INFECTION AND IMMUNITY, May 1983, p. 464-471 Vol. 40, No. 20019-9567/83/050464-08$02.00/0Copyright © 1983, American Society for Microbiology

Inhibition of Chlamydia psittaci in Oxidatively ActiveThioglycolate-Elicited Macrophages: Distinction BetweenLymphokine-Mediated Oxygen-Dependent and Oxygen-

Independent Macrophage ActivationGERALD I. BYRNE* AND CYNTHIA L. FAUBION

Department of Medical Microbiology, University of Wisconsin Medical School, Madison, Wisconsin 53706

Received 14 September 1982/Accepted 25 January 1983

Immune sensitization of spleen cells was required to generate lymphokines(LK) that activated thioglycolate-elicited peritoneal macrophages (thio MACs) torespond via both oxygen-dependent and oxygen-independent systems. LK pro-duced by incubating spleen cells from immunized A/J and LAF mice withconcanavalin A stimulated a response by thio MACs to phorbol-12-myristate-13-acetate (PMA)-induced chemiluminescence and activated these cells to inhibitintracellular Chlamydia psittaci replication. Concanavalin A-incubated spleen cellpreparations from unimmunized animals stimulated neither PMA-induced chemi-luminescence nor antichlamydial activity. Activated thio MACs demonstrated arapid chemiluminescence response to the intracellular protozoan Toxoplasmagondii, but C. psittaci did not induce chemiluminescence in LK-activated thioMACs, although cells exposed to C. psittaci retained their responsiveness toPMA-induced chemiluminescence. The PMA-induced response was inhibited bythe addition of exogenous superoxide dismutase and catalase and was thereforerelated to the production of superoxide anion (02f-) and H202 by these cells. LKpreparations incubated at 56°C before macrophage treatment retained antichla-mydial activity, but heated preparations no longer stimulated thio MACs torespond in the chemiluminescence assay. These data provide evidence thatmacrophage oxygen-dependent and oxygen-independent systems are simulta-neously activated by LK, and these preparations comprise at least two distinctactivities. The portion responsible for activating oxygen-dependent systems(PMA-induced chemiluminescence) is heat labile, whereas the portion responsiblefor activating oxygen-independent systems is heat stable. It is the latter systemthat results in restriction of chlamydial growth and in vitro parasite persistence.

Macrophages play an essential role in elim-inating a wide variety of pathogenic microorga-nisms from the body and are essential for themaintenance of resistance to infection (28). It iswell established that immunologically commit-ted lymphocytes (29) and products derived fromthese cells (11) activate macrophages. This phe-nomenon has been assessed by a multitude offunctional criteria, yet the mechanisms wherebylymphokines (LK) activate macrophages to ex-hibit enhanced antimicrobial and antitumor ef-fects are incompletely characterized. We havepreviously shown (5) that thioglycolate-elicitedmouse peritoneal macrophages (thio MACs)support growth of the obligate intracellular pro-caryotic pathogen Chlamydia psittaci. Whenthio MACs were activated in vitro by mitogen-induced LK produced in spleen cell suspensionsfrom C. psittaci-immune A/J mice, C. psittaci

replication was inhibited for as long as the thioMACs were under the influence of LK (6).A variety of mechanisms have been proposed

to account for the antimicrobial capabilities ofLK-activated macrophages. A role for toxicintermediates of oxygen metabolism (38, 41) hasbeen linked to the killing of intracellular Toxo-plasma gondii (34), intracellular Trypanosomacruzi (35), and extracellular cytolysis of tumorcells (39). Oxygen-independent systems havealso been implicated in the microbicidal activityof macrophages and granulocytes. Cohen et al.(9) reported that macrophage oxidative activitywas unrelated to tumor cytotoxicity. Cationicpeptides are produced by activated macro-phages (26) and granulocytes (25, 50). Thesesubstances kill fungi (25, 26), gram-positive bac-teria (26), and gram-negative bacteria (50) invitro by mechanisms that increase membrane

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permeability (14, 49). Uptake of intracellularpathogens may also be inhibited in LK-activatedmacrophages and monocytes. Ingestion of Rick-ettsia tsutsugamushi was reported to be inhibit-ed when macrophages were activated beforeinfection, and an additional microbicidal activitywas seen when macrophages were activatedafter infection (37). Horwitz and Silverstein (20)found that activated human monocytes restrict-ed the uptake of Legionella pneumophila, andgrowth of those organisms that were taken up byLK-activated monocytes was diminished ascompared with growth in untreated monocytes.We have shown (5) that, although uptake of C.psittaci was not appreciably inhibited in LK-activated thio MACs, growth was curtailed bymechanisms apparently unrelated to oxygen-dependent systems. We did not, however, dem-onstrate whether thio MACs were capable ofresponding oxidatively after LK-mediated acti-vation.Thio MACs have been shown to exhibit only

minimal antibody-dependent cell-mediated cy-tolysis (46). Spitalny (48) reported that thioMACs were impaired in their ability to killListeria monocytogenes when compared withresident peritoneal macrophages. We found thatthio MACs were more responsive to LK-mediat-ed stimulation that resident peritoneal macro-phages (5). Thio MACs have also been reportedto have poor oxygen-dependent activity (40),although Cohen et al. (8) demonstrated thatadherent thio MACs secreted large amounts ofsuperoxide anion (02' ) and H202.

In this report, we provide evidence that spleencells from either A/J or LAF mice produced LKonly when animals were first immunized againstC. psittaci challenge. Thio MACs were activatedin vitro by LK in two ways. In our hands, thecells were normally poor in oxidative activity;they exhibited enhanced release of oxygen me-tabolism intermediates and were also stimulatedin an oxygen-independent manner to inhibit C.psittaci replication. The LK components re-sponsible for each activation sequence werefunctionally separated according to their differ-ential sensitivities to treatment at elevated tem-peratures.

MATERIALS AND METHODS

Animals. Male A/J and LAF (Jackson Laboratories,Bar Harbor, Maine) 6- to 12-week-old mice weremaintained under routine conditions. Animals wereimmunized by subcutaneous inoculation with lowdoses of viable C. psittaci as previously described (5,6).

Macrophages. Mice were injected intraperitoneallywith 3 ml of 3% (wt/vol) thioglycolate broth (DifcoLaboratories, Detroit, Mich.). Cells were lavagedfrom the peritoneal cavities 3 days after injection inDulbecco phosphate-buffered saline. Macrophages

were collected by centrifugation and resuspended inEagle minimum essential medium supplemented with10%o heat-inactivated fetal bovine serum (Flow Labo-ratories, Inc., Rockville, Md.), 2 mM L-glutamine, 10,ug of gentamicin sulfate (garamycin; Schering Corp.,Bloomfield, N.J.) per ml, and 100 pg of streptomycinsulfate (standard medium) per ml. The cells were thenplated onto either round glass cover slips (15-mmdiameter) in Linbro plates (16-mm-diameter well) orround glass cover slips (12-mm diameter) in tissueculture dishes (35-mm diameter) (three cover slips perdish) at densities of 5 x 105 or 4 x 105 cells per coverslip, respectively. Plated cells were incubated for 60min at 37°C in a humidified atmosphere of 5% CO2 inair, washed free of the nonadherent portion, andreturned to the incubator with or without LK, mockLK, or heat-treated LK. A final volume of 1 ml perwell or 2 ml per dish was added.LK. LK were prepared by incubating erythrocyte-

free spleen cells from immunized animals with 5 ,ug ofconcanavalin A (ConA) per ml. Cells were adjusted toa density of 5 x 106 per ml in medium containingConA, and 6-ml portions were incubated for 24 h intissue culture dishes (100-mm diameter). After incuba-tion, cell-free supernatant fluids were passed throughfilters (pore-size, 0.45 pm) and then added at a finalconcentration of l0o (vol/vol) to the macrophagecultures. These methods have been described in great-er detail elsewhere (5). Mock LK supernatant fluidswere prepared in exactly the same way as LK prepara-tions, except that spleen cells from unimmunized(normal) animals were used.For some experiments, LK were subjected to ele-

vated temperatures. Plastic tubes containing 1-ml por-tions of LK preparations were incubated in waterbaths set at either 56 or 100°C for specified times andthen immediately cooled in crushed ice and added at afinal concentration of 10% (vol/vol) to macrophages instandard medium.

In all experiments, cells were incubated overnight inthe presence of LK, mock LK, or standard medium,and then infected with approximately 1 50% infectivedose (ID50) of C. psittaci or processed for chemilumi-nescence as described below.

Preparation of chlamydiae and infection of thioMACs. Chlamydial harvests were prepared by infect-ing mouse fibroblast (L cell) monolayers with 10 timesthe ID50 of C. psittaci 6BC. Infected cells were incu-bated for 48 h at 37°C in standard medium containing 2jig of cycloheximide per ml. After incubation, the cellswere collected and sonicated. Chlamydiae were sepa-rated from cell debris by differential centrifugation andresuspended in phosphate-buffered saline supplement-ed with 0.2 M sucrose and 2% heat-inactivated fetalbovine serum. They were then stored in small portionsat -70°C until needed. These methods have beendescribed previously in greater detail (5). Infectivitytiters were determined by the ID50 method describedbest by Hatch (19). Thio MACs that were in culture for24 h in either standard or LK-containing medium werewashed and then infected with a dilution of C. psittaciadjusted to infect approximately 50% of the cells in atotal volume of 0.2 ml for cells in Linbro plates and 1ml for cells in culture dishes. Fresh standard mediumwas added (1 ml for plates and 2 ml for dishes) after 90to 120 min of inocula adsorption. Evidence of parasitereplication was measured 18 to 20 h after infection by

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TABLE 1. Comparisons of C. psittaci growth in A/Jand LAF thio MACs after incubation of host cells in

standard, mock, or LK-containing media

Mouse strain and % Inclusion- % Inhibitionincubation condition bearing cells growtha

Strain A/JStandard medium 25 ± 2 010% (vol/vol) 22 ± 3 10mock LKb

10% (vol/vol) LKC 7 ± 1 72

Strain LAFStandard medium 26 ± 3 2010% (vol/vol) 33 ± 4 0mock LKb

10% (vol/vol) LKC 10 ± 3 70

a [(Percent maximum inclusion-bearing cells - per-cent inclusion-bearing cells in sample)/Percent maxi-mum inclusion-bearing cells] x 100.

b Supernatant fluids generated by incubating spleencells from normal mice for 24 h in the presence of 5 ,ugof ConA per ml.

c Supernatant fluids generated by incubating spleencells from C. psittaci-injected mice for 24 h in thepresence of 5 ,Lg of ConA per ml.

determining the fraction of Giemsa-stained inclusion-containing cells.

Preparation of T. gondii. T. gondii RH was harvested3 days after intraperitoneal infection of outbred (CF1)male mice. Parasites were partially purified by differ-ential centrifugation, resuspended in phosphate-buff-

ered saline, and quantitated by hemacytometercounts.

Chemiluminescence assay. The oxidative activity ofLK- or standard medium-incubated thio MACs wasdetermined by a standard luminol-enhanced chemilu-minescence assay (32). Macrophage-containing coverslips were first incubated overnight in standard medi-um with or without LK or heat-treated LK. Afterincubation, cover slips were washed twice in Hanksbalanced salt solution (HBSS) without color indicatorand placed in prescreened glass scintillation vialscontaining HBSS and 0.1 p.M (10 ,ul of a 0.02 M stocksolution in dimethyl sulfoxide) luminol (Aldrich Chem-ical Co., Milwaukee, Wis.). Phorbol-12-myristate-13-acetate (PMA) (Sigma Chemical Co., St. Louis, Mo.)at a final concentration of 200 ng/ml (20 ,J of a 0.1dilution of 200 ,ug/ml in dimethyl sulfoxide) was addedto some samples. Other samples were incubated with50 ID50 of C. psittaci or T. gondii at a ratio of 10parasites to each host cell (0.2 ml of the appropriatedilution) in HBSS. All samples contained a final vol-ume of 2 ml of overlay medium. Chemiluminescencewas measured by quantitating light emission with aliquid scintillation spectrophotometer (Isocap 300;Searle & Co., Chicago, Ill.) by using the tritiumwindow with the coincidence circuit in the off (single)mode. Chemiluminescence was correlated to the re-lease of intermediates of oxygen metabolism by ablat-ing the response in the presence of exogenous super-oxide dismutase (100 ,ug/ml) and catalase (200 ,ug/ml).Equivalent concentrations of boiled enzyme prepara-tions were included as controls for nonspecific inter-ference. Endogenous chemiluminescence was mea-sured in vials containing 0.1 ,uM luminol in a finalvolume of 2 ml of HBSS.

It)

8 ~~~~~~~~8

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4 4

2 2

0 r

0 5 10 15 0 5 10 15MINUTES

FIG. 1. PMA-induced chemiluminescence in A/J (A) and LAF (B) thio MACs after incubation with mock LK(0) or LK (0). Responses of untreated thio MACs to PMA (solid lines) and endogenous activity of immune LK-activated thio MACs (broken lines) are shown.

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FIG. 2. Chemiluminescence in LK-activated A/J (A) and LAF (B) thio MACs to PMA (0), PMA in thepresence of exogenous catalase (200 ,ug/ml) and superoxide dismutase (100 ,ug/ml) (A), and PMA in the presence

of boiled enzyme preparations (A).

RESULTS

Replication of C. psittaci in LK-treated anduntreated thio MACs. When C. psittaci stockswere diluted and added to either A/J or LAF thioMACs incubated in standard medium, evidenceof parasite replication was detected 18 to 20 hafter infection. Chlamydiae replicate by binaryfission within membrane-bound cytoplasmicvesicles of the host cell (27), and these microcol-onies were present in Giemsa-stained prepara-tions in proportion to the inoculum added. Thedata presented in Table 1 contrast the portion ofstandard medium-incubated, mock LK-incubat-ed, and LK-incubated thio MACs that supportedchlamydial replication to an extent that permit-ted inclusion development. When A/J and LAFthio MACs were treated with mock LK, chla-mydial replication proceeded unencumbered.When similar thio MAC populations were treat-ed with LK, there was a marked decrease in theinclusion-bearing fraction 18 to 20 h after infec-tion. We have previously shown (5, 6) that theobserved decrease was not a result of differ-ences in the number of adherent host cellsamong the various preparations. Not only were

there fewer inclusion-containing cells in the im-mune LK-treated thio MACs, but also the ob-served inclusions were much smaller than thoseobserved in either mock LK-incubated or stan-dard medium-incubated cells. We have previ-ously shown (5) that chlamydial uptake is notappreciably impaired in LK-activated thioMACs; thus, the same fraction of each host cell

population was initially infected. Therefore, thepercent inhibition given (Table 1) actually repre-sents minimal values because of the inclusionsize differences observed among the variouspopulations. The reported antimicrobial defect(4) inherent in the A/J mouse strain was notobserved in these studies.

Oxidative activity in LK-stimulated and un-treated thio MACs. Standard medium-incubatedA/J and LAF thio MACs did not respond toPMA-induced chemiluminescence (Fig. 1).Therefore, these cells were inactive in responseto an agent that induces chemiluminescence bymembrane perturbations, possibly involving

TABLE 2. Chemiluminescence response of standardor LK-containing media incubated with A/J thio

MACs to T. gondiiCounts per minute

Incubation(min)a Standard mediumb 10%o LK-containing

mediumc

1 42,191 81,4255 51,683 191,64010 73,740 551,15715 66,789 626,63120 74,949 549,167

a Macrophages were prepared for chemilumines-cence as described in the text. T. gondii were added ata ratio of approximately 10:1 to the sample vials, andrepeat interval counts per minute were recorded afterT. gondii addition. Selected time points were reported.

b Mean values of two independent determinations.c Mean values of three independent determinations.

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020

x

0.15

10

5

0 5 10 15 20 0 5 10 15 2

MINUTESFIG. 3. Chemiluminescence in LK-activated A/J (A) and LAF (B) thio MACs to PMA (0) or C. psittaci (A)

added at the start of the assay and to PMA added 9 min (arrow) after endogenous (0) or C. psittaci (A) exposure.

fluxes in Ca2+ concentrations (12). When thioMACs were first incubated overnight in mockLK-containing medium and then tested forPMA-induced chemiluminescence, there wasalso no response. In contrast, thio MACs incu-bated in LK-containing medium exhibited amarked PMA-induced chemiluminescence re-sponse. No response was observed when cellswere incubated overnight in ConA-containingmedium, nor did ConA induce chemilumines-cence in LK- or standard medium-incubated thioMACs (data not shown).The PMA-induced chemiluminescence ob-

served in LK-treated thio MACs was found tobe related to the release of 02'- and H202 bythese cells (Fig. 2). When LK-treated A/J thioMACs were assayed for PMA-induced chemilu-minescence in the presence of exogenous super-oxide dismutase and catalase, enzymes that re-duce, respectively, 02- and H202, themagnitude of the response was decreased by96%. Residual activity was still observed whenLK-treated LAF thio MACs were tested, but theresponse was nevertheless diminished by 74%.This inhibition of chemiluminescence after enzy-matic reduction of secreted O2 and H202 pro-vided evidence that the observed response wasin some way related to an increase in the oxida-tive activity of LK-treated thio MACs. Relation-ships between respiratory activity and chemilu-minescence have been drawn since this assaywas first used to measure granulocyte responsesto various stimuli (3). Incubation of oxidatively

active thio MACs in the presence of boiledenzyme preparations did not alter their responseto PMA induction (Fig. 2).We have previously provided indirect evi-

dence (5) that LK-mediated inhibition of C.psittaci replication did not involve oxygen-de-pendent activity. When C. psittaci (50 ID50) wasadded to LK-treated A/J or LAF thio MACs, nochemiluminescence was detected. These cellswere capable of rapidly responding to T. gondii(Table 2), an intracellular protozoan that hasbeen reported to be inhibited via oxygen-depen-dent mechanisms in LK-activated macrophages(34), but, despite the fact that chlamydial uptakeoccurred and parasite replication was inhibitedin these cells, there was no associated evidenceof increased respiratory activity (Fig. 3), evenwhen the incubation time was extended for 40min. The addition of C. psittaci did not, howev-er, directly inhibit secretion of O2- and H202 byLK-activated thio MACs. When PMA was add-ed to C. psittaci-containing cells, a responseequal to that seen in cells incubated for equiva-lent times in HBSS alone was observed. Thesedata provided direct evidence that oxidativeactivity, as assessed by chemiluminescence,was unrelated to the LK-mediated antichlamydi-al activity observed in thio MACs.

Effect of elevated temperatures on immune LK-mediated antichlamydial and chemiluminescentactivity in A/J thio MACs. LK preparations wereincubated at either 56 or 100°C for specifiedtimes before treatment of thio MACs. Thio

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FIG. 4. Replication of C. psittaci in thio MACs activated by LK subjected to 56°C (0) or 100°C (A) for theindicated times. Results are expressed as means ± the standard error of the mean of chlamydial growth withrespect to the control population as follows: 100 x [(percent inclusion-bearing cells in control [cross-hatchedarea] - percent inclusion-bearing cells in sample)/percent inclusion-bearing cells in controls]. The cross-hatchedarea reflects the amount of chlamydial replication ± the standard error of the mean in untreated thio MACs.

MACs treated with LK incubated at 56°C re-mained activated to inhibit C. psittaci replica-tion (Fig. 4). LK preparations subjected to 100°Cfor brief periods no longer stimulated thio MACsto inhibit chlamydial growth. In contrast, LKincubated at 56°C did not activate A/J thioMACs to respond in the PMA-induced chemilu-minescence assay (Fig. 5). The decrease in re-sponsiveness was found to be proportional tothe time ofLK incubation at 56°C. Incubation ofLK at 100°C for 2 min caused a complete abla-tion of activity. These data provide evidencethat LK preparations comprise two distinct ac-tivities. One resulted in thio MAC activation toinhibit chlamydial replication, and the otherresulted in enhanced oxidative activity, as as-sessed by PMA-induced chemiluminescence.The former activity was stable at 56°C, whereasthe latter was heat labile.

DISCUSSIONMacrophage activation is a complex event

that requires a coordinated sequence of steps(31) culminating in enhanced antimicrobial andcytotoxic activity (1) accompanied by alter-ations in cell morphology (13). David (10) initial-ly reported that substances produced by antigen-stimulated lymphocytes were capable ofactivating macrophages in vitro. We now recog-nize that these LK stimulate an entire spectrumof activities, ranging from induction of B cell

proliferation (21) to inhibition of fibroblastgrowth (42). LK-mediated macrophage activa-tion also results in acquired antimicrobial activi-ty. It is apparent that, although specific sensiti-zation may be required to generate antigen- ormitogen-responsive lymphocytes, the effects ofthese mediators on macrophages are not selec-tive, and, once activated, macrophages mayexhibit enhanced resistance to a variety of mi-crobes (43). Despite this apparent generalizedconcept of the activated macrophage, severaldistinct antimicrobial and cytotoxic mechanismshave been proposed to account for the activitywhereby macrophages exert their effects (2, 16).Anaerobic systems have been recognized (30) inwhich acidification across a pH gradient, vari-ous hydrolases, or cationic proteins may play arole (16). Oxygen-dependent mechanisms havealso been established as important for microbi-cidal and tumoricidal responses in the activatedmacrophage (35). Evidence has also been pre-sented to implicate endocytosis inhibition ofcertain obligate intracellular bacteria by activat-ed macrophages (20, 37).Given the variety of proposed activation

mechanisms and the nonspecific nature of theactive macrophage, it is apparent that either LKmediate several distinct metabolic alterations orseveral species of LK act together during thecomplex macrophage activation process. Wehave provided evidence here that implicates at

v v

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FIG. 5. PMA-induced chemiluminescence in thioMACs activated by LK (x) or LK incubated at 56°Cfor 10 min (0), 30 min (A), or 60 min (l), or at 100°Cfor 2 min (0) before treatment of macrophage cultures.

least two distinct LK activities operating intandem to stimulate two separate mechanisms inthio MACs. Production of these activities re-

quired the presence of immunologically sensi-tized spleen cells. Although specificity of macro-phage-mediated cytotoxicity has been reported(17), we do not stipulate that immunity to C.psittaci is specifically required, nor do we implythat LK produced by sensitized spleen cellsspecifically activates macrophages to restrictchlamydial growth. We found that chlamydiaewere inhibited by an oxygen-independent mech-anism that was not affected by incubating LK at56°C. Oxygen-dependent activity was also stim-ulated but was labile when subjected to elevatedtemperatures. The data reported here do notdistinguish between either a single LK moietywith two active sites or two distinct LK sub-stances, but techniques have been developed toseparate and characterize various LK and cyto-kines (21, 22, 36, 47) and can now be exploited toidentify specific mediators in terms of theireffector cell activity.We have previously provided evidence (5)

that macrophages activated by soluble spleencell mediators may provide an environment thatpromotes intracellular chlamydial persistence.Many naturally occurring human (44) and animal(15) chlamydial infections tend to run a chronicor persistent course, but the underlying mecha-

nisms for chlamydial persistence are not wellunderstood, despite the development of severalinteresting in vitro models (19, 23, 24, 33, 45).Macrophages may contribute to viral persis-tence (18), and a similar role, especially inresponse to immune intervention in the form ofLK, should be considered for chlamydial infec-tions. A report (7) that demonstrated that LK-mediated activation of somatic cells (fibroblastsand kidney cells) resulted in the inhibition of T.gondii and Besnoitia jellisoni lends credence tothe concept that immune intervention may alsobe important during chlamydial infection of cellsother than those of the mononuclear phagocytesystem.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant Al-16459 from the National Institute of Allergy and InfectiousDiseases.The expert secretarial assistance of Alice Stapp and Patricia

Geschwent is gratefully acknowledged.

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Lk-noLA

,X24 - /

xoX/

X Lk- 560 30min

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