INFECTION AND IMMUNITY, Oct. 1981, p. 274-284 Vol. 34, No. 10019-9567/81/100274-11$02.00/0

Dissociation of Bactericidal Activity from Other Functions ofActivated Macrophages in Exudates Induced by Thioglycolate

MediumGEORGE L. SPITALNY

Trudeau Institute, Inc., Saranac Lake, New York 12983

Received 13 April 1981/Accepted 19 June 1981

Macrophages displayed increased spreading, increased Fc-receptor-mediatedphagocytosis, and increased secretion of plasminogen activator when collectedfrom the peritoneal cavities of either Listeria-immune mice challenged intraper-itoneally 3 days earlier with Listeria or nonimmune mice injected intraperitone-ally 3 days earlier with fluid thioglycolate medium. In contrast, macrophagesfrom the thioglycolate-induced peritoneal exudates were severely impaired invitro in their ability to destroy Listeria. Injection of thioglycolate markedlyinterfered with the destruction of sublethal intraperitoneal challenge of Listeria,which resulted in nonimmune animals dying of an overwhelming systemic infec-tion. In animals immune to Listeria, injection of thioglycolate delayed the onsetof the expression of immunity to an intraperitoneal challenge of bacteria. Thethioglycolate-induced suppression of bactericidal activity was determined to beconfined to the site of injection. Results of experiments indicated that the colloidalagar in thioglycolate medium was the cause of the impairment of macrophagebactericidal activity. In addition to the impairment of bactericidal activity inducedby agar, additional studies showed that an intraperitoneal injection of colloidalagar (0.075% wt/vol) by itself was a sufficient inflammatory stimulus for theaccumulation of a large number of host phagocytic cells.

The acquisition of cell-mediated immunity tobacterial (3, 23, 25), protozoal (27), or neoplasticdisease (26, 31) has been shown to result in thesystemic activation of macrophages. This hasbeen demonstrated by an enhanced ability ofinfected mice to nonspecifically destroy a chal-lenge with facultative intracellular bacteria suchas Listeria monocytogenes. In addition to in-creased bactericidal functions, activated macro-phages have been shown in vitro to display avariety ofmorphological (12), ultrastructural (3),and physiological (9, 22) changes. Althoughthere is no definitive evidence of a direct corre-lation between bactericidal functions and otherproperties of activated macrophages, it has beengenerally concluded that macrophages whichdisplay morphological or physiological charac-teristics of activation or both are also likely toexhibit enhanced bactericidal activity.The studies reported in this paper were de-

signed to determine whether the enhancementof certain functions associated with macrophageactivation coincides with an increase in bacteri-cidal activity. The results of these studies showthat intraperitoneal injection of the phlogogenicagent thioglycolate results in the local accumu-lation of macrophages that appear activated by

morphological and certain functional criteria,but are markedly impaired in their ability todestroy a challenge of Listeria in vitro and invivo. Evidence is also presented that the colloi-dal agar in thioglycolate medium is the cause ofthe impairment of macrophage bactericidal ac-tivity.

MATERIALS AND METHODSAnimals. Female AB6F, mice (A/Tru x C57BL/6

Tru) 8 to 12 weeks of age were used throughout.Animals were supplied by the Trudeau Institute Ani-mal Breeding Facility. Mice were free of 11 commonmurine viruses in tests conducted by MicrobiologicalAssociates Viral Testing Service (Cockeysville, Md.).Mice were determined to be free of lactic dehydrogen-ase virus by the failure of an injection of AB6F, serumto raise the level of lactic dehydrogenase enzyme inthe serum of germfree mice (29). Levels of lactic de-hydrogenase were measured in serum with a diagnostickit (no. 500) from Sigma Chemical Co. (St. Louis,Mo.).

Bacteria. L. monocytogenes, strain EGD, wasgrown to log phase in Trypticase soy broth (TSB; BBLMicrobiology Systems), dispensed in 1.0-ml samples,and frozen at -70°C. An inoculum of bacteria wasprepared for immunization or challenge by thawingone vial, diluting bacteria in saline, and injecting 0.2ml of the appropriate dilution. To prepare a log-phase

274

on June 13, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

FUNCTIONS OF ACTIVATED MACROPHAGES 275

culture of Listeria for use in the in vitro bactericidalassay, several dilutions of bacteria were added to sep-arate 5.0-ml tubes of TSB and grown at 37°C. Astandard dose of 2 x 103 Listeria was used for intra-venous (i.v.) immunization; challenge doses consistedof 5 x 103 i.v. and 5 x 105 or 5 x 106 intraperitoneally(i.p.).Measurement of bacterial growth in vivo. The

ability of immune and nonimmune mice to destroy ani.p. and i.v. challenge of Listeria was determined. Tofollow the fate of an i.p. challenge of Listeria, theperitoneal contents were collected in 3 ml of heparin-ized phosphate-buffered saline (PBS), and the wash-ings were subjected to sonication to release cell-asso-ciated bacteria. The number of Listeria was quanti-tated by plating 10-fold dilutions onto Trypticase soyagar (TSA; BBL Microbiology Systems). The fate ofa 5 x 103 inoculum ofListeria injected i.v. was followedin the livers of experimental and control mice. Thenumber of bacteria in the liver was determined byhomogenizing the whole organ in 8 ml of saline andplating 0.1 ml of 10-fold serial dilutions onto TSA.

Preparation of macrophages for in vitro as-says. Macrophages were collected from the peritonealcavities of the following groups, 3 days after i.p. injec-tion of the appropriate material. Mice immunized 6days earlier with 2 x 103 Listeria i.v. (referred to asimmune) were challenged i.p. with 5 x 106 bacteria(referred to as immune-boosted). Another group ofimmune and nonimmune mice were injected i.p. with1.0 ml of thioglycolate medium. A group ofnonimmunecontrols was injected i.p. with 1.0 ml of PBS. Cellswere collected by infusing 1.5 ml of tissue culturemedium i.p. The medium consisted of RPMI 1640(GIBCO Laboratories) supplemented with 5% fetalbovine serum (GIBCO) and 5 U of preservative-freeheparin (Sigma Chemical Co.) per ml. Typically 107 orgreater total cells/animal were collected from the im-mune-boosted and thioglycolate-injected mice. ThePBS-injected controls yielded between 5 x 106 and7 x 10' total cells, of which 20 to 40% were identifiedas macrophages by morphology and phagocytosis ofheat-killed Listeria as previously described (30).

Bactericidal assay. The ability of macrophages todestroy a challenge ofListeria in vitro was determinedas previously described (30). In brief, cells collectedfrom the peritoneal cavities of immune-boosted orthioglycolate-injected animals were diluted in tissueculture medium to 4 x 106 total cells/ml, and 1.0 mlwas added to petri dishes containing three 13-mmcircular glass cover slips. In the case of PBS-injectedcontrols, 6 x 106 to 8 x 106 total cells were added topetri dishes in 1.0 to 1.5 ml. Cells were allowed toadhere for 2 h, nonadherent cells were then rinsed offthe cover slips, and the remaining cells were chal-lenged 1 h later with 5 x 106 Listeria in 1.0 ml of tissueculture medium. After 20 min, extracellular bacteriawere washed off the cover slips. When all the coverslips were washed (designated as the To time point),the number of cell-associated viable bacteria wasquantitated. Three cover slips were removed fromseparate dishes at 30-min intervals (designated as theTi, T2, T3, etc. time points) and sonicated to releasecell-associated bacteria. Bacteria were enumerated byplating 10-fold dilutions on TSA. Data are presented

as the percentage of viable bacteria detected at eachtime point. This was calculated according to the for-mula:

number of colony-forming unitsTTI,T2,T3, * X 100

number of colony-forming units To

Macrophage spreading. To measure macrophagespreading, cells were collected from the peritonealcavities of mice and allowed to adhere to circular glasscover slips for 30 min at 22°C. Nonadherent cells werewashed off, the cover slips were placed in warm me-dium, and spreading was allowed to proceed by placingthe dishes at 37°C in a humidified atmosphere of 5%CO2. Cover slips were removed at timed intervals,rinsed in warm PBS, and fixed in glutaraldehyde di-luted to 2% in 37°C PBS. At least 50 cells on eachcover slip were photographed with a Zeiss photomi-croscope. From the developed negative, the image ofthe cells was projected onto graph paper, and theoutline of each cell was traced. From a knowledge ofthe magnification of the microscope and enlarger, thearea of spreading of each cell was calculated.

Fc-receptor-mediated phagocytic activity.The phagocytic activity of macrophages was assessedby their ability to ingest opsonized sheep erythrocytes(SRBC). Anti-SRBC antibodies (immunoglobulin G[IgG]) were purchased from Cordis Labs (Miami, Fla.),and the agglutination titer was determined to be 1:500.SRBC were washed in PBS, suspended to 5 x 107/mlin tissue culture medium containing a 1:5,000 dilutionof anti-SRBC antibody. This suspension was incu-bated for 20 min at 4°C and then at 37°C for 20 min.The medium overlaying the macrophage monolayerson the glass cover slips was replaced with 1.0 ml of theSRBC suspension. Cover slips were removed at inter-vals and rinsed in warm saline, and uningested SRBCwere lysed in hypotonic PBS. The cells were thenfixed in 2% glutaraldehyde, and the number of SRBCingested per 100 cells was counted using a phase-contrast microscope.

Fibrinolytic assay. The secretion of plasminogenactivator from macrophages was quantitated by therelease of "I from labeled fibrinogen as describedelsewhere (21, 33). Fibrinogen was labeled with carrier-free "I (Amersham Corp.) by the chloramine Tmethod (13). To each 16-mm well of a 24-well Linbroplate (Flow Laboratories, Inc.), 10 ug of fibrinogen wasadded which contained 50 x 103 to 100 x 103 trypsin-releasable cpm. Peritoneal macrophages were har-vested in tissue culture medium containing 10 .tg ofgentamicin per ml (Schering Corp.) and 50 jg of soy-bean trypsin inhibitor (fraction VI; Miles Laboratories,Inc., Kankakee, Ill.) per ml, the latter to inhibit spon-taneous fibrinolysis. Cells (5 x 105 to 2 x 106) wereadded to the wells and allowed to adhere for 2 h.Nonadherent cells were washed off the plate, and theadherent cells were cultivated for 24 h in medium withsoybean trypsin inhibitor. The cells were then washedthree times to remove inhibitors, as well as dead cells,and the assay was started by adding 1.0 ml of RPMI1640 containing 5% acid-treated dog serum. At theindicated times, 0.01 ml of medium was withdrawnfrom triplicate wells of each group and assayed for the

VOL. 34, 1981

on June 13, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

276 SPITALNY

release of radioactivity in a Searle gamma counter.The actual number of adherent cells in each well atthe time of the assay was determined by direct micro-scopic counts or by removing all cells with cetrimideand pronase (32) and counting in a hemacytometer.

Reagents. Fluid thioglycolate medium (0256-01)was prepared in accordance with the manufacturer'sinstructions (Difco Laboratories, Detroit, Mich.). Thi-oglycolate medium was also prepared without agar.All ingredients were purchased from Difco Laborato-ries and prepared according to the proportions on thefluid thioglycolate medium bottle without the additionof agar (0.75 g/liter) as found in thioglycolate medium.Granulated agar was purchased from BBL Microbiol-ogy Systems, (Cockeysville, Md.).

RESULTSThioglycolate and immunologically in-

duced enhancement of macrophage func-tion. Experiments were designed to study mac-rophage spreading, Fc-receptor-mediated phag-ocytosis, and plasminogen activator activities.Cells were collected from the peritoneal cavitiesof either Listeria-immune mice 3 days after an

INFECT. IMMUN.

i.p. challenge of bacteria (immune-boosted) orfrom normal mice injected i.p. 3 days earlierwith thioglycolate or PBS. Injection of thiogly-colate into normal mice or bacteria into immunemice induced a cellular exudate which amountedto a three- to fivefold increase in total cell num-ber and consisted mainly of macrophages (60 to85%) at the time of collection. Examination ofcell spreading (Fig. 1A), Fc-receptor-mediatedphagocytosis (Fig. 1B), and release of plasmin-ogen activator (Fig. 1C) revealed that macro-phages from the immune-boosted and thiogly-colate-injected groups showed enhancement ineach of these functions which greatly exceededthat of macrophages from PBS-injected con-trols. In fact, macrophages from the thioglyco-late-induced exudate exhibited activities abovethat of macrophages from immune-boostedmice. Macrophages from both of these groupsalso displayed the morphological features of ac-tivation (Fig. 2).Thioglycolate-induced impairment of

macrophage bactericidal activity. In con-

A

ilkCa

-A

-j

-I.

p.

a

C

MUS MNUTES HURSFIG. 1. Comparison of macrophage (A) spreading, (B) Fc-receptor-mediated phagocytosis of opsonized

sheep erythrocytes (EA), and (C) release ofplasminogen activator. Macrophages were harvested from theperitoneal cavities of Listeria-immune mice challenged i.p. 3 days earlier with bacteria (IMMUNE-BOOSTED) or from the peritoneal cavities ofnonimmune mice 3 days after injection of thioglycolate (THIO)or PBS (PBS CONTROL). (A) Each time point represents the geometric mean of at least 50 determinations± SEM. (B [± SEMI and C) Each time point represents the mean of triplicate samples assayed.

on June 13, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

FIG. 2. Phase-contrast photomicrograph of glass-adherent macrophages. Macrophages were fixed in 2%oglutaraldehyde after 1 h in culture. Macrophages were harvested from the peritoneal cavities 3 days afterinjection of (A) thioglycolate into nonimmune mice, (B) 10; Listeria in immune mice, and (C) PBS intononimmune mice. Macrophages in (A) and (B) show conspicuous features of activation, including highlyruffledplasma membrane, greater tendency to spread, large numbers of lysosomes andpinocytic vesicles.

277

on June 13, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

278 SPITALNY

trast to the enhancement of macrophage func-tions shown in the preceding section, results ofin vitro studies revealed that macrophages fromthe thioglycolate-injected mice were impaired intheir ability to destroy Listeria compared withmacrophages from PBS-injected or immune-boosted mice (Fig. 3). Further studies revealedthat an injection of thioglycolate interfered withthe destruction of an i.p. challenge of Listeria(Fig. 4). The impairment of bactericidal activitywas so severe in nonimmune mice injected withthioglycolate that Listeria growth progressedrapidly and ultimately led to an overwhelminginfection. In immune mice injected with thiogly-colate, the onset of expression of anti-Listeriaimmunity was delayed for at least 2 h and wasthen expressed more slowly than in immune-boosted controls. Thus, by 24 h after challenge,the immune-boosted group had completely de-stroyed the challenge, whereas the thioglycolate-injected mice still contained large numbers ofListeria in their peritoneal cavities. The rateand extent of destruction of an i.p. Listeria

Lai

1--a

eLm

/

0

I

270

challenge were similar in Listeria-immune andimmune-boosted animals (data not shown). Itshould be noted that although immune-boostedmice rapidly destroyed an in vivo challenge in-fection (Fig. 4), macrophages from immune-boosted mice were certainly no better than thosefrom PBS-injected mice in their ability to de-stroy a bacterial challenge in vitro (Fig. 3).Evidence that thioglycolate-induced sup-

pression is confined to the site of injection.To determine whether the impairment of bac-tericidal activity induced by thioglycolate wasconfined locally to the site of injection, growthof an i.v. challenge infection was followed onsuccessive days, in the livers of mice injected i.p.3 days earlier with PBS or thioglycolate. Theresults show (Fig. 5A) that animals injected pre-viously with thioglycolate or PBS displayed typ-ical expression of nonspecific resistance duringthe first 12 h of infection (23). Thereafter, bac-terial growth was similar until day 4 when thio-glycolate animals showed an increase in thenumber of Listeria in their livers. These studies

A

I/

270MINUTES

FIG. 3. Comparison ofthe in vitro bactericidal activities ofmacrophages. (A) Macrophages harvested fromtheperitoneal cavities ofnonimmune mice injected 3 days earlier with thioglycolate or PBS. (B) Macrophageswere harvested from the peritoneal cavities ofListeria-immune mice injected 3 days earlier with thioglycolateor bacteria. Each time point is the geometric mean of triplicate samples.

INFECT. IMMUN.

on June 13, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

FUNCTIONS OF ACTIVATED MACROPHAGES 279

A

-4-

24 48

B

H

24 48

HOURS of LISTERIA INFECTIONFIG. 4. Effect ofan i.p. injection of thioglycolate on the animals' ability to destroy a sublethal challenge of

Listeria injected into the same site. Appropriate groups were injected with either thioglycolate, PBS, orbacteria 3 days before Listeria challenge. (A) Growth of a 105 inoculum of Listeria in nonimmune mice. (B)Growth of a similar inoculum in Listeria-immune animals. Geometric mean of five mice per time point ±SEM.

ALIVER

CONTROL

I * * i

PERIT. CVITY B

DAYS OF LISTERIA INFECTIONFIG. 5. Growth ofan i.v. challenge infection. A 5 x 103 inoculum ofListeria was injected i.v., and its growth

was simultaneously followed in the livers and peritoneal cavities of mice injected i.p. 3 days earlier with

either thioglycolate or PBS. Geometric mean offive mice per time point + SEM.

also revealed that large numbers of bacteriawere present in the peritoneal cavities of the i.v.infected, thioglycolate-treated mice (Fig. 5B).This shows that after i.v. challenge, bacteriaspread to the peritoneal cavity where they wereable to grow virtually unchecked because of theprevious injection of thioglycolate. Hence, under

these circumstances it is conceivable that Lis-teria in the peritoneal cavity could, in turn, havespread to the liver, again leading to a fatal infec-tion. This latter possibility is supported by theresults in Fig. 6A, which show that a sublethalbacterial challenge grew progressively in the per-itoneal cavities of mice injected 3 days earlier

(n

-aC=cm

-j

5.

I4-0-W4C=

<10

VOL. 34, 1981

on June 13, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

280 SPITALNY

~5f4 4 PBS

+,,CONTROL3 PBS 3/'

CONTROLT2 2

012~~~~301 3DAYS OF LISTERIA INFECTION

FIG. 6. Growth ofa sublethal bacterial challenge. A 1l' inoculum ofListeria was injected i.p., and bacterialgrowth was simultaneously followed in the peritoneal cavities and livers of mice injected i.p. 3 days earlierwith either thioglycolate or PBS. Geometric mean offive mice per time point ± SEM.

with thioglycolate. From the initial site of infec-tion in the peritoneal cavity, bacteria spread tothe livers of thioglycolate and control mice (Fig.6B). However, control mice limited bacterialmultiplication, whereas in the livers of thiogly-colate mice, Listeria growth progressed until thedeath of the mice.Evidence that agar in thioglycolate me-

dium is the cause of the impairment ofbactericidal activity. Experiments were per-formed to determine whether the suppressioninduced by thioglycolate could be attributed tothe presence of colloidal agar in the medium.The results in Fig. 7 show that an injection ofeither complete thioglycolate medium or PBSprepared with a similar concentration of agar(0.075% wt/vol) impaired the expression of an-tibacterial resistance. Injection of agar-free thi-oglycolate or PBS failed to interfere with thedestruction of the Listeria challenge. In vitrostudies revealed that macrophages harvestedfrom the peritoneal cavities of the agar-injectedmice were severely impaired in their ability todestroy bacteria (Fig. 8). Injection of agar-freePBS or thioglycolate failed to inhibit macro-phage bactericidal activity.Cellular inflammatory property of colloi-

dal agar. In addition to the impairment ofbactericidal activity induced by colloidal agar,

the results in Table 1 show that i.p. injection ofeither thioglycolate or PBS prepared with agarinduced a cellular exudate that was similar bothin the number and types of cells present. In theagar-free preparations, injection of thioglycolateor PBS caused little, if any, change in the com-position of peritoneal cells and resulted in a lessthan twofold increase in total number. Theseresults implicate colloidal agar as being the com-ponent of fluid thioglycolate medium able toinduce a cellular exudate.

DISCUSSIONThe concept that macrophages become acti-

vated as part of the host immune response toinfection was developed by Mackaness (11, 12,24) to explain the enhancement in nonspecificbactericidal activity of macrophages from in-fected mice (3, 24). Since these early studies,enhancement of a variety of physiological orbiochemical functions or both has been attrib-uted to activated macrophages (4, 8). The stud-ies presented in this paper show, however, thatincreases in certain functions associated withactivation are not always accompanied by anenhancement in bactericidal activity. Similarly,a previous study (30) demonstrated that mac-rophages which accumulated in a progressive

INFECT. IMMUN.

on June 13, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

FUNCTIONS OF ACTIVATED MACROPHAGES 281

Lhi.-

q-I

cm

CMJM

HOURS of LISTERIA INFECTIONFIG. 7. Comparison of the growth of a lo' inocu-

lum of Listeria in the peritoneal cavities of miceinjected i.p. 3 days earlier with 1.0 ml of either com-

plete thioglycolate medium, PBS containing the sameconcentration (0.075% wt/vol) of agar as in thiogly-colate, or agar-free thioglycolate or PBS. Geometricmean offive mice per time point ± SEM.

syngeneic peritoneal ascites tumor were far bet-ter than normal in adherence, spreading, andphagocytosis, but were severely impaired in theirability to destroy a challenge of Listeria in vivo.Several other reports have also shown that "ac-tivated" macrophages sometimes exhibit de-creases in their ability to phagocytose or degradecertain particles (2, 16, 28, 35). Such studies are

valuable because they challenge the stereotypeof an activated macrophage and are promptinga reevaluation of the concept of macrophageactivation.

After local injection of either Listeria intoimmune mice or thioglycolate into nonimmunemice, macrophages collected from the peritonealcavities of these mice consistently displayed en-

hancement of spreading, Fc-receptor-mediatedphagocytosis, and release of plasminogen acti-vator. Despite these increases, i.p. injection ofthioglycolate interfered with antibacterial resist-

ance in vivo, and macrophages from the perito-neal cavities of these mice were severely im-paired in their ability to destroy Listeria in vitro.Thioglycolate-induced exudate macrophageshave also been shown to be no better thannormal macrophages in their ability to destroyTrypanosoma cruzi in vitro (18). The destruc-tion of T. cruzi has been shown to require mac-rophages activated in vivo in conjunction withthe host's response to infection (20) or in vitroby lymphokines (19). In the case of Listeria,there is ample evidence that normal macro-phages possess some capacity to destroy bacteriain vivo (17, 23) and in vitro (30). The injectionof thioglycolate into nonimmune mice virtuallyabolishes this measure of nonspecific resistance,and the animals ultimately die of an otherwisesublethal infection. On the other hand, the in-jection of thioglycolate into Listeria-immunemice delayed the ultimate onset and expressionof antibacterial immunity against an i.p. chal-lenge. The reason immunity was capable ofbeing expressed is not known, but it could per-haps be attributed to either the influx of a newpopulation of cells or the activation of the exist-ing local population via the release of lympho-kines. An observation that deserves commentpertains to the finding that immune-boostedmice rapidly destroyed an i.p. challenge of Lis-teria and yet peritoneal macrophages from thesemice were no better than normal in their bacte-ricidal activity in vitro. No explanation of thisapparent discrepancy is immediately available.The finding that an i.p. injection of thiogly-

colate impairs bactericidal activity in vivo andin vitro is in agreement with published studiesfrom other laboratories (1, 14). In our studies,the suppression was shown to be confined to thesite of injection, similar to that previously dem-onstrated in mice bearing peritoneal ascites tu-mors (30). Hence, i.p. injection of tumor cells orthioglycolate did not interfere with the expres-sion of antibacterial resistance in the liver andspleen. Published studies have shown that, ir-respective of the route of infection, bacteria canspread to virtually all tissues and cavities of thebody (5). In view of this fact, it was not surprisingto find that after i.v. injection of Listeria, bac-teria were detected in the peritoneal cavities ofall mice and were growing rapidly in those ani-mals previously injected with thioglycolate.Consistent with the finding of widespread dis-semination of a bacterial infection, our resultsalso showed that the large numbers of bacteriain the peritoneal cavities of the thioglycolate-injected mice rapidly spread to the liver andmust have contributed to the overwhelming sys-temic infection. If this was so, then the reported

VOL. 34,1981

on June 13, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

282 SPITALNY

L&J

-

LAJ-J

ae

e

II

O0

J:I:I.

I

360

MINUTESFIG. 8. Comparison of the in vitro bactericidal activity of macrophages collected from the peritoneal

cavities ofmice injected i.p. 3 days earlier with 1.0 ml ofeither complete thioglycolate medium, PBS containingthe same concentration (0.075% wt/vol) of agar as in thioglycolate, or agar-free thioglycolate or PBS. Eachtime point is the geometric mean of triplicate samples.

reduction in the i.v. 50% lethal dose and theimpaired ability of thioglycolate-treated mice tocontrol the growth of Listeria in their livers (1)were probably not due to the thioglycolate caus-

ing a generalized systemic impairment of bacte-ricidal activity.Agar or some contaminant in the agar is con-

sidered to be the most likely single componentof thioglycolate medium which caused the in-duced suppression of bactericidal activity. Mac-rophages isolated from the peritoneal cavities ofanimals injected with either thioglycolate or

PBS containing agar exhibited characteristics ofcells which had engaged in extensive phagocy-tosis. This was shown by the large number of

vacuoles and lysosomes in the cytoplasm. Thisfinding could be interpreted to suggest that mac-rophages may have endocytosed the suspensionof colloidal agar. If macrophages engulfed theagar, this could have provided sufficient condi-tions for impairing bactericidal activity. Thispossibility is not without precedent since otherstudies have shown that macrophage ingestionof heterologous erythrocytes inhibited the de-struction of bacteria (6, 10). Indeed, more recentstudies have shown that the ingestion of eryth-rocytes inhibits the tumoricidal activity of acti-vated macrophages (34). The exposure of acti-vated macrophages to thioglycolate is known toinhibit antibody-dependent cell-mediated cyto-

INFECT. IMMUN.

on June 13, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

FUNCTIONS OF ACTIVATED MACROPHAGES 283

TABLE 1. Cellular composition ofperitoneal exudates after i.p. injection of thioglycolate medium or PBS,prepared with or without agar

Cellular compositiona on day:

1 ~~~~~~~~23Groupb

Mononu- Mononu- Mononu-paclears Lympho- PMNd clear phag- Lympho- PMN clear phag- cytes PMpaoye yeocytes cytes ocytes cye

Thioglycolate me- 2.3 ± 0.5 2.7 ± 0.5 6.9 ± 0.6 11.0 ± 1.1 4.0 ± 0.6 7.9 ± 1.5 11.0 ± 0.3 3.0 ± 0.8 2.4 ± 0.8dium

PBS' with agar 7.0 ± 1.0 4.9 ± 0.5 18.0 ± 2.9 10.0 ± 2.3 3.6 ± 0.3 7.7 ± 1.0 9.9 ± 0.9 2.0 ± 0.7 2.7 ± 0.93Thioglycolate with- 2.6 ± 0.7 3.2 ± 0.2 1.9 ± 0.5 2.8 ± 0.1 6.7 ± 1.0 0.6 ± 0.02 2.0 ± 0.5 7.3 ± 0.7 0.5 ± 0.03

out agarPBS without agar 1.3 ± 0.06 3.4 ± 0.5 0.48 ± 0.07 3.1 ± 0.06 4.5 ± 0.3 0.47 ± 0.3 3.0 ± 0.4 6.2 ± 1.4 0.7 ± 0.3Control uninjected 1.7 ± 0.2 3.8 ± 0.9 0.1 ± 0.08 1.8 ± 0.5 5.0 ± 1.3 0.4 ± 0.09 1.9 ± 0.7 3.9 ± 0.3 0.5 ± 0.09

aResults are expressed as the number of cells x 10-6 ± standard error of the mean (SEM).'Three mice/group per time point.C Mononuclear phagocytes were identified by morphology, as well as by their ability to ingest heat-killed Listeria.d PMN, Polymorphonuclear leukocyte.PBS was prepared with the same concentration of agar (0.075% wt/vol) as in thioglycolate medium.

toxicity and the release of H202 (15). However,thioglycolate-induced exudate macrophageshave been shown to produce greater than normalamounts of superoxide anion (7). From thesefindings, it is tempting to postulate that theingestion of agar acted somehow to divert thebiochemical pathways or that the agar acted as

a scavenger of H202, which would have resulted,in either case, in a reduction of the bactericidalpotential of macrophages.

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Servicegrants CA-21360 and RRO5705 from the National CancerInstitute and the Division ofResearch Resources, respectively.

The excellent technical assistance of Patricia Riemensniderand Susan Hutcelseider is gratefully acknowledged.

LfTERATURE CITED

1. Baker, L., and P. A. Campbell. 1980. Thioglycolatemedium decreases resistance to bacterial infection inmice. Infect. Immun. 27:455-460.

2. Bar-Eli, M., and R. Gallily. 1975. The effect of macro-

phage hydrolytic enzyme levels on the uptake and deg-radation of antigen and immune complexes. RES J.Reticuloendothel. Soc. 18:317-328.

3. Blanden, R. V., M. J. Lefford, and G. B. Mackaness.1969. The host response to Calmette-Guerin bacillusinfection in mice. J. Exp. Med. 129:1079-1107.

4. Cohn, Z. A. 1978. The activation of mononuclear phago-cytes: fact, fancy and future. J. Immunol. 121:813-816.

5. Collins, F. M. 1968. Effect of specific immune mouse

serum on the growth of Salnonella enteritidis in non-

vaccinated mice challenged by various routes. J. Bac-teriol. 97:667-675.

6. Gill, F. A., D. Kaye, and E. W. Hook. 1966. The influ-ence of erythrophagocytosis on the interaction of mac-

rophages and Salmonella in vitro. J. Exp. Med. 124:

173-183.7. Johnston, R. B., Jr., C. B. Godzik, and Z. A. Cohn.

1978. Increased superoxide anion production by immu-

nologically activated and chemically elicited macro-

phages. J. Exp. Med. 148:115-127.

8. Karnovsky, M. L., and K. J. Lazdins. 1978. Biochemicalcriteria for activated macrophages. J. Immunol. 121:809-813.

9. Karnovsky, M. L, J. Lazdins, D. Drath, and A. Har-per. 1975. Biochemical characteristics of activated mac-rophages. Ann. N.Y. Acad. Sci. 256:266-274.

10. Kaye, D., and E. W. Hook. 1963. The influence ofhemolysis on susceptibility to Salmonella infection. J.Immunol. 91:65-75.

11. Mackaness, G. B. 1962. Cellular resistance to infection.J. Exp. Med. 116:381417.

12. Mackaness, G. B. 1970. Cellular immunity, p. 461477.In R. van Furth (ed.), Mononuclear phagocytes. Black-well Scientific Publications, Oxford.

13. McConahey, P. J., and F. J. Dixon. 1966. A method oftrace iodination of proteins for immunologic studies.Int. Arch. Allergy Appl. Immunol. 29:185-189.

14. Miake, S., K. Takeya, T. Matumato, Y. Yoshikai, andK. Nomoto. 1980. Relation between bacterial and phag-ocytic activities of peritoneal macrophages induced byirritants. RES J. Reticuloendothel. Soc. 27:421427.

15. Nathan, C., and Z. Cohn. 1980. Role of oxygen-depend-ent mechanisms in antibody-induced lysis of tumor cellsby activated macrophages. J. Exp. Med. 152:198-208.

16. Nathan, C. F., and W. D. Terry. 1977. Decreased phag-ocytosis by peritoneal macrophages from BCG treatedmice. Induction of the phagocytic defect in normalmacrophages with BCG in vitro. Cell. Immunol. 29:295-311.

17. Newborg, M. F., and R. J. North. 1980. On the mecha-nism of T-cell-independent anti-Listeria resistance innude mice. J. Immunol. 124:571-576.

18. Nogueira, N., and Z. Cohn. 1976. Trypanosoma cruzi:mechanism of entry and intracellular fate in mamma-lian cells. J. Exp. Med. 143:1402-1420.

19. Nogueira, N., and Z. Cohn. 1978. Trypanosoma cruzi:in vitro induction of macrophage microbiocidal activity.J. Exp. Med. 148:288-300.

20. Nogueira, N., S. Gordon, and Z. Cohn. 1977. Trypa-nosoma cruzi: modification of macrophage functionduring infection. J. Exp. Med. 146:157-171.

21. Nogueira, N., S. Gordon, and Z. Cohn. 1977. Trypa-nosoma cruzi: the immunological induction of macro-phage plasminogen activator requires thymus-derivedlymphocytes. J. Exp. Med. 146:172-183.

22. North, R. J. 1969. Cellular kinetics associated with thedevelopment of acquired cellular resistance. J. Exp.Med. 130:299-314.

VOL. 34, 1981

on June 13, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

284 SPITALNY

23. North, R. J. 1974. T-cell dependence of macrophageactivation and mobilization during infection with My-cobacterium tuberculosis. Infect. Immun. 10:66-71.

24. North, R. J. 1978. The concept of the activated macro-phage. J. Immunol. 121:806-809.

25. North, R. J., and J. F. Deissler. 1975. Nature of "mem-ory" in T-cell-mediated antibacterial immunity: cellularparameters that distinguish between the active immuneresponse and a state of "memory." Infect. Immun. 12:761-767.

26. North, R. J., and D. P. Kirstein. 1977. T-cell-mediatedconcomitant immunity to syngeneic tumors. I. Acti-vated macrophages as the expressors of nonspecificimmunity to unrelated tumors and bacterial parasites.J. Exp. Med. 145:275-292.

27. Ruskin, J., and J. Remington. 1968. Immunity andintracellular infection: demonstration of resistance toListeria and Salmonella in mice infected with a pro-tozoan (toxoplasma). Science 160:72-74.

28. Salvin, S. B., and S. L. Cheng. 1971. Lymphoid cells indelayed hypersensitivity. II. In vitro phagocytosis andcellular immunity. Infect. Immun. 3:548-552.

29. Snodgrass, M. J., D. S. Lowrey, and M. G. Hanna, Jr.1972. Changes induced by lactic-dehydrogenase virus in

INFECT. IMMUN.

thymus and thymus-dependent areas of lymphatic tis-sue. J. Immunol. 108:877-892.

30. Spitalny, G. L. 1980. Suppression of bactericidal activityof macrophages in ascites tumors. RES J. Reticuloen-dothel. Soc. 28:223-235.

31. Spitalny, G. L., and R. J. North. 1977. Subversion ofhost defense mechanisms by malignant tumors: an es-tablished tumor as a privileged site for bacterial growth.J. Exp. Med. 145:1264-1277.

32. Stewart, C. C., H. Lin, and C. Adles.1975. Proliferationand colony-forming ability of peritoneal exudate cells inliquid culture. J. Exp. Med. 141:1114-1132.

33. Unkeless, J. C., S. Gordon, and E. Reich. 1974. Secre-tion of plasminogen activator by stimulated macro-phages. J. Exp. Med. 139:834-850.

34. Weinberg, J. B., and J. B. Hibbs, Jr. 1977. Endocytosisof red blood cells or haemoglobin by activated macro-phages inhibits their tumoricidal effect. Nature (Lon-don) 269:245-247.

35. Weiner, E., and A. Bandieri. 1975. Modification in thehandling in vitro of 1251-labelled Keyhole limpet hae-mocyanin by peritoneal macrophages from mice pre-treated with the adjuvant Corynebacterium parvum.Immunology 29:265-274.

on June 13, 2020 by guesthttp://iai.asm

.org/D

ownloaded from