INFECTION AND IMMUNITY, Oct. 1977, p. 52-59Copyright i 1977 American Society for Microbiology

Vol. 18, No. 1Printed in U.S.A.

Interactions Between Salmonellae and Macrophages ofGuinea Pigs

IV. Relationship Between Migration Inhibition and Antibacterial Actionof Macrophages

DONALD R. MAYO,t H. S. HSU,* AND FRANKLIN LIM

Department ofMicrobiology* and Department of Pathology, Medical College of Virginia, VirginiaCommonwealth University, Richmond, Virginia 23298

Received for publication 28 February 1977

The in vitro macrophage migration inhibition test was used to detect thedevelopment of delayed-type hypersensitivity in guinea pigs infected with Sal-monella typhimurium. Four different preparations from supernatants of S. typhi-murium cultures were used as the antigens in this test. They included theconcentrated bacterial antigens, the high-molecular-weight (>50,000) antigens,the ammonium sulfate-precipitated antigens, and the ribonuclease-treated anti-gens. All four antigen preparations were shown to inhibit the migration ofperitoneal macrophages of salmonella-infected (immune) guinea pigs from capil-lary tubes, in comparison with cells of normal control animals. By use of thehigh-molecular-weight antigens and the ammonium sulfate-precipitated antigens,the production of the migration inhibition factor(s) was elicited from cultures oflymphocytes obtained from the peripheral blood of immune guinea pigs. Theactivity of the migration inhibition factor(s) was demonstrated by its ability toinhibit the migration of peritoneal macrophages of normal guinea pigs fromcapillary tubes. In contrast, normal peritoneal macrophages exposed to productsof antigen-stimulated immune lymphocytes did not exhibit an enhanced phago-cytic or bactericidal action against virulent S. typhimurium as compared withthose of the normal control. The present study indicated that the bacterialantigens responsible for the elicitation of the production of the migration inhibi-tion factor from Iymphocytes ofimmune guinea pigs are inactivated by proteolyticenzymes, but not by ribonuclease, and have molecular weights of >50,000.

Experimental evidence in this laboratory hasconsistently shown that virulent salmonellaeare effectively destroyed by macrophages ofguinea pigs and mice (11, 16, 22). Macrophagesfrom immune animals were not endowed withan enhanced capacity to accelerate the intracel-lular killing of virulent Salmonella typhimu-rium (11, 16), as found by other investigators (2,6, 17, 24, 30). In contrast, the opsonizing effectof immune serum appears to play an importantrole in the acquired resistance of the host tosalmonellosis (12, 14, 16, 19, 23). On the otherhand, it is generally agreed that delayed-typehypersensitivity to bacterial antigens is a con-comitant manifestation of the host in salmonel-losis (4, 7, 27).

Current knowledge in immunobiology favorsthe concept that the antibacterial cellular im-munity expressed by macrophages is mediated

t Present address: Department of Microbiology, GraduateSchool of Public Health, University of Pittsburgh, Pittsburgh,PA 15213.

by products elicited from T-lymphocytes eitherspecifically with antigens (5, 15, 21, 25) or non-specifically with mitogens (8). In this context,the production of lymphokines from sensitizedlymphocytes requires the elicitation with spe-cific antigens, whereas the expression of en-hanced antibacterial action in macrophages me-diated by lymphokines is directed nonspecifi-cally against different bacteria.The present paper is intended to demonstrate

the manifestation of delayed-type hypersensi-tivity in salmonella-infected guinea pigs by useof the in vitro macrophage migration inhibitiontest and to determine whether the antibacterialactivities of macrophages can be altered byproducts of immune lymphocytes stimulatedwith salmonella antigens, using a cell cultureprocedure.

MATERIALS AND METHODSS. typhimurium. The stock virulent strain SR-11

(13) was used in this study. The mean lethal dose of52

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MACROPHAGE AND SALMONELLA INTERACTIONS

this strain was approximately 10 bacteria upon intra-peritoneal injection into mice. The preparation of anoptically standardized bacterial suspension in saline(containing approximately 2.5 x 109 bacteria/ml) wasdescribed previously (11, 13).

Preparation of bacterial antigens. Cultures ofS. typhimurium were grown in tubes of 5 ml of trypticsoy broth (Difco) placed in a rotating drum at 37°Cfor 48 h. They were heated in a 60°C water bath for30 min and then centrifuged at 2,000 x g for 30 min.The supernatant fluid was passed through a mem-brane filter (0.45,m; Millipore Corp.), checked forsterility, and then concentrated to approximately 1/10of its original volume with Lyphogel (Gelman Instru-ment Co.). This preparation is referred to as the con-centrated bacterial antigens. When this preparationwas ultrafiltered at 550 x g for 10 min through aCentriflo ultrafiltration membrane (Amicon Corp.)that eliminated substances of molecular weights<50,000, it is then referred to as the high-molecular-weight antigens.Ammonium sulfate-precipitated antigens, hereafter

referred to as the precipitated antigens, were preparedby treating 40 ml of heated S. typhimurium culturesupernatant with an equal amount of a saturatedsolution of ammonium sulfate. After overnight refrig-eration at 5°C, the precipitates were sedimented bycentrifugation at 2,000 x g for 20 min at 5°C. Thesediment was dissolved in 5 ml of phosphate-bufferedsaline (PBS; pH 7.3) and ultrafiltered at 550 x g for 10min through a Centriflo ultrafiltration membrane toremove the ammonium sulfate. The antigenic prepa-ration retained by the membrane was adjusted to atotal volume of 2 ml with PBS.

Alternatively, 20 ml of the heated culture superna-tant was treated with insoluble ribonuclease (RNase-agarose beads, Sigma Chemical Co.) prior to the pre-cipitation with ammonium sulfate. The insolubleRNase was washed twice in distilled water beforebeing added to the culture supernatant and agitatedcontinuously for 30 min at room temperature. Afterremoval of the insoluble RNase by centrifugation at550 x g for 10 min, the ammonium sulfate extractionprocedure was then followed as described above. Thispreparation is referred to as the RNase-treated anti-gens.

Tryptic soy broth was treated similarly as in thepreparation of concentrated bacterial antigens andused as a control. All final preparations were filteredthrough a membrane filter (0.22 Mm; Millipore Corp.)and checked for sterility before use.Treatment of bacterial antigens with proteo-

lytic enzymes. The insoluble trypsin (trypsin-poly-acrylamide, Sigma Chemical Co.) or insoluble protease(protease-carboxymethyl cellulose, Sigma ChemicalCo.) was suspended by adding 5 mg of each compoundto 0.5 ml of PBS, pH 7.3. The suspensions were leftat room temperature for 2 h with occasional shakingand then washed twice with PBS. Seven-tenths of 1ml of the precipitated antigenic preparation was mixedwith 0.35 ml of the soaked and washed insolubleenzyme preparations for 30 min, each in successionat room temperature. At each time, the insolubleenzyme was removed by centrifugation at 1,000 x g for10 min. The final preparation was passed through

membrane filters (0.22 ,um; Millipore Corp.).Guinea pigs. Male albino guinea pigs were pur-

chased from commercial sources and fed with guineapig pellets, supplemented with vitamins (Poly-Vi-Sol,Mead Johnson) in their drinking water. Each animalweighed between 500 and 800 g.

Infection of guinea pigs with S. typhimuriumGuinea pigs were injected intradermally in two siteswith 105 virulent S. typhimurium. After 2 to 4 weeks,they were given an intraperitoneal injection of 2 xI0W bacteria of the same strain. These animals werereferred to as immune guinea pigs and used 1 to 3weeks later as donors of peritoneal exudate cells orcirculating lymphocytes.

Procedures for the isolation and cultivation oflymphocytes. Lymphocytes were separated from theperipheral blood of guinea pigs. Blood was drawn bycardiac puncture with a 10-ml syringe containing hep-arin (The Upjohn Co.). A sterile Hanks solution con-taining 6% dextran (molecular weight, approximately184,000; Pharmchem Corp.) was added to the heparin-ized blood in a volume ratio of one part of dextran tothree parts of blood. This mixture was drawn up in asyringe that was held in an inverted position in a ringstand for approximately 20 min. After the sedimenta-tion of the erythrocytes, the upper layer of turbidplasma containing leukocytes was pushed out fromthe syringe through a bent, 18-gauge needle. Theleukocytes were sedimented by centrifugation at 150x g for 10 min. This cell pellet was resuspended in 0.4ml of the original supernatant fluid. Equal portionsof this suspension were then layered onto two tubes,each containing 2.5 ml of 20% sodium diatrizoate (OralHypaque Sodium Powder, Winthrop Laboratories)and centrifuged at 220 x g for 10 min. The buffy coatwas transferred into 5 ml of medium 199 (Microbiolog-ical Associates, Inc.) containing 2% homologous serum.The leukocytes were then collected by centrifugationat 320 x g for 10 min. The sedimented cells wereresuspended in culture medium, composed of 20%normal homologous serum, 70% medium 199, and 10%isotonic 1.4% sodium bicarbonate (CO2 saturated). Theleukocytic population in the suspension was quanti-tated in a hemocytometer. Peripheral blood so treatedyielded between 90 to 95% lymphocytes and approxi-mately 1% erythrocytes.The lymphocyte suspension was adjusted to 1.5 x

106 cells/ml and dispensed in 1-ml volumes into Vac-utainer tubes, to which 0.02 ml of the antigenic prep-aration or the control broth and 20 Mg of kanamycinper ml were added. The tubes were flushed with 5%CO2 in air before incubation in an Eberbach waterbath at 37°C with a horizontal shaking movement atapproximately 72 cycles/min. After 48 h of incubation,the lymphocytes were removed by centrifugation at1,100 x g for 15 min. The supernatant fluid was usedas the culture medium in the macrophage migrationtest or for the peritoneal macrophages.Technique for macrophage migration inhibi-

tion test. The procedure for macrophage migrationwas adapted from the in vitro test for delayed hyper-sensitivity described by Bloom and Glade (3). Perito-neal exudate cells, containing primarily monocytes,were harvested 3 to 5 days after an intraperitonealinjection of 10 ml of sterile mineral oil into guinea

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54 MAYO, HSU, AND LIM

pigs, as described in detail previously (13). Suspensionsof peritoneal macrophages were drawn up in heparin-ized, microhematocrit capillary tubes (Fisher Scien-tific Co.) that were then sealed at one end with Plas-ticine. The cells were packed into one end of thecapillary tubes by centrifugation at 150 x g for 5 min.The portion of the capillary tube containing the cellpellet was cut off at the cell-medium interface undera dissecting microscope and placed in Lexy culturechambers (Mini-Lab Co., Laval, Quebec, Canada). Thechambers consisted of round wells with a diameter of20 mm that held a volume of 0.5 ml of culture mediumcontaining 2% by volume of the test antigens or ofthe control broth when applicable. Two capillary tubesof packed macrophages (6 to 8 mm long) were an-

chored to the side of each well with silicone grease.The wells were covered on the top with a prefittedround cover slip and sealed with a paraffin-petrolatummixture. There were two openings at one side of thewells, through one of which the appropriate cell cul-ture medium containing the bacterial antigens andkanamycin or the supernatant fluid from the antigen-stimulated lymphocyte culture was injected with a

syringe and needle, while air was expelled throughthe other opening. The openings were then sealedwith paraffin-petrolatum. After 24 h of incubation at370C, the distance of macrophage migration was ob-served under a dissecting microscope with x30 mag-nification. The distance of migration was measuredfrom the center of the capillary tube to the outermargin of migrating cells with a calibrated ocularmicrometer. The percent inhibition of migration was

determined by use of the following formula: % inhibi-tion = 1 - (mean distance of migration in the presenceof antigen/mean distance of migration in the absenceof antigen) x 100.

Procedure for the determination of the fate ofS. typhimurium within macrophages cultured inmedium containing stimulated lymphocyte prod-ucts. Peripheral lymphocytes of normal and immuneguinea pigs were isolated and cultured in the presenceof the 10-3 dilution of the precipitated antigens for 2days after the procedure described above. The culturesupernatant fluid was recovered by removing cells anddebris at 1,100 x g for 15 min. Two volumes of freshlycollected peritoneal macrophages from one normalguinea pig containing approximately 4.5 x 107 cellseach were centrifuged at 150 x g for 10 min at 5°C.The cell sediment was resuspended in 7 ml of mediumcomposed of65% freshly prepared cell culture medium,35% antigen-stimulated lymphocyte culture superna-tant fluid prepared from either a normal or immuneguinea pig, and 20 ,ug of kanamycin per ml of medium.The cell suspension was placed in a 25-ml silicone-coated Erlenmeyer flask and incubated in an Eberbachshaking water bath at 370C for 16 to 18 h. The cellswere then recovered by centrifugation at 150 x g for10 min at 5°C and resuspended in Hanks solutioncontaining 6 U of heparin per ml and 2% homologousserum. The supernatant medium was centrifuged at1,400 x g for 15 min to remove culture debris and thenused again in the cultivation of infected macrophages.The macrophage population was determined witheosin staining in a hemocytometer. Only macrophagesuspensions containing over 90% unstained cells wereinfected with virulent S. typhimurium.

INFECT. IMMUN.

The procedures for the infection and cultivation ofmacrophages and for the determination of the fate ofintracellular salmonellae by quantitative recovery ofbacteria by sonic treatment were all described in detailpreviously (11). Briefly, after an overnight incubationin culture supernatant fluid derived from antigen-stim-ulated lymphocytes of either normal or immune guineapigs, 3 x 107 macrophages of a normal guinea pig wereresuspended in 4 ml of Hanks solution containing 20%normal homologous serum and mixed with S. typhi-murium in a test tube at a ratio of 10 bacteria/cell.After 15 min of rotation at 370C, the contents of eachtube of infected cells were transferred separately intotwo 40-ml silicone-coated centrifuge tubes, each con-taining 32 ml of chilled Hanks solution with 40,ujg ofkanamycin per ml. The cells were collected by centrif-ugation at 150 x g for 10 min at 50C and resuspendedin 4 ml of corresponding culture supernatant in whichthey were previously maintained overnight and towhich 0.4 ml of normal homologous serum was added.The kanamycin concentration in the culture mediumwas adjusted to approximately 40 ,tg/ml. The infectedcell cultures were left in 25-ml Erlenmeyer flasks inan Eberbach shaking water bath at 370C. Samples ofinfected cells were removed at 0-, 2-, 4-, and 6-hintervals. The ratio of viable bacteria per cell wasdetermined for each interval.

RESULTSEffect of bacterial antigens on the migra-

tion of normal and immune peritoneal ex-udate cells. Peritoneal exudate cells derivedfrom normal or immune guinea pigs were packedin capillary tubes, placed in duplicates in theLexy chamber, and cultured in medium contain-ing either the concentrated bacterial antigensor the high-molecular-weight antigens. In pre-liminary experiments, the original preparationsof the antigens and their 10-fold serial dilutionsin PBS were tested in the cell culture medium.A substantial amount of inhibition of macro-phage migration was seen in both the normaland immune cells in the presence of the undi-luted antigen preparations, as compared withthose in the medium containing the controlbroth. When 10-' and 10-2 dilutions were used,there was a noticeable difference in the migra-tion inhibition of the normal versus the immunecells, despite an appreciable migration inhibitionin the nornal cells. When the antigens werediluted to lo-, an apparent migration inhibitionwas seen in the immune cells, whereas the extentof migration in the normal cells did not appearto be significantly affected by the presence ofthe antigens.Table 1 shows the average results from three

experiments in which a 10-3 dilution of eitherthe concentrated bacterial antigens or the high-molecular-weight antigens was used. The aver-age percentage of migration inhibition in normalcells was 4 ± 3 (standard deviation [SD]) forthe concentrated bacterial antigens and 1 + 2

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MACROPHAGE AND SALMONELLA INTERACTIONS 55

TABLE 1. Migration inhibition in peritonealmacrophages derived from normal and immuneguinea pigs and cultured in medium containing

bacterial antigensMigration inhibition (%)a

Antigen Normal Immune

cells cells

Control 0 0Concentrated bacterialb 4 ± 3 28 ± 10High molecular weightb 1 ± 2 37 ± 3Precipitatedc 10 ± 4 48 ± 6RNase treatedc 13 ± 3 47 ± 4

a Data are expressed as mean ± SD.b A 10-' dilution of the antigenic preparation was

used.cA 10' dilution of the antigenic preparation was

used.

A B

_' C D _

FIG. 1. (A) Migration of peritoneal exudate cellsof a normal guinea pig cultured in the presence ofthe concentrated bacterial antigens; (B) migrationinhibition ofperitoneal exudate cells ofa salmonella-infected guinea pig cultured in the presence of theconcentrated bacterial antigens; (C) migration ofper-itoneal exudate cells of a normal guinea pig main-tained in the supernatant fluid ofa culture ofantigen-stimulated lymphocytes derived from a normalguinea pig; (D) migration inhibition of peritonealexudate cells of a normal guinea pig maintained inthe supernatant fluid of a culture of antigen-stimu-lated lymphocytes derivedfrom a salmonella-infectedguinea pig (original magnifications 8 5x).

for the high-molecular-weight antigens, whereasthe average migration inhibition in immune cellswas 28 + 10 and 37 3%, respectively. Photo-graphs of the comparative migration of normaland immune peritoneal macrophages from cap-illary tubes challenged with the concentratedbacterial antigens are shown in Fig. 1A and B.

In an initial attempt to characterize the chem-ical nature of the antigens that elicited the in-hibition of macrophage migration, the bacterialculture filtrate was treated with ammonium sul-fate. Also, a portion of the culture filtrate was

pretreated with RNase before the precipitationwith ammonium sulfate. Both preparations weretested in preliminary experiments, using 10-foldserial dilutions. In both cases, 102 and 10-dilutions failed to inhibit the migration of im-mune cells. Table 1 also shows the average re-

sults of three experiments in which a 10-1 dilu-tion of either the precipitated antigens or theRNase-treated antigens was used. The average

percentage of migration inhibition was 48 ± 6for immune cells as compared with 10 ± 4 fornormal cells when cultured in the presence ofthe precipitated antigens. When RNase-treatedantigens were used, the comparative data were

47 ± 4% for immune cells and 13 ± 3% fornormal cells.Effect of lymphocyte products on the mi-

gration of normal peritoneal exudate cells.

To detect the production of the migration inhib-itory factor(s) (MIF) from antigen-stimulatedimmune lymphocytes, lymphocytes were iso-lated from peripheral blood of normal and im-mune guinea pigs and cultured in the presence

of antigenic preparations or control broth for 48h. The supernatant fluid was used as the testmedium for the migration inhibition of perito-neal exudate cells harvested from normal ani-mals.Table 2 shows data from three experiments

in which the 10-3 dilution of the high-molecular-weight antigens was used in the lymphocytecultures. An average migration inhibition of 31± 16% was imposed on the peritoneal cells bythe culture fluid of antigen-stimulated immunelymphocytes as compared with an average of 85% by that of the normal lymphocytes.When serial dilutions of the precipitated an-

tigens were tested in lymphocyte cultures, the10-3 dilution was the optimum concentration

TABLE 2. Migration inhibition in peritonealmacrophages derived from normal guinea pigs andcultured in medium containing lymphocyte products

elicited by bacterial antigensMigration inhibition (%)(

Antigen Normal Immunelymphocyte lymphocytesupernatant supernatant

Control 0 0High molecular weightb 8 ± 5 31 + 16Precipitatedb 9 + 2 26 ± 5

a Data are expressed as mean ± SD.bA 10-3 dilution of the antigenic preparation was

used.

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56 MAYO, HSU, AND LIM

that elicited the production of specific MIF fromimmune lymphocytes. Table 2 also shows theresults of three experiments in which the super-natant from antigen-stimulated immune lym-phocytes inhibited the migration of normal mac-rophages by an average of 26 ± 5% as comparedwith that of the normal lymphocytes by anaverage of 9 ± 2%. Figures 1C and D are photo-graphs of the comparative migration of normalmacrophages from capillary tubes cultured inthe supematant fluid of normal and immunelymphocytes stimulated with the precipitatedantigens.

Effect of lymphocyte products on the an-tibacterial action of normal peritonealmacrophages. Peritoneal macrophages fromnormal guinea pigs were cultured in mediumcontaining antigen-stimulated lymphocyte prod-ucts overnight. They were removed from theirmedium, infected with virulent S. typhimurium,and then returned to their original culture su-pernatant fluid for maintenance according tothe procedure described above. The fate of sal-monellae within macrophages cultured in thelymphocyte supernatant fluid derived fromeither normal or immune animals was comparedover a 6-h period. Figure 2 shows the rate ofintracellular destruction of these organisms plot-ted from the average of three sets of experi-ments. The phagocytic indexes, as defined pre-viously (31), were 0.4 ± 0.19 (SD) for the infectedmacrophages exposed to the antigen-stimulatedlymphocyte products from normal animals and0.4 ± 0.17 for those exposed to antigen-stimu-

0

k-I-1

(.t)

14I

(.Z)

K1

0 2 4 6

INTERVAL IN HOURS

FIG. 2. Comparative rate of destruction of S. ty-phimurium within normal macrophages maintainedin the supernatant fluid of a culture of antigen-stim-ulated lymphocytes derived from a normal guineapig (N) or from a salmonella-infected guineapig (LS).

INFECT. IMMUN.

lated lymphocyte products from immune ani-mals. Under the condition of cell culture de-scribed here, exposure to products of antigen-stimulated immune lymphocytes did not en-hance the bactericidal capacity of the normalmacrophages against S. typhimurium, nor did italter the phagocytic efficiency of these macro-phages.Chemical nature of bacterial antigens.

Table 1 shows that the migration inhibitoryaction of the precipitated antigens against im-mune peritoneal cells was not diminished bypretreatment with RNase. The precipitated an-tigens were also digested with trypsin and pro-tease as described above and tested in cell cul-ture against peritoneal exudate cells of immuneguinea pigs. In two separate experiments, theaverage migration inhibition by the precipitatedantigens was 46% as compared with cells exposedto the control broth. The treatment with prote-olytic enzymes reduced the migration inhibitionof the immune peritoneal cells to an average of17%.

DISCUSSIONDelayed hypersensitivity to bacterial antigens

was shown to be a concomitant developmentalong with the emergence of acquired antibacte-rial immunity in murine salmonellosis (7). Pre-vious studies (12) in the nature of cutaneouslesions generated by S. typhimurium in guineapigs also suggested the involvement of delayedhypersensitivity in the development of second-ary lesions. After a preliminary report on thepresent study from this laboratory (D. R. Mayoand H. S. Hsu, Va. J. Sci., 25:105, 1974), usingthe macrophage migration inhibition test toshow delayed hypersensitivity in guinea pigsinfected with S. typhimurium, Cameron andvan Rensburg (4) related host resistance to sal-monellosis in guinea pigs by using a similartechnique, except that their protein antigenswere extracted from bacterial cells. The firstobjective of the present investigation was todemonstrate the presence of delayed hypersen-sitivity to salmonella antigens in salmonella-in-fected guinea pigs by use of the macrophagemigration inhibition test and to deternine thenature of the antigens responsible for this reac-tion.

Experimental data presented here showedthat antigens prepared from culture superna-tants of S. typhimurium could elicit the specificmigration inhibition of peritoneal cells derivedfrom previously infected guinea pigs. However,when peritoneal exudates from normal or sal-monella-infected guinea pigs were initiallytested with low dilutions of the concentratedbacterial antigens or of the high-molecular-

,-

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MACROPHAGE AND SALMONELLA INTERACTIONS

weight antigens, there was a consistentlygreater percentage of migration inhibition onthe immune cells than on the normal cells, al-though the migration of the normal cells wasalso significantly inhibited. It would thereforebe reasonable to presume that the migrationinhibition of the normal cells was a result oftheir reaction with a variety of nonspecific anti-gens in the preparations, such as the commonantigens among the gram-negative bacteria,rather than the result of the toxicity of theantigenic preparations. A subsequent dilution ofthe antigenic preparations to 10' removed theeffect of the nonspecific antigens and showed aspecific migration inhibition of the immune cells(Table 1).The elimination of molecules with molecular

weights of <50,000 from the bacterial culturefiltrate by ultrafiltration did not diminish themigration inhibition activity of the antigenicpreparations (Table 1). Protein was separatedfrom the bacterial culture filtrate by ammo-nium-sulfate precipitation and tested in the cul-ture medium for macrophage migration. A 10-fold dilution of the precipitated antigen prepa-ration was also shown to elicit specific inhibitionof cellular migration (Table 1). The bacterialculture filtrate was also treated with RNasebefore being subjected to the procedure of saltprecipitation. No change in the antigenic activityof the treated antigenic preparation was de-tected (Table 1). On the other hand, when thesame antigenic preparation was digested withtrypsin and protease, its ability to induce migra-tion inhibition of immune peritoneal cells wasessentially eliminated.To demonstrate the production of MIF by

lymphocytes derived from guinea pigs infectedwith the virulent S. typhimurium, normal andimmune lymphocytes were incubated with ap-propriate dilutions of the high-molecular-weightor the salt-precipitated antigens. The cell-freemedium from immune lymphocyte cultures wasshown to contain MIF, as evidenced by its abil-ity to inhibit the migration of peritoneal macro-phages of normal guinea pigs (Table 2).The in vitro observations presented here are

in agreement with those of Smith and Bigley(26), who reported that protein-rich fractions ofS. typhimurium were responsible for the elicita-tion of MIF production by immune lympho-cytes of mice in vitro. They also support thefindings of Collins and Mackaness (7), whodemonstrated a delayed hypersensitive reactionin footpads of salmonella-infected mice wheninjected with a protein-containing fraction ob-tained from cultures of several species of Sal-monella.The second objective of this investigation was

to determine whether specific antigen-stimu-lated lymphocytes could produce lymphokinesthat might alter the antibacterial activities ofmacrophages against salmonellae. When a cellculture procedure similar in principle to thoseused successfully by other investigators in thistype of experiments (8, 15, 18, 21, 25) was em-ployed, the result shows that the supernatantfluid of antigen-stimulated lymphocytes of im-mune guinea pigs, despite its ability to inhibitthe migration of macrophages, did not altertheir bactericidal activity against S. typhimu-rium (Fig. 2), nor did it enhance their phago-cytic capacity against these organisms as deter-mined by the phagocytic indexes.The failure to demonstrate enhanced antibac-

terial activities of macrophages against salmo-nellae under the influence of antigen-stimulatedlymphocyte products does not necessarily ne-gate the prevalent thesis regarding the role oflymphokines in the antibacterial action of mac-rophages (5, 8, 18, 25), nor does it dispute thefunctional capacity of lymphokines in acquiredimmunity against certain facultative intracellu-lar bacteria such as Mycobacterium tuberculo-sis (21) and Listeria monocytogenes (15).Rather, the innate ability of macrophages todestroy S. typhimurium may indicate a maxi-mum capacity for the bactericidal action of themacrophages against this pathogen and is there-fore no longer subject to further activation bylymphokines. This is in agreement with pre-vious observations (11, 16) that macrophagesderived from immunized animals were not en-dowed with an enhanced capacity to destroy S.typhimurium.

Unlike M. tuberculosis (9, 10), authenticatedexperimental evidence to establish that S. ty-phimurium multiples within macrophages ofthe host is still lacking. Other investigators havealso provided evidence of killing of S. typhimu-rium within mouse peritoneal macrophages (29)and by fractions of rabbit granulocytes (1). Ex-aminations of histopathological specimens (H.S. Hsu and I. Nakoneczna, Proc. Natl. Meet.Reticuloendothel. Soc., 12th, Abstr. no. 81,1975) revealed that the primary lesions in thereticuloendothelial system in murine salmonel-losis consisted essentially of polymorphonuclearleukocytes at the initial stage of the infection,whereas the peripheral infiltration of mononu-clear cells did not appear until 6 or 7 days afterthe infection. The latter coincided with theemergence of delayed hypersensitivity to bacte-rial antigens (7) and also with the terminal stageof a fatal primary infection. In contrast, thecharacteristics of a secondary lesion weremarked by an early appearance of mononuclearcells and the formation of a granuloma. In the

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58 MAYO, HSU, AND LIM

final analysis, it is questionable whether themacrophages are in fact the crucial site of theinitial bacterial proliferation in vivo. Hence,pathogenic salmonellae should not be classifiedas facultative intracellular parasites (6, 28).Rather, the antiphagocytic nature of the viru-lent S. typhimurium (31) would promote a rapidextracellular propagation of the pathogen in thetissue before the onset of acquired humoral im-munity by the active production of specific op-sonins (31) and cytophilic antibodies (11, 16).The beneficial role of delayed hypersensitivityis most likely manifested by an accelerated in-flux of the cellular and humoral elements intothe site of infection. The view of an extracellularpropagation rather than an intracellular inva-sion of salmonellae has largely been ignored bymany investigators as the basic mechanism ofpathogenesis in murine salmonellosis.There is a general tendency to equate delayed

hypersensitivity with acquired resistance in cer-tain infectious diseases and to refer to theirbasic mechanisms as cell mediated or cellularimmunity. It has been recognized, however, thatthese two phenomena are not synonymous, al-though they frequently emerge simultaneouslyafter an infection. The dissociation of delayedhypersensitivity and acquired immunity in tu-berculosis was discussed in detail recently byYoumans (32). In listeriosis, Osebold et al. (20)presented experimental evidence that separatedacquired immunity from delayed hypersensitiv-ity. Thus, there is now an increasing awarenessin the literature to define clearly cellular hyper-sensitivity as the basis for the delayed hypersen-sitivity to antigens and cellular immunity as thebasis for an enhanced antibacterial action ofimmune macrophages (18, 25). They are appar-ently two independent and distinguishableexpressions of the altered functions of macro-phages of the host in infectious diseases, al-though they may both be dependent on media-tors produced by antigen-stimulated lympho-cytes.

ACKNOWLEDGMENTSD.R.M. was supported by Public Health Service training

grant 2T01AI00382 from the National Institute of Allergy andInfectious Diseases.

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P. Elsbach. 1974. Early and discrete changes in perme-ability of Escherichia coli and certain other gram-neg-ative bacteria during killing by granulocytes. J. Exp.Med. 140:396-409.

2. Blanden, R. V. 1968. Modification of macrophage func-tion. RES J. Reticuloendothel. Soc. 5:179-200.

3. Bloom, B. R., and P. R. Glade. 1971. In vitro methodsin cell mediated immunity, p. 234-312. Academic PressInc., New York.

4. Cameron, C. M., and J. J. van Rensburg. 1975. Inhi-bition of macrophage migration in Salmonella immu-nity. Onderstepoort J. Vet. Res. 42:15-24.

5. Campbell, P. A. 1976. Immunocompetent cells in resist-ance to bacterial infections. Bacteriol. Rev. 40:284-313.

6. Collins, F. M. 1974. Vaccines and cell-mediated immu-nity. Bacteriol. Rev. 38:371-402.

7. Collins, F. M., and G. B. Mackaness. 1968. Delayedhypersensitivity and Arthus reactivity in relation tohost resistance in Salmonella-infected mice. J. Immu-nol. 101:830-845.

8. Fowles, R. E., I. M. Fajardo, J. L. Leibowitch, andJ. R. David. 1973. The enhancement of macrophagebacteriostasis by products of activated lymphocytes. J.Exp. Med. 138:952-964.

9. Hsu, H. S. 1965. In vitro studies on the interactionsbetween macrophages of rabbits and tubercle bacilli.II. Cellular and humoral aspects of acquired resistance.Am. Rev. Respir. Dis. 91:499-509.

10. Hsu, H. S. 1971. The fate of Mycobacterium tuberculosiswithin macrophages of guinea pigs. Am. Rev. Respir.Dis. 103:607-611.

11. Hsu, H. S., and D. R. Mayo. 1973. Interactions betweenmacrophages of guinea pigs and salmonellae. III. Bac-tericidal action and cytophilic antibodies of macro-phagesof infected guinea pigs. Infect. Immun. 8:165-172.

12. Hsu, H. S., and V. M. Piper. 1972. Acquired resistanceto and comparative virulence of Salmonella typhimu-rium demonstrated by cutaneous lesions in guinea pigs.RES J. Reticuloendothel. Soc. 11:343-357.

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