Pertanika 13(1), 45-52 (1990)

The In Vitro Bactericidal Activity of Normal and ActivatedMouse Macrophages against Salmonella typhimurium C5sr

M. MUSA and D. ROWLEYDepartment of ImmunoLogy,SchooL of MedicaL Sciences,Universiti Sains MaLaysia,

11800 Minden, PuLau Pinang, MaLaysia.

Key words: In vitro bactericidal activity, mouse macrophages, Salmonella typhimurium C5sr

ABSTRAKPerbandingan aktiviti bakterisidaL in vitro makrojaj normaL dan teraktif teLah menunjukkan bahawa pem­bunuhan strain viruLen Salmonella typhimurium C5sryang beropsonin oLeh makrojaj normaL ditingkatkandengan kehadiran serum segar di daLam medium kuLtur. SebaLiknya, kehadiran serum tersebut tidak diperlukanuntuk aktiviti bakterisidaL makrojaj teraktif. Pembunuhan bakteria yang meningkat oLeh makrojaj teraktifdiperoLehi sama ada dengan kehadiran atau ketiadaan serum, dan ini tidak disebabkan oLeh kewujudanimunogLobuLin terikat makrojaj. Faktor di daLam serum amab yang meningkatkan pembunuhan bakteriaoLeh makrojaj normaL berkemungkinan besar terdiri daripada komponen-komponen sistem kompLemen. Faktor­jaktor tahan haba (imunogLobuLin) di daLam serum amab normaL hanya berkesanjika ia imunogLobuLin terikatpermukaan.

ABSTRACTA comparison oj the in vitro bactericidaL activity oj normaL and activated macrophages indicated that killingoj the viruLent strain oj opsonized Salmonella typhimurium C5sr by normaL macrophages was enhancedby the presence ojjresh serum in the cuLture medium. In contrast, there was no such requirement jor thebactericidaL activity ojactivated macrophages. The enhanced killing oj the bacteria by activated macrophagesoccurred both in the presence and absence ofserum, and this was not accountedjor by the existence ofmacrophage­bound immunogLobuLin. The jactors in normaL rabbit serum which potentiated the killing oj bacteria bynormaL macrophages were suspected to be components oj the compLement system. Heat-stabLe jactors(immunogLobuLins) in normaL rabbit serum were effective only if they were surface bound immunogLobuLins.

I TRODUCTIOStudies have established the participation ofnormal immunoglobulin G(IgG) in enhancing thephagocytosis and intracellular killing of bacteriasuch as Staphylococcus aureus and Escherichia coli bynormal human monocytes and granulocytes (Leijhet al. 1981; van Fu rth and Leijh 1980) andresident mouse peritoneal macrophages (Leijh etal. 1984). These studies indicared that the en­hancing effect of IgG was due to the interation ofIgG with the Fc receptor on the membrane of thephagocyte. Other serum proteins such as comple­ment components and plant lectins have beenshown to have similar effect as IgG following

binding of receptors to phagocytes (Leijh et a/.1981). It was shown from their studies that acomplement component, probably C3b, wasrequired for maximum intracellular killing byphagocytes. Similar results were reported by otherworkers on the enhancing effect of normal IgG andcomplement on the bactericidal activity of humanmacrophages against Listeria monoeytogenes (Mac­Gowan et at. 1983). In vitro studies on thebactericidal activity of both normal and activatedmacrophages were carried out to determine thecell requirements for maximum bactericidalactivity.

M. MUSA AND D. ROWLEY

MATERIALS AND METHODSTest BacteriaFor these studies, a virulent strain of S. typhimuriumC5sr (streptomycin-resistant) was chosen as the testorganism. It has been shown previously thatactivation of macrophages was an importantprerequisite for optimal killing of this organism(Blanden et at. 1966). This strain is also notkilled in the presence of specific antibody and com­plement (Reynolds and Pruul 1971). The strainof S. enteritidis 11RX was used for macrophageactivation. The bacterial strains were obtainedfrom the Department of Microbiology andImmunology, University of Adelaide, Australia.Prior to the experiments, the log-phase cultureswere washed once with 10ml tissue culturemedium RPMI-1640 (Flow Labs) and resuspend­ed in a similar volume of the medium at a con­centration of 2 x 10 5 bacterial/m!. The bacteriawas then opsonized at room temperature for 15min with purified rabbit IgG to S. typhimuriumC5sr (Donated by Dr. Reynolds of the Depart­ment of Microbiology and Immunology, U niver­sity of Adelaide). The bacterial suspension wasthen divided into three aliquots (or otherwiseindicated), to which a final concentration 10% v/vof either fresh or heat-inactivated (56°C/1h)normal rabbit serum or tissue culture medium wasadded.

Lymphokine- Containing Supernatant

Ten ml of mouse spleen cell suspensions (5-6 x106 cells/ml) in tissue culture medium containing2% v/v heat- inactivated foetal calf serum (FCS)was dispensed into tissue culture flasks (FlowLabs). Concanavalin A (Con A) (Pharmacia) ata final concentration of 3.0ug/ml or an equivalentvolume or tissue culture medium was added to theflasks. After incubation for 24 hours, cell-freesupernantants were obtained by centrifugationand filtration through a Millipore membrane(0.45um). To the control supernantant harvestedfrom unstimulated cell cultures, an equal concen­tration of Con A was added after harvesting. Thesupernatants were stored in aliquots at - 70°C.

Removal of Complement Activity from Normal RabbitSerumZymosan-adsorbed rabbit serum: Fresh rabbitserum was adsorbed with washed zymosan(Sigma) (2mg/ml) at room temperature for 1

hour. Zymosan and adsorbed material were thenremoved by centrifugation.

Aggregated IgG-treated rabbit serum: Anaggregated rabbit IgG was prepared by heatingnormal rabbit serum at 63°C for 25 min. Freshnormal rabbit serum was incubated at 37°C for30 min with the dilution (1/8) of aggregated rabbitIgG.

Collection of Mouse Peritoneal Cells

Normal resident macrophages were collected frommice immediately upon arrival from the CentralAnimal House, University of Adelaide. Activatedmacrophage referred as 11 RX-activatedmacrophages were obtained from mice which hadbeen previously (6-8 days) injected intra­peritoneally with 105 live S. enteritidis 11 RX (LaPosta et al, 1982). Peritoneal cells obtained werewashed by centrifugation and finally resuspendedin tissue culture medium to a concentration of 2x 10 6 cells/m!. An aliquot of the suspension wasstained with Giemsa to determine the percentageof macrophages in the population of cells. Trypanblue exclusion revealed that the viability of thecells was more than 95 %.

Bactericidal Assay

The bactericidal assay was carried out in micro­culture trays by the following steps. The cells(ei ther normal or 11 RX-activated macrophages,2 x 10 5/well) adhered to the wells after an initialincubation of 30 min at 37°C in 5% C02/air.

on-adherent cells were removed by washing thewell once with 0.3 ml RPMI -1640. Themonolayers were then incubated in 0.3mlRPMI-1640 for a further 30 min. The mediumwas removed from the monolayers prior to theaddition of bacteria. Fifty /11 of the opsonizedbacterial suspension was added to the monolayersin tiplicate. To ensure close contact between thecell and bacteria, the micro-culture trays werecentrifuged at 1500g for min at 4°C in a Coolspincentrifuge (MSE). The trays were then incubatedat 43°C for 2 min, follow by incubation at 37°Cin 5 % C02/air. Control wells containing onlybacteria in the presence of the three differentculture media used above were included andtreated in a similar manner. Viable counts of thebacteria were made on nutrient agar at time zeroand 60 min. Fifty /11 of Triton-XlOO (1 % v/v in

46 PERTA IKA VOL. 13 NO.1, 1990

IN VITRO BACTERICIDAL ACTIVITY OF NORMAL AND ACTIVATED MOU E MACROPHACES

RESULTS

The Bactericidal Activity ojNormal and 11RX-ActivatedMacrophagesResults (Figure I) show that killing of the virulentstrain of preopsonized S. typhimurium C5sr bynormal macrophages was enhanced in the presenceoffresh serum. Bacteria incubated in control wellsin the absence of macrophages increased innumber by a factor of 1.5 to 2. The l1RX­activated macrophages showed an enhancedbactericidal capacity compared to normalmacrophages, and were able to kill opsonizedbacteria equally well in the presence or absenceof erum.

Survival qf preopsonized S. typhimurium C5srincubated with normal (0, 0 ,~) and 11RX­

lUtivated (e, • , &) mlUTOphages in the presenceqf10% vlvfresh rabbit serum (0, e), 10% vlv heat­inlUtivated rabbit serum (~, &), or tissue culturemedium (0, .). Each point represents the meanpercentage survival ± S. D obtainedfrom jive replicateexperiments.

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saline) was added to each well prior to samplingin order to disrupt the macrophages and releaseintracellular bacteria. The survival of bacteria wascalculated by dividing the number of bacteria inthe experimental wells by the number of bacteriain the control wells and was expressed aspercen tage.

Removal oj Cell- Bound Antibody on 11RX-ActivatedM acrophagesTwo methods were employed to removemacrophage-bound immunoglobulins. In the first,llRX-activated macrophages in siliconized glasstubes were washed three times with lOml aliquotsof 0.005M EDTA in tissue culture medium andonce with the same medium but without EDTAbefore being cultured and their bactericidalpotency assayed as described before. The secondmethod involved treatment of the cells with aneutral protease (Dispase, grade II, Mannheim,Boehringer) 1.5mg/ml at 37°C for 45 min. Afterincubation, the cells were washed once withmedium before being cultured as above.

The Expression ojthe Bactericidnl Activity ojMacrophagesActivated in Vitro with Lymphokines.

ormal macrophage monolayers were incubatedin 30% v/v Iymphokine-containing supernatantderived from Con-A-stimulated spleen cellcultures. For control, macrophage monolayerswere also incubated in a culture supernatantderived from unstimulated spleen cells to whichan equivalent concentration of Con-A had beenadded following harvesting. The cells wereincubated at 370C in 5% C02/air overnight. Afterincubation, the monolayers were washed onceprior to bactericidal assay. The bactericidal assaywas performed as described before in the presenceof either fresh or heat-inactivated normal rabbitserum or medium alone.

Coating of Tray with Purified Rabbit IgGPurified rabbit IgG was prepared according to themethod described by Ey et al. (1978). The wellswere coated by adding 50f.11 of purified rabbit IgG(Img/ml) after which the micro-culture trays werekept at 4°C overnight and washed once withmedium prior to use.

Statistical AnafysisStatistical analyses were performed usmg theStudent's t-test.

The Effect oj the Removal oj Cell- Bound Antibody onthe Bactericidnl Activity oj11RX-Activated MacrophagesThe enhanced bactericidal activity of l1RX­activated macrophages may be due to immuno­globulins already bound to the cell surface whichresult in maximal stimulation in the absence ofadded serum. To investigate this possibility, thebactericidal activity of llRX-activated macro­phages was determined after removal ofthe possible

PERTANIKA VOL. 13 NO.1, 1990 47

M. MUSA AND D. ROWLEY

TABLE 1Survival of preopsonize S. typhimurium C5sr incubated with 11 RX-macrophages

before and after treatment with EDTA, in the presence of10% v/v rabbit serum or culture medium alone.

Treatment Percentage survival'with EDTA

Serum 30 min 60 min

Before + 9.1 ± 1.8 5.6 ± 1.411.2 ± 1.9 6.1 ± 1.8

After + 10.5 ± 1.9 6.4 ± 1.311.5 ± 0.9 10.3 ± 1.8

'Mean ± S.D. of triplicate cultures.

TABLE 2Survival of preopsonized S. typhimurium C5sr incubatd with 11 RX-activated macrophages

before and after treatment with Dispase, in the presence of10% v/v fresh rabbit serum or culture medium alone.

Treatment Percentage survival'with dispase

Serum 30 min 60 min

Before + 13.9 ± 2.4 12.0 ± 0.416.4 ± 1.0 13.8 + 1.0

After + 15.3 ± 2.9 13.2 ± 2.315.2 ± 1.2 14.5 ± 1.1

• Mean ± S.D. of triplicate cultures.

cell-bound immunoglobulins and addition ofeither fresh rabbit serum or medium alone. Thisactivity was compared with that of untreatedmacrophage cultures under similar conditions(Tables 1 and 2). The ability of llRX-activatedmacrophages to kill the bacteria was still retainedalthough cell-bound immunoglobulins on theirsurface had been removed. In addition, theexpression of enhanced bactericidal capacity wasnot dependent on the presence of serum in themedium.

The Serum Requirement for Expression of the Bact';'icidal

Activity of Macrophages Activated In Vitro with

LymphokinesTo support further the possibility that thebactericidal capacity of activated macrophages isindependent of both cell- bound immunoglobulinsand the presence of serum, the bactericidal activityof macrophages activated in vitro by lymphokines

was determined. As in the earlier studies, thekilling of the bacteria by normal macrophages wasenhanced in the presence of fresh serum (Fig. 2.1).However, for lymphokine-activated macrophages,the presence of serum was not necessary forexpression of bactericidal potential (Fig. 2.2).These findings substantiate earlier results indicatingthat cell- bound antibody was not necessary forexpression of enhanced bactericidal capacity inactivated macrophages.

The Effect of Various Dilutions of Fresh Rabbit Serum

on the Bactericidal Activity of Normal Macrophages

To estimate the optimal amount of fresh serumrequired for the killing of virulent preopsonizedS. typhimurium C5sr by normal macrophages, thebacteria and the cells were incubated together inthe presence of various dilutions of fresh rabbitserum and the bactericidal assay was performedas previously described. It may be seen from the

48 PERTANlKA VOL. 13 O. I, 1990

IN VITRO BACTERICIDAL ACTIVITY OF NORMAL AND ACTIVATED MOUSE MACROPHACES

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The Nature oj the Factor(s) in Fresh Serum Involved in

Stimulating the Bactericidal Activity oj Normal

MacrophagesIn order to delineate the relative roles of IgG andcomplement in the killing of the virulent strain ofS. typhimurium CSsr by normal macrophages,rabbit serum lacking certain complement compo-

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Fig 2 Survival oj preopsonize S. typhimurium C5srincubal£d with (I) normal control macrophages or (2)

lymphokine-activated macrophages in the presence of10% vlv fresh rabbit serum (A), 10% vlv heat­intu:tivated rabbit serum (B) or culture medium alone(C). Each histogram represents the mean percentagesurvival ± S. D obtainedfromJour separale experiments.

ABC 0 E F G H

data (Table 3) that there was a correlation (r =

0.96 for serum concentration 0-10%) between thenumber of bacteria killed and the concentrationof fresh rabbit serum in the wells. Optimal killingof the bacteria by normal macrophages wasobserved when 10% fresh rabbit serum waspresent in the medium. Concentrations greaterthan this did not increase significantly (P < 0.001)the number of bacteria killed.

Fig 3 Survival oj preopsonized S. typhimurium C5srincubated with normal macrophages in the presence ifvarious sera in the culture mediuim (10% vlv). (A)fresh rabbit serum, (B) Jresh guinea-pig serum, (C)fresh C6-deficient rabbit serum, (D) fresh C4-deficientguinea-pig serum, (E) heat-inactivated rabbit serum,(F) zymosan-adsorbed rabbit serum, (G) aggregatedIgG-treatpd rabbit serum, (H) tissue culture mediumalone. Each histogram represents the mean percentagesurvival ± S. D. oj three separate experiments.

TABLE 3The effect of the concentration of fresh rabbit serum on the killing of preopsonized.

S. typhimurium C5sr by normal macrophages.

Concentration ofserum (%v/v) 30 min

Percentage survival'

60 min

0.00.11.0

10.030.0

54.0 ± 1.753.2 ± 1.044.2 ± 0.827.2 ± 0.926.8 ± 1.3

61.6 ± 1.9564 ± 0.747.5 ± 0.625.2 ± 1.422.4 ± 1.2

'Mean ± S.D. of triplicate cultures.

PERTANIKA VOL. J:l I O. 1, 1990 49

M. MUSA AND D. ROWLEY

nents and purified rabbit IgG were used tostimulate the bactericidal activity of normal

macrophages. The bactericidal assay was per­formed as previously described by adding pre­opsonized bacteria to the normal macrophagemonolayers in the presence of the following sera(10% v/v): (a) zymosan-adsorbed rabbit serum;(b) aggregated IgG-treated rabbit serum; (c) heat­inactivated (56C / 1h) rabbit serum; (d) freshC6-deficient rabbit serum and (e) freshC4-deficient guinea-pig serum (provided by Dr.S. Neoh). The killing of the bacteria by normalmacro phages was reduced in the presence ofnormal rabbit serum from which more than oneof the complement components had been removed(Fig. 3). Since effective killing could still bedemonstrated in the presence of serum lacking asingle component such asC4 and C6, it is suggestedthat neither of these components was involved inthe stimulation of the bactericidal activity ofnormal macrophages. The ability of normalmacrophages to kill the bacteria in the presence

100

of purified rabbit IgG was also studied. The data(F~I{. 4) show that the killing of the bacteria wasno different from that observed using heat­inactivated rabbit serum. In addition, no additiveeffect was shown when purified rabbit IgG andheat - inactivated rabbit serum were presenttogether in the culture medium. It seemed clearthat the IgG did not significantly potentiate thekilling of the bacteria by normal macrophages.Maximal potentiation was achieved only whenheat-liable factors were present; these factors beinglikely components of the complement system.

The Effect of Surface- Bound fgC on the Bactericidal

Activity of Normal Macrophages

It seemed possible that surface-bound IgG maybe more efficient than soluble IgG in stimulatingthe bactericidal activity of normal macrophages.In order to test this possibility, normalmacrophage monolayers were prepared either inwells of micro-culture trays which had been pre­coated with purified rabbit IgG or in uncoatedwells. Monolayers on the uncoated wells were

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Survival oj preopsonized S. typhimurium C5srincubated with normal macrophages in the presence of10% v/v fresh rabbit serum (A), 10% v/v heat­inactivated rabbit serum (B), purified rabbit IgG(lmg/ml) (C), 10% v/v heat-inactivaled rabbit serum+ purified rabbit IgG (1 mg/ml) (D), tissue culture

medium (E). Each histogram represents the meanpercentage survival ± S D. of triplicate cultures.

Uncoated wellsFig 4

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o L--~A-'--'-;B::-'---'-C;:;-'--{I-"-;C::-'--­

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coated wells

Survival oj preopsonized S. typhimurium C5srincubated with normal macrophages in the presence of10% v/vfresh rabbit serum (A), soluble purified IgG(B), tissue culture medium alone (C). Each histogramrepresents the mean percentage survival ± S D ojthreeseparate experiments.

50 PERTANIKA VOL. 13 NO.1, 1990

IN VITRO BACTERICIDAL ACTIVITY OF NORMAL AND ACTIVATED MOUSE MACROPHACES

infected with preopsonized bacteria in the presenceof either 10% v /v fresh rabbit serum, purifiedrabbit IgG (1 mg/ml) or medium alone. Whereasmonolayers on the rabbit IgG-coated wells wereinfected with preopsonized bacteria in the presenceof medium alone. The bactericidal assay wasperformed as previously described. Normal mouseperitoneal macrophages, upon incubation withpurified rabbit anti-BSA IgG, adsorbed to ahydrophobic polymer, polystyrene have beenshown to generate superoxide anion (Kasai etai. 1982). In this studies, it was also shown thatsoluble IgG did not stimulate the production ofthis oxygen radical by normal macrophages. Fromthese results, it seemed possible that surface-boundIgG may be more efficient than soluble IgG instimulating the bactericidal activity of normalmacrophages. Our results (Fig. 5) show thatpurified rabbit IgG bound to the polystyrene trayenhanced the killing of the bacteria by normalmacrophages to a greater extent than did solublerabbit IgG (p < 0.02).

DISCUSSIONStudies carried out in vitro by Leijh and othersshowed that factors in normal serum influencedthe bactericidal activity of normal phagocytic cellssuch as granulocytes, monocytes and macrophages(Solberg and Helium 1973; Leijh et ai. 1979,1980, 1984; MacGowan et al. 1983). Theseserum factors not only enhanced phagocytosis butwere also necessary to allow the cells to exhibittheir maximal bactericidal potential. We decidedto investigate whether the bactericidal activityof macrophages from both normal and S.enteritidis 11RX-infected mice was enhanced in thepresence of serum, and our data show that thebactericidal activity of macrophages from normalanimals is enhanced. Thus, despite the fact thatthe bacteria were preopsonized, their intracellularfate was also determined to some extent by thecontinual presence of serum in the extracellularenvironment.

This observation was confirmed by ourfindings that the number of bacteria killed inthe time period under study correlated well withthe concentrations of fresh rabbit serum in themedium. It should be pointed out that the en­hancing property of rabbit serum was not speciesspecific since similar results were obtained usingguinea-pig or rat serum (data not shown). With

regard to the in vitro killing activity of macrophagesobtained from Salmonella-infected mice, our dataindicate that the presence of serum extracellularlywas not required. The opsonized bacteria werekilled by these cells equally well both in thepresence or absence of serum. In view of thefinding that the enhanced bactericidal activity ofactivated macrophagef was independent of serum,we investigated whether this could be accountedfor by immunoglobulins bound to the surface ofthese cells.

Other studies have shown that macro­phages from mice previously infected withsalmonellae possessed cytophilic antibodies(Turner et ai. 1964; Rowley et ai. 1964). Thepresent investigation showed that removal ofsurface immunoglobulins from macrophages usingtwo different methods did not alter their abilityto kill bacteria in the absence of serum. These datawere confirmed using macrophages activated bya supernatant obtained from Con A-stimulatedspleen cells. Again the enhanced bactericidalactivity was serum independent. It seemed possiblethat the factor in serum which enhanced thebactericidal activity of normal macrophages wasimmunoglobulins together with the complementcomponent C3b (Leijh et al. 1984). However,results obtained in the present study indicate thatcomplement components are the essential factors,since the bactericidal activity of normalmacro phages was not enhanced in the presenceof heat-inactivated serum.

The importance of complement wasstrengthened further by the observation thatthe presence of IgG in the culture mediumalone was not sufficient to stimulate thebactericidal activity of normal macrophages.However, some stimulation was observed if themacrophages were in contact with surface-boundIgG. These findings are in agreement with thosereported by Kasai et al. (1982) who showed thatIgG adsorbed to a hydrophobic polymer, poly­strene or protein A stimulated mouse macrophagesto generate superoxide anion whereas soluble IgGdid not. This may be due to the binding of Fereceptors by, in the former case, sui tably orientatedimmunoglobulin molecules. The importance ofcomplement was' substantiated when it was foundthat its removal by adsorption with zymosan oraggregated IgG abolished the enhancing effect ofthe serum. Both C6 and C4 deficient sera were

PI::RTANIKA \,()L. J:l :\U I. 1990 51

M. MUSA ADD. ROWLEY

able to enhance the bacteridical capacity of normalmacrophages indicating that these componentswere not necessary for this effect. Absence of C4prevents classical (immunoglobulin mediated)complement activation. A C6 deficiency abrogatesformation of the terminal membrane attackcomplex of complement. Thus activation of com­plement must have occurred by the alternatepathway, and the component most likely to beinvolved in macrophages stimulation is C3b.Although our results have not shown definitivelythat the component responsible is C3b, data byother workers suggests that this might be so.Schorlemmer and Allison (1976) have shown, forexample, that C3b binding to its receptor on thesurface membrane of mouse macrophages effectsthe secretion of certain lysosomal enzymes.

In conclusion, the present study stronglysupports the contention that both heat stable andheat labile components of serum enhance thebactericidal activity of normal macrophages.Activation of these cells either in vivo, or in vitro,

however, negate the requirement for such serumcomponents in allowing full expression of theirbactericidal potential and it would be of interestto see if this reflect changes in the number of Fcand C3b receptors on their membrane.

ACK OWLEDGEME TSWe thank Dr. S. Neoh of the Clinical Immuno­logy Department, Finders Medical Centre andDr. Reynolds of the Department of Microbiologyand Immunology, University of Adelaide for thesera.

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(Received 10 October, 1988)

52 PERTA IKA VOL. 13 NO. 1,1990