalSAA-stimulating doses of the protein-poor S. minnesotaLPS were 0.1 and 50 p.g, respectively, in endotoxin-sensitiveCF1 mice and 100 and 1,000 to 5,000 p.g in endotoxin-resistant HeJ mice. The protein-poor phenol-water LPS

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FIG. 2. Induction of SAA synthesis in C3H/HeJ mice by EAP.Each point represents the mean SAA concentration for a group offour C3H/HeJ mice 18 h after the intraperitoneal injection of 0.5 mlof either cell-free supernatants from EAP plus C3H/HeJ peritonealexudate cells (@) or the equivalent amount of EAP in RPMI medium(0).

preparation from S. minnesota S218 (the species used as a

source of EAP) induced only moderate increases in SAAconcentration after intraperitoneal injection of as much as

250 pig (Fig. 1) into C3H/HeJ mice. In contrast, supernatants(initially containing 0.25, 2.5, or 25 pig of LPS and SAAinducer which had been produced) of human macrophagesincubated with the same LPS for 24 h were 100- to 1,000-foldmore active in inducing SAA. Supernatants from humanmacrophages incubated with 2.5 pg of S. minnesota S218LPS resulted in SAA elevations in HeJ mice to 100 ,ug/ml, a

level comparable to that observed after intraperitoneal injec-tion of 250 pig of the same LPS.

Induction of SAA synthesis by EAP. (i) C3H/HeJ mice. Incontrast to the negligible activity of LPS in inducing SAA inHeJ mice, EAP was found to be a potent stimulus for SAAproduction in these mice (Fig. 2). Preliminary studies indi-cated that the minimal and maximal SAA-stimulating dosesof EAP were similar in both endotoxin-resistant HeJ mice(0.5 and 50 pig, respectively) and endotoxin-sensitive CF1mice (0.5 and 5.0 pig). Intraperitoneal doses of EAP as smallas 0.5 pg produced a small but definite increase in SAA 18 hafter injection, whereas doses of 250 pig (the maximumamount tested) resulted in SAA concentrations of greaterthan 200 pig/ml (Fig. 2). Concomitant studies evaluated therole of macrophages in SAA induction in HeJ mice bycomparing levels of SAA observed 18 h after intraperitonealinjection of EAP alone versus levels observed after injectionof cell-free supernatants from peritoneal exudate cells of HeJmice incubated with an equivalent amount of EAP. Quanti-ties of SAA induced by intraperitoneal injection of EAPalone, at each dose tested, were similar to those found afterinjection of supernatants (containing the same amount ofEAP) of peritoneal exudate cells preincubated with EAP, ateach dose tested (Fig. 2). This contrasts strikingly with thepreviously described observations made with protein-freeLPS. Further studies of the SAA response of HeJ mice toEAP injection, using two subsequent lots of EAP, revealedsimilar dose-response relationships, with 0.5 pig of EAPbeing a threshold dose of EAP for SAA induction and 200 pig

resulting in the maximal SAA induction.(ii) CF1 mice. The SAA-inducing activity of EAP was also

evaluated in CFi mice. Intraperitoneal injection of EAPinduced elevations of SAA in endotoxin-susceptible CF1mice similar to those observed in endotoxin-resistant HeJ

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FIG. 3. Effect of EAP on SAA inducer production by macro-phages from responder CF1 mice. Each point represents the meanSAA concentration for a group of four C3H/HeJ mice 18 h after theintraperitoneal injection of 0.5 ml of either EAP alone in RPMImedium (0) or cell-free supernatant of CF1 peritoneal exudate cellsincubated with EAP (0).

mice (Fig. 3), except that the dose-response curve wasshifted towards greater increases in SAA levels in the 1- to10-pig dose range, which presumably was the result of theslightly greater base-line activity of macrophages elicited bythioglycolate. There was no evidence of increased SAAresponse to supernatants of CF1 peritoneal exudate cellsincubated with EAP, in contrast to the results observed withLPS, suggesting that EAP was directly responsible for theSAA response, rather than the production of SAA inducerby macrophages.Enzyme and heat stability of EAP. The effects of two

proteolytic enzymes, trypsin and pronase, and heat (100°C,30 min) on the SAA-inducing activity of EAP in endotoxin-resistant HeJ mice were also evaluated (Table 1). Neithertrypsin nor pronase treatment affected the SAA-inducingactivity of EAP. Heating of EAP to 100°C for 30 minappeared to increase the SAA-inducing activity of EAPalmost twofold. Similar resistance of the mitogenic effects ofEAP to heat and proteolytic treatment has been reportedpreviously (10, 18).LAF activity of EAP. The demonstrations that administra-

tion of EAP produced an increase in SAA concentration inendotoxin-resistant HeJ mice and that incubation of EAPwith peritoneal macrophages from either HeJ or CF1 micedid not enhance this SAA-inducing activity suggested thatEAP itself might have IL-1-like activity. The LAF assay isgenerally considered a sensitive and relatively specific indexof IL-1 activity, and hence the ability of EAP to stimulate

TABLE 1. Effects of enzyme and heat treatment of EAP onSAA-inducing activity

Treatment Mean SAA concn

(,ug/ml of serum) ± SD

EAP (untreated) ................................. 25.8 ± 3.7EAP + trypsin................................. 25.0 ± 4.2EAP + pronase ................................. 18.2 ± 2.8Trypsin (alone) ................................. 1.8 ± 0.2Pronase (alone) ................................. 2.1 ± 0.3Phosphate-buffered saline control .............. 2.5 ± 0.2EAP heated at 100°C for 30 min................ 41.9 ± 5.0

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FIG. 4. LAF-stimulating activity of 50 jig of EAP compared with that of S. minnesota LPS (at concentrations similar to that contaminatingEAP and at larger quantities), using CF1 thymocytes. The LAF-stimulating activity of EAP in these cells exceeded that of LPS, was notinhibited by addition of polymyxin B, and was not enhanced by incubation with macrophages (MO) from CF1 or HeJ mice. 3H TdR,[3H]thymidine; se, standard error.

PHA-sensitized thymocytes was evaluated by this means.Since our preparation of EAP was contaminated by a smallamount of LPS (0.007%), the LAF activity of EAP wasevaluated in both endotoxin-sensitive (CF1) and endotoxin-resistant (HeJ) mice with and without the addition of suffi-cient quantities of polymyxin B to inactivate any endotoxinpresent. In addition, controls utilizing the amount of con-taminating LPS were included.

(i) LAF assay with thymocytes from CFl mice. Figure 4illustrates [3H]thymidine incorporation in CF1 thymocytesstimulated by suboptimal doses of PHA and EAP (50 jig/ml),LPS (3.5 ng/ml [the quantity contaminating EAP] and 10,ug/ml), and human IL-1 standard (diluted 1:100). In addition,supernatants of macrophages from CF1 and HeJ mice,incubated with the same test materials, were also evaluatedfor activity in the LAF assay. Polymyxin B was added at aconcentration of 100 jig/ml to EAP and LPS to inhibit theLAF-stimulating activity of LPS. EAP alone exhibited amarked stimulatory effect on CF1 thymocytes greater thanthat observed with the IL-1 standard. The addition ofpolymyxin B to EAP did not reduce its LAF activitymaterially. Supernatants from either CF1 or HeJ macro-phages incubated with EAP were not as active producers ofLAF activity as EAP alone. S. minnesota LPS, at a concen-tration of 10 ,ug/ml, induced some IL-1 activity in macro-

phages from both CF1 and HeJ mice, but this activity was

abrogated by the addition of polymyxin B to the LPS. NoLAF activity could be detected in supernatants of both typesof macrophages incubated with 3.5 ng of LPS per ml.

(ii) LAF assay with thymocytes from C3H/HeJ mice. Re-sults with the LAF assay carried out with thymocytes fromHeJ mice (Fig. 5) paralleled those seen with thymocytesfrom CF1 mice except that the reactivity of HeJ cells to allstimuli appeared less than that of CF1 cells (note different

scales of [3H]thymidine in Fig. 4 and 5). EAP alone was a

potent stimulus of [3H]thymidine incorporation by HeJ thy-mocytes, and this effect exceeded that observed with super-natants of macrophages from HeJ mice. LPS at a concentra-tion of 10 ,ug/ml, but not 3.5 ng/ml, did induce LAF activitywith CF1 macrophages, but to a lesser degree than thatobserved with EAP alone, and it did not induce stimulatoryactivity after incubation with HeJ macrophages. Resultssimilar to these were observed with two additional prepara-tions of S. minnesota EAP.

(iii) LAF response to various doses of EAP. Figure 6 depictsthe response of PHA-stimulated thymocytes from CF1 andHeJ mice to doses of EAP ranging from 0.1 to 50 pug/ml. PeakLAF assay activity was observed at EAP concentrations of30 to 40 ,ug/ml, with a decrease in stimulatory activity at 50,ug/ml with thymocytes from both strains of mice.

Stimulation of resting T cells by EAP. Activation of restingT lymphocytes has been shown to require both properantigen presentation and the presence of cytokines (12, 17,34, 39). Anti-CD3 monoclonal antibody 64.1 that has beenbound to Sepharose beads (Sepharose-64.1) can serve toprovide the necessary antigenic stimulation, while IL-1functions as the required cytokine component (39). Toassess the ability of EAP to replace the function of IL-1,purified T lymphocytes were incubated with Sepharose-64.1and various concentrations of EAP (Table 2). EAP alonefailed to induce stimulation, and Sepharose-64.1 alone re-sulted in only minimal proliferation of T lymphocytes. Incontrast, EAP, in combination with Sepharose-64.1, stimu-lated proliferation of resting T lymphocytes in a dose-dependent manner. The optimal T-cell-stimulating concen-tration ofEAP was approximately 5 ,ug/ml, with larger dosesinducing less optimal response. The stimulatory activity of 5

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FIG. 5. LAF-stimulating activity of 50 jig of EAP compared with that of S. minnesota LPS (at concentrations similar to that contaminatingEAP and at larger quantities), using endotoxin-resistant HeJ thymocytes. The LAF-stimulating activity of EAP in these cells exceeded thatof LPS, was not inhibited by addition of polymyxin B, and was not enhanced by incubation with macrophages (M+) from CF1 or HeJ mice.3H TdR, [3H]thymidine; se, standard error.

jig of EAP per ml was comparable to or slightly exceededthat observed with 2.5 U of IL-1 per ml.

DISCUSSION

The present study demonstrates that components of gram-negative bacilli other than LPS may exhibit IL-1-like effectsin three different biological systems, namely, induction of

10000 CFI Thymocytes

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responsiveness of HeJ thymocytes in comparison with CF1 thy-mocytes. 3H TdR, [3H]thymidine; se, standard error.

SAA and stimulation of T lymphocytes as demonstrable bythe LAF and resting T-cell costimulation assays. LPS hasbeen used extensively in elucidation of the mechanismsinvolved in induction of SAA synthesis and LAF activity,and these studies have indicated that LPS does not actdirectly on the effector cells but that its activity is mediatedthrough production of IL-1 by macrophages. The ability ofLPS to induce SAA synthesis and LAF activity as well asother effects has complicated delineation of the biologicactivity of other components of gram-negative bacilli be-cause of the difficulty in obtaining preparations free of LPScontamination and the extreme sensitivity of the assays toLPS. The use of the endotoxin-resistant HeJ mouse strainand the ability of polymyxin B to abrogate the effects of LPSprovide methods of circumventing these problems and dem-onstrating that the IL-1-like activity of EAP is not explicableon the basis of its contamination with minute amounts ofLPS.Three types of bacterial cell wall components have been

shown to induce synthesis of the acute-phase reactant SAAwhen injected into mice: protein-free and protein-containingLPS (EAP) (16) and the low-molecular-weight immunoadju-vant N-acetylmuramyl-L-alanyl-D-isoglutamine (15). Thisimmunoadjuvant and LPS appear to be of comparable activ-ity, with induction thresholds between 1 and 10 ,ug. EAPexhibited potency similar to that of LPS, with a thresholddose of approximately 0.5 ,ug. Since EAP is composed of amixture of proteins, the activity of the individual effectorprotein may be even greater.These findings indicate that SAA synthesis in C3H/HeJ

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IL-1-LIKE ACTIVITY OF EAP 1599

TABLE 2. Resting T-cell costimulation by EAP'

[3H]thymidine uptakeb (103 cpm)Culture (concn of addition)

Expt 1 Expt 2 Expt 3

T cells 4.5 ± 0.8 4.5 ± 0.7 6.8 ± 1.4T + EAP (5 ,ug/ml) 4.1 ± 1.9 4.7 ± 1.0T + PHA (50 ,ug/ml) 5.3 ± 0.4 7.4 ± 2.9 9.8 ± 1.6T + Sepharose-64.1 (anti-CD3) 20.1 ± 1.2 29.6 ± 2.7 8.8 ± 1.3T + Sepharose-64.1 + IL-1 (2.5 p,/ml) 33.7 ± 2.0 97.9 ± 15.5 27.4 ± 2.2T + Sepharose-64.1 + EAP (50 p.g/ml) 38.1 ± 5.3 64.3 ± 4.9 67.6 ± 7.1T + Sepharose-64.1 + EAP (10 jig/ml) 36.2 ± 4.2 62.1 ± 0.5 51.4 ± 5.6T + Sepharose-64.1 + EAP (5.0 ,ug/ml) 100.7 ± 5.7 69.7 ± 11.9T + Sepharose-64.1 + EAP (1.0 ,ug/ml) 81.8 ± 8.3 48.2 ± 3.7T + Sepharose-64.1 + EAP (0.5 ,ug/ml) 40.2 ± 1.2 73.2 ± 7.9 34.0 ± 7.2T + Sepharose-64.1 + EAP (0.1 ,ug/ml) 76.9 ± 4.8 21.4 ± 2.7T + Sepharose-64.1 + EAP (0.05 ,ug/ml) 25.6 ± 1.9T + Sepharose-64.1 + EAP (0.01 jg/ml) 20.3 ± 2.9

a T cells were prepared as described and cultured for 72 h with the indicated additions.b [3Hlthymidine was added to each culture for the final 4 h of incubation at 1 ,Ci per well. Each value is the mean ± standard error for four replicate cultures.

mice is a biological response that clearly discriminatesbetween LPS and EAP. The SAA response to LPS (likeother endotoxin responses in mice) has been shown to beunder genetic control, apparently mediated by a single geneon chromosome 4, designated lps (28, 36). It was initiallyrecognized (16) that protein-containing LPS preparationswere capable of eliciting a small SAA response in thenonresponder HeJ mouse strain, with a threshold of 10 to100 ,ug of LPS. This was in contrast to protein-depleted LPSextracted by the hot phenol-water procedure, which resultedin a smaller rise in SAA concentration, with a threshold of100 to 1,000 ,ug of LPS. Thus, with respect to SAA induc-tion, many LPS preparations consist of two biologicallyactive moieties. The first is lipid A; C3H/HeJ andC57BL/1OScCR strains have undergone a mutation thatrenders them refractory to the physiological effects of lipidA, and its activity thus necessitates production of SAAinducer by stimulated macrophages. The second, EAP, wasas active as LPS, or more, in SAA induction in CF1 mice,and it was vastly more active in HeJ mice, indicating that theminute contamination by LPS was not responsible for theseeffects. In addition, the SAA-inducing activity of EAPdiffered from that of LPS in that incubation with macro-phages did not enhance SAA-inducing activity over thatseen with EAP alone, suggesting a direct effect of EAP onhepatocytes to induce SAA production.

Additional evidence for the IL-1-like activity of EAP issuggested from studies of the LAF assay utilizing CF1 andHeJ thymocytes and macrophages. Although LPS hasproven to be one of the most potent stimuli for induction ofLAF activity (5), EAP demonstrated a similar degree ofactivity in the present studies using both CF1 and HeJthymocytes, and LPS, in the concentration present as acontaminant of EAP, could not be shown to induce LAFactivity by acting directly on thymocytes in the absence ofmacrophages. In addition, incubation of EAP with poly-myxin B failed to diminish LAF activity, and incubation ofEAP with either CF1 or HeJ macrophages did not increaseLAF activity over that observed with EAP alone. In additionto the stimulation of T lymphocytes by EAP as illustrated inthe LAF assay, further evidence of IL-1-like activity wasalso demonstrated in the resting T-cell costimulation assay.These studies demonstrated that EAP can substitute for IL-1in the activation of resting T lymphocytes obtained fromnormal human donors and appears to be as effective as IL-1in this regard. It is assumed that the addition of EAP to cell

cultures results in the generation of IL-2 to support prolifer-ation and that EAP itself does not substitute for IL-2.Furthermore, the data shown in Table 2 indicate that EAPalone, in the absence of antigen, is not sufficient to induceT-cell proliferation. These studies are consistent with thecurrent model of T-lymphocyte activation, which requiresboth processed antigen and a cytokine (supplied by EAP inthese experiments).

Overall, these experiments strongly suggest that a compo-nent of EAP functions as a cytokine with IL-1-like activity.It might be postulated that EAP acts as an inducer of IL-1,but no enhancement of the activity of EAP alone in the SAAor LAF assay was observed with supernatants of macro-phages incubated with EAP. While these observations donot absolutely exclude IL-i-inducing activity, they tend tomake this relatively unlikely since recognized IL-1 inducershave been identified in this manner. Also, since IL-2 andother cytokines can exhibit activity in the LAF assay, EAPcould mimic the activity of one of these other cytokines.Studies are currently in progress to evaluate whether EAPinduces IL-2 release or exhibits activities similar to those ofIL-2. However, the activity of EAP mimicking IL-1 in threedifferent assays of IL-1-like activity suggests that it doesshare some of the biological activities of IL-1. Irrespective,however, of whether the activities of EAP most closelyparallel those of IL-1 or other cytokines, these studiesprovide unique evidence that components of procaryotesmay exhibit cytokinetic effects on eucaryotic cells. Otherstudies of the effects of EAP have demonstrated humangranulocyte colony-stimulating activity which apparentlydoes not require the presence of macrophages or lympho-cytes for mediation (1). In addition, in collaborative studiesof W. R. McCabe and M. Ho, infection of EAP has beenshown to serve as a potent inducer of interferon in both miceand rabbits (unpublished data), as also observed by Sultzer(31).More complete definition of the active component of EAP,

its characterization, and more precise delineation of itsbiologic activity will be possible if the active component canbe isolated. Preliminary studies have indicated that theactive component is resistant to trypsin digestion and ispartially resistant to treatment with pronase. Similar resis-tance to proteolytic digestion is characteristic of the porinOMPs (9), suggesting these may be the effector componentsof EAP. The extreme insolubility of the porins has handi-capped their separation from other OMPs. More recently,

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1600 JOHNS ET AL.

the detergent Zwittergent 3-14 has been utilized successfullyto isolate gonococcal OMPs (2). Although it was possible tosolubilize EAP from S. minnesota in Zwittergent 3-14, 0.05%was the lowest concentration capable of maintaining solubil-ity of these proteins, and this concentration proved toxic tothymocytes and hepatocytes. These attempts to isolate thespecific protein(s) responsible for the IL-1-like activity ofEAP are continuing, but other investigators working withEAP have also not been able, to date, to isolate the activecomponent.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grants fromthe National Institute of Allergy and Infectious Diseases (Al 14789and 121783) and the National Institute of Arthritis, Diabetes, andDigestive and Kidney Diseases (AM 07104) and by grants from theArthritis Foundation and the Kroc Foundation.

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