INFECTION AND IMMUNITY, July 1978, p. 10-160019-9567/78/0021-0010$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 21, No. 1

Printed in U.S.A.

Acquired Cellular Resistance, Delayed Hypersensitivity, andAltered Macrophage Migration in Listeria monocytogenes-

Infected Guinea PigsMEHER M. DUSTOOR AND ANDREW A. BLAZKOVEC*

Department ofMedical Microbiology, University of Wisconsin, Madison, Wisconsin 53706

Received for publication 16 January 1978

A Listeria monocytogenes infection in guinea pigs was used to study theinterrelationship between antigen-induced macrophage migration inhibition, de-layed-type hypersensitivity, and acquired cellular resistance. Early after infection(at 2 and 7 days), very significant enhancement of macrophage migration was

observed. Migration inhibition was detected beginning on day 14 and was uni-formly observed only on day 21 of the infection, after which a shift again toenhancement was seen. The early detection (by day 2) of migration enhancementsuggested that this assay may be more sensitive than assessment of delayed typehypersensitivity in vivo, which in this system was first detectable only on day 4.Acquired cellular resistance, as measured by enhanced survival following a highdose challenge with Listeria, was present from day 7 after infection until at leastday 60. By splenic clearance studies, however, acquired cellular resistance was

present only until day 14 after infection, suggesting that in this system splenicclearance was not a very reliable criterion for measuring acquired cellular resist-ance.

The relationship between acquired cellularresistance (ACR) and delayed-type hypersensi-tivity (DTH) remains an area of conflict (3-5,15-17). A Listeria monocytogenes infection inguinea pigs has been used in this laboratory tostudy the interrelationships between ACR andDTH (8, 11). The results obtained from in vivostudies showed that infected animals were re-sistant to a small challenge dose of Listeriagiven 2, 7, or 14 days after the primary infection.DTH, however, was not detectable until 5 or 6days postinfection, which suggested that, for thisspecies, ACR was not dependent on the devel-opment of DTH. In the two studies cited above,the only criterion of ACR employed was en-hanced splenic clearance and, furthermore, thechallenge dose used was low and hence no defi-nite conclusions could be reached. In the presentstudy, enhanced survival was used in addition tosplenic clearance as a criterion of ACR, and ahigh dose of Listeria was used for challenge.The macrophage migration inhibition assay is

considered an in vitro correlate of DTH (6, 10).It was, hence, of interest to study whether itcould be used as an indicator of DTH in thissystem.

(The research described in this paper wassubmitted in part to the Graduate School of theUniversity of Wisconsin by M.M.D. in partialfulfillment of the requirements for the Ph.D.degree in Medical Microbiology.)

MATERIALS AND METHODSAnimals. Randomly bred albino guinea pigs of

both sexes were obtained from local suppliers. Theanimals weighed 500 to 700 g at the time of infection.

Bacteria. The L. monocytogenes strain used andthe method of cultivation have been described else-where (11).Immunization and challenge. For immunization,

approximately one-half of a 50% lethal dose in a totalvolume of 2 ml was injected intracardially into eachguinea pig. Ten times the 50% lethal dose of Listeriawas used for challenge via the intracardial route. Theexact number of viable organisms injected was deter-mined by plate counts.Spleen assays. The number of viable Listeria in

brain heart infusion broth homogenates of spleen wasdetermined by the method of Mackaness (13). Appro-priate decimal dilutions were plated in duplicate onwell-dried brain heart infusion plates. Colonies werecounted after 24 to 48 h of incubation at 370C.

Antigen. A water-soluble extract of sonically dis-rupted Listeria was prepared by using a modificationof the method of Hinsdill and Berman (12) as de-scribed previously (11). Before use, the lyophilizedmaterial was reconstituted and sterilized by passagethrough a 0.45-gm membrane filter (Millipore Corp.,Bedford, Mass.).

Delayed hypersensitivity tests. Guinea pigs wereinjected intradermally on the shaved dorsal flank with30 jg of the water-soluble extract in 0.1 ml of saline.The skin tests were read at 2, 4, 8, 12, 24, and 48 h.Cutaneous reactivity was evaluated as previously de-scribed (11). Maximum skin reactivity was alwaysrecorded at 24 h after testing. Thus skin reactivity

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CELLULAR IMMUNITY TO L. MONOCYTOGENES

data presented in Table 2 are comprised of only the24-h diameter of erythema. Histological examinationof skin test sites at various time intervals after infec-tion revealed a classical tuberculin-type infiltrate,thereby confirming previously published findings ofthis laboratory (8).PE cells. Peritoneal exudate (PE) cells were in-

duced in guinea pigs by the intraperitoneal injectionof 25 to 30 ml of sterile no. 31 heavy paraffin oil(American Oil Co., Chicago, Ill.). Three days later, theexudates were harvested with 300 ml of Eagle minimalessential medium (Grand Island Biological Co., GrandIsland, N.Y.) containing 200 fig of penicillin per ml and40 ug of streptomycin per ml. The PE cells werewashed twice, and equal volumes were distributed intoseparate 15-ml centrifuge tubes. These were centri-fuged and a 7.5% suspension of the pelleted cells wasmade in Eagle minimal essential medium containing15% normal guinea pig serum and the appropriateconcentration of Listeria antigen. For some experi-ments, purified protein derivative (Parke-Davis andCo., Detroit, Mich.) served as a specificity control.Migration inhibition assay. The 7.5% cell suspen-

sions were drawn up to 75 mm in 100-mm capillarytubes (Kimax no. 34502, 0.8 to 1.1 by 100 mm; KimbleProducts, Toledo, Ohio), which were sealed at one endwith a mixture of paraffin wax and petroleum jelly. Sixtubes were filled with each cell suspension. They werecentrifuged at 35 x g for 4 min and cut at the cell-fluidinterface. The tubes were placed in Sykes-Moorechambers (Bellco Glass, Inc., Vineland, N.J.), whichwere then filled with medium containing the sameconcentration of antigen as was used to suspend thecells and incubated for 24 h at 370C in an atmosphereof 5% CO2. The areas of cell migration were magnifiedwith a Microprojector (Bausch and Lomb, Rochester,N.Y.), traced, cut out, and weighed. For each animal,the mean areas of migration from capillary tubes withantigen and without antigen were then used in thefollowing formula: percentage of migration = (meanarea of migration in presence of antigen/mean area ofmigration without antigen) x 100.

RESULTSACR in guinea pigs infected with ListerwL

(i) Splenic clearance. Guinea pigs infectedwith a sublethal dose of Listeria, together withuninfected controls, were challenged at 2, 7, 14,21, and 28 days with an intracardial injection oflOx the 50% lethal dose of Listeria. Groups ofanimals were sacrificed on days 0, 1, 2, and 3following challenge.

Preliminary studies utilizing a streptomycin-resistant mutant of Listeria as the challengeorganism have been performed. There was, how-ever, a relative lack of multiplication of themutant organisms in outbred guinea pigs. Theuse of mutants in further studies was, therefore,discontinued.At the 2-day challenge interval (Fig. 1), al-

though there was no multiplication of the chal-lenge organisms in the infected animals withinthe first 24 h, by 48 and 72 h there was no

difference in the numbers of Listeria recoveredfrom infected and control guinea pigs. Thus nodefinite conclusion could be reached regardingthe presence of ACR in the animals at 2 days,since the fate of the challenge organisms alonewas not followed.At 7 days after infection, splenic clearance by

the infected animals was markedly enhancedover that by controls. On day 14, it appeared tobe on the wane, and by 21 and 28 days theclearance by infected animals almost paralleledthat by the controls.

(ii) Survival. Outbred guinea pigs were in-fected with one-half of a 50% lethal dose ofListeria. On days 2, 4, 7, 14, 21, 28, and 60, theseanimals, together with a group of uninfectedcontrols, were challenged with an intracardialinjection of approximately 1Ox the 50% lethaldose of Listeria. Daily deaths were recorded,and these data are given in Table 1. There wasno significant difference between the number ofdeaths recorded for the controls and the 2-daychallenge group. After 7 days, the mortality inthe infected groups was significantly reduced.From these data, ACR did not appear to bepresent at 2 days, but was present from day 7through at least day 60.Macrophage migration inhibition in

guinea pigs infected with Listeria. Outbredguinea pigs were infected with one-half of a 50%lethal dose of Listeria, and 2, 7, 14, 21, and 28days later PE cells from these animals were usedin the migration inhibition assay. A controlgroup of normal animals was also used. Animalsused in these studies were skin tested 24 h earlierwith Listeria antigen.Migration inhibition of normal PE cells.

Figure 2 shows the percentage of migration ofnormal cells in the presence of various concen-trations of Listeria antigen. A concentration of1,000 Itg/ml was clearly toxic, inhibiting migra-tion by 40%. The other four concentrations werenontoxic. In all subsequent studies, concentra-tions ranging from 0.1 to 100 tg/ml were utilized.PE cells obtained from six normal guinea pigs

and from six animals at 21 days after infectionwith Listeria were incubated with 10 and 1 ,g ofpurified protein derivative per ml and served asspecificity controls. With 10 jig of purified pro-tein derivative, the mean migration of cells frominfected and normal animals was 91.13 ± 5.66and 104.71 + 7.41%, respectively; with 1 ug ofpurified protein derivative, migration values of108.38 + 10.45 and 92.58 + 8.04%, respectively,were obtained. These findings in relationship tospecific Listeria antigen-induced alterations ofimmune PE cell migration established the spec-ificity of the latter.Alteration of macrophage migration

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12 DUSTOOR AND BLAZKOVEC

CHALLENGE AT

2 30 1 2 30 1DAYS AFTER CHALLENGE

21 DAYS 28 DAYS

FIG. 1. Clearance ofa challenge dose ofListeria 2, 7, 14, 21, and 28 days after a primary Listeria infection.Normal (0) and Listeria-infected (0) outbred guinea pigs were challenged intracardially with lOx the 50%lethal dose of Listeria. Five animals from each group were sacrificed at 0, 1, 2, and 3 days after challenge,and the numbers ofListeria present in the spleen were determined. Bars indicate standard error.

TABLE 1. Resistance ofguinea pigs previouslyinfected with one-half ofa 50% lethal dose of

Listeria, upon challenge with lOx the 50% lethaldose of Listeria

Time of No. dead/challenge Mortality record' no. chal- P value"(days) lenged

Controls 1(7), 2(9), 1(10), 2(12), 10/17C1(13), 1(14), 1(15),1(21)

2 1(7), 1(15), 1(17), 1(19), 6/13 0.27451(22), 1(23)

7 1(8), 1(9), 1(30) 3/14 0.0402

14 0/11 0.00001

21 0/11 0.00001

28 1(13) 1/8 0.0378

60 1(23) 1/11 0.0104a Number of animals (day of death).b Comparison of experimental groups with the con-

trol group made using the Fisher Exact Test.c Three more animals appeared sick, but later re-

covered.

after infection. The mean percentage of migra-tion of PE cells harvested at various time inter-vals after infection is shown in Fig. 3. Each barrepresents the group mean ± standard errorbased on the minimum and maximum percent-age of migration for each animal. At 2 days,significant enhancement of migration (154.66 +1.01%) over control values was obtained. On day7, a maximum percentage of migration of 183.44± 17.07% was seen. At 14 days, two animalsdisplayed significant enhancement and threeothers showed significant inhibition, but the val-

160.0!-

120.0 F

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I 60.0

z 60.0'a'"i

40.01F

20.0 F

1000 100 10 1.0 0.1

LISTERIA ANTIGEN (pg)FIG. 2. Percentage ofmigration ofnormalPE cells

in the presence of Listeria antigen. PE cells were

harvested from normal outbred guinea pigs. Eachpoint represents the mean percentage of migrationobtained with 10 animals. Bars indicate standarderror.

ues for the group as a whole did not differsignificantly from the controls. At 21 days, mi-gration inhibition was routinely observed withall the animals. None of the animals showed anysignificant enhancement. By 28 days, however,only one animal displayed inhibition, and en-

hancement was again observed with three ani-mals.

Preliminary experiments had indicated that

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CELLULAR IMMUNITY TO L. MONOCYTOGENES

200.0

160.0 +

160.0

140.0-

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60.0 +

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20.0

0

Oi

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UNINFECTED 2 7 14 21CONTROLS

DAYS AFTER LISTERIA INFECTION

FIG. 3. Alteration ofPE cell migration in thepres-ence of Listeria antigen after infection with Listeria.PE cells were harvested from outbred guinea pigs atvarious time intervals after a primary Listeria infec-tion. Each bar represents the group mean based onthe lowest (0) and the highest (a) percentage ofmigration for each animal obtained in dose-responseexperiments (0.1 to 1(X)pg). Groups consisted of six toseven animals. Stand&d error is indicated on eachbar. The mean migration inhibition or enhancementobtained with each experimental group was com-pared to that of the control group by means of Stu-dent's t test. The level of significance is indicatedabove each bar (NS, not significant).

no single antigen concentration was uniformlyinhibitory or enhancing towards cells from in-fected donors. It was therefore necessary to em-ploy a range of antigen concentrations.DTH and macrophage migration pat-

terns. The onset and persistence ofDTH follow-ing infection with Listeria has been reportedpreviously by this laboratory (8, 11). Resultsobtained throughout the present study paral-leled the previously reported data and for thisreason were not detailed in the present report.Table 2 shows the DTH skin test response andthe percentages of migration for individualguinea pigs at various time intervals after infec-tion. It can be seen that after 7 days all theanimals were uniformly skin test positive. Onday 7, when the skin reactions were at theirpeak, no migration inhibition was seen and, infact, enhancement was routinely observed. In

general, all animals showing significant migra-tion inhibition were also skin test positive exceptin the case of animal no. 1 on day 2, whichshowed migration inhibition in the absence of apositive skin test. On day 2, in four of the fiveanimals, significant migration enhancement wasobserved in the absence of a positive skin test.No correlation existed between the intensity ofskin reactivity and the level of macrophage mi-gration inhibition or enhancement. Similar ob-servations have been reported for the BCG-rab-bit system by others (9).

DISCUSSIONThe present experiments, as well as a study of

lymphocyte blastogenesis by Fulton et al. (8),clearly establish that a state of specific cell-me-diated immunity develops in guinea pigs follow-ing infection with L. monocytogenes.The results of the splenic clearance of 1Ox the

50% lethal dose of Listeria used as a challengedose at various time intervals after an immuniz-ing dose were essentially similar to those re-ported by Halliburton and Blazkovec (11). It ispossible that the inactivation of the primaryinfection, which was seen to occur at about day2 (11), may not have been immunological butcould have been due either to an influx ofinflam-matory cells or to direct activation of residentmacrophages by listerial products. Hence, thedecline in numbers of the 2-day challenge inoc-ulum seen over the first 24 h may have been dueto such nonspecific phenomena. The fact that,after 48 h, clearance was not enhanced in the 2-day challenge animals when compared with con-trol values was in marked contrast to the clear-ance patterns of animals challenged at 7 and 14days. Petit and Unanue (17) have shown thatproducts released by Listeria are B-cell mito-gens and also enhance resistance to virulentListeria in mice. Listeria cell walls have alsobeen shown to possess similar mitogenic andadjuvant properties (1, 2). An early nonspecificphase of resistance may, therefore, be operativeprior to the onset of specific resistance. Passivetransfer studies would provide more clear-cutanswers to this question.The clearance studies at the other time pe-

riods suggested that at 7 and 14 days after infec-tion sufficient numbers of sensitized lympho-cytes were present for a state of immunity to bemanifested. At 14 days, however, these may havebeen on the decline, necessitating the 24-h lagperiod during which time there was some mul-tiplication of the Listeria. In addition, it alsoappeared that there was no generation or entryof memory cells in the spleen on day 21 andafter.

It should be noted that injection of 1Ox the

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14 DUSTOOR AND BLAZKOVEC

TABLE 2. Comparison of Listeria antigen-specific macrophage migration and delayed hypersensitivity ofguinea pigs after infection with L. monocytogenesMigration inhibition Migration enhancement 24-h skin

Time reactinafter in- Guinea Optimal Optimal a

P reactivityfection pig no. antigen PercentMc nSignificance'timl an Percent mi- (diam oferS-(days) concna gration' Signficancec tigen gration them in(2101) 605g/ml) mm)

2 1 0.1 65.69 0.01 100 154.65 0.001 Neg2345

1.0 92.57NoneNone

0.1 80.71

7 67891011

NoneNoneNoneNoneNoneNone

NS 100 152.94100 155.0010 152.50

NS 100 158.22

100100

1.0100100100

143.64169.23147.22189.66257.70193.17

0.0010.0010.0010.01

0.0010.0010.0010.0010.0010.001

NegNeg4Neg

17.515.52015.51415.5

14 12 0.113 0.114 1.015 0.116 1.017 100

21 18 0.119 1.020 1021 10022 1023 1024 0.1

28 25262728293031

83.8661.2888.2260.5160.7798.09

79.0869.7447.0924.5360.5076.2470.16

0.1 65.84None

10 88.080.1 99.210.1 95.30

None1.0 78.03

NS0.01NS0.0010.01NS

10 120.54None

100 110.10100 168.20100 144.6210 126.28

0.010.010.010.050.05NS0.05

0.001100

NS 10NS 1.0NS 10

10NS 100

NS

NS0.0010.010.001

NoneNoneNoneNoneNoneNoneNone

None202.39127.99115.55122.80157.16161.55

0.0010.010.02NS

0.010.01

10.512.515131317

101419161314.57

13681110.51516.5

a Concentration at which maximum inhibition/enhancement, if any, occurred (range, 0.1 to 100 gg/ml).b The value given represents the lowest/highest percentage of migration obtained with cells from a particular

animal. "None" signifies that at no antigen concentration was the percentage of migration below or above 100%.'Antigen-stimulated cultures compared with unstimulated control cultures by two-tailed Student's t test.

NS, Not significant.

50% lethal dose of Listeria into control animalsdid not lead either to progressive growth oforganisms in the spleen or to enhanced multi-plication. Over the first 72 h at least, the growthpattern appeared to be independent of the doseused. It thus appeared that, for the guinea pigmodel, splenic counts did not constitute a reflec-tion of the degree of resistance.The results of survival experiments also sup-

ported this concept. Using the survival criterion,resistance was present when measured on day 7and persisted until at least day 60, at which timethe last assessment was made. Because injection

of Listeria results in an acute short-term infec-tion in guinea pigs, it is very likely that the highlevel of resistance to reinfection seen on days 21,28, and 60 was due to the persistence of memorycells which, presumably, rapidly generated astate of immunity. This, however, was not re-flected in the 21- and 28-day splenic clearanceresults. The length of time that these memorycells persisted is not known, since survival stud-ies were not extended beyond 60 days. In allstudies carried out in this laboratory to date,titrations of sera by the passive hemagglutina-tion method have shown that little or no anti-

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CELLULAR IMMUNITY TO L. MONOCYTOGENES

body can be demonstrated after either a primaryor a secondary infection of guinea pigs with L.monocytogenes (8). It is, therefore, unlikely thatantibodies play a significant role in terms ofprotection against Listeria in this species.

In this system, peripheral DTH has beenshown to be present from day 4 onwards, peak-ing at 7 days and persisting for almost 1 year(11). The results of the present study are ingeneral agreement with these findings, althoughskin testing was not carried out beyond 84 days.These results, in conjunction with those ob-tained from the survival studies, suggest thatDTH precedes the expression of ACR.Lymphocyte-derived mediators such as mi-

gration inhibition factor have been implicated inthe expression of cellular resistance to infection.Although cell types other than T cells have beenreported to produce migration inhibition factor(18, 22), the use in these studies of PE cellpopulations, which represent a rich source of Tcells (19), has made it possible to assess theinhibition factor-producing capacity of T cellsprimarily.The results in Table 2 and Fig. 3 make it

evident that the macrophages were being sub-jected to opposing influences. Many reports havedocumented the enhancement of macrophagemigration and the secretion of a distinct migra-tion enhancement factor by antigen-stimulatedlymphocyte cultures (7, 20, 21, 23). In a fewinstances, the existence of migration enhance-ment factor was demonstrable only after migra-tion inhibition factor activity was first removed.It is possible that the enhancement observed inthe present study was also due to the presenceof such a factor. This phenomenon is immuno-logical, since enhancement was observed onlywhen using cells from hypersensitive donors.

It is not known whether the same or differentcell types secrete migration inhibition and en-hancement factors. In the present study, thecells harvested from 2 animals at 14 days afterinfection were shown to be both enhanced andinhibited, depending on the antigen concentra-tion employed, suggesting that both activitiescan be released in measurable quantities by agiven cell population. Early after sensitization,enhancing activity was predominant, with highlevels of enhancement demonstrable on days 2and 7. It was not clear what the significance ofthe shift to migration inhibition and the returnto migration enhancement at 28 days was. Thepresence of inhibition factor demonstrable at 21days constituted a real phenomenon, since allseven of the animals assayed were positive andenhancement of cell migration was never ob-served at any of the antigen concentrations em-ployed.

Results reported by Fulton et al. (8) derivedfrom the use of PE cells for the study of lym-phocyte blastogenesis during the course of aListeria infection in guinea pigs revealed thatsignificant blastogenesis was first measurable onday 7 in about 40% of the animals. Skin testsbecame positive on day 4. The results of thepresent study showed that enhancement ofmac-rophage migration was evident as early as day 2,suggesting that this assay may be more sensitivethan assessment of levels of DTH in vivo. Allanimals that were DTH positive also tested pos-itive for migration inhibition or enhancement,except for animals no. 12 and 14.The studies reported here strongly suggest

that for guinea pig listeriosis, a positive correla-tion exists between ACR, DTH, and migrationinhibition/enhancement. Passive transfer stud-ies, together with dissection of the in vitro re-sponse by assaying purified lymphocyte super-natant fractions, are warranted to clarify theseinterrelationships further.

ACKNOWLEDGMENTSThis investigation was supported by grant no. 5-R22AI-

08608 from the United States-Japan Cooperative MedicalScience Program, administered by the U.S. Department ofHealth, Education and Welfare; by funds from AmericanCancer Society Institution Grant no. IN-35022; by funds fromPublic Health Service General Research Support Grant no.G-474-17 to the University of Wisconsin Medical School fromthe Division of Research Facilities and Resources, NationalInstitutes of Health; by funds from the University ofWisconsinGraduate School Research Committee; and by funds fromPublic Health Service training grant no. AI-00451 from theNational Institute of Allergy and Infectious Diseases.

LITERATURE CITED1. Campbell, P. A., C. Schuffler, and G. E. Rodriguez.

1976. Listeria cell wall fraction: a B cell adjuvant. J.Immunol. 116:590-594.

2. Cohen, J. J., G. E. Rodriguez, P. D. Kind, and P. A.Campbell. 1975. Listeria cell wall fraction: a B cellmitogen. J. Immunol. 114:1132-1134.

3. Collins, F. M. 1971. Mechanisms in antimicrobial immu-nity. RES J. Reticuloendothel. Soc. 10:58-99.

4. Collins, F. M., and G. B. Mackaness. 1970. The rela-tionship of delayed hypersensitivity to acquired anti-tuberculous immunity. I. Tuberculin sensitivity andresistance to reinfection in BCG vaccinated mice. Cell.Immunol. 1:253-265.

5. Collins, F. M., and G. B. Mackaness. 1970. The rela-tionship of delayed hypersensitivity to acquired anti-tuberculous immunity. II. Effect of adjuvant on theallergenicity and immunogenicity ofheat-killed tuberclebacilli. Cell. Immunol. 1:266-275.

6. David, J. R. 1975. Macrophage activation by lymphocytemediators. Fed. Proc. 34:1730-1736.

7. Fox, R. A., D. S. Gregory, and J. D. Feldman. 1974.Migration inhibition factor (MIF) and migration stim-ulation factor (MSF) in fetal calf serum. J. Immunol.112:1861-1866.

8. Fulton, A. M., M. M. Dustoor, J. E. Kasinski, and A.A. Blazkovec. 1975. Blastogenesis as an in vitro cor-relate ofdelayed hypersensitivity in guinea pigs infectedwith Listeria monocytogenes. Infect. Immun. 12:647-655.

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9. Galindo, B., and Q. N. Myrvik. 1970. Migratory re-sponse of granulomatous alveolar cells from BCG-sen-sitized rabbits. Infect. Immun. 1:227-237.

10. George, M., and J. H. Vaughan. 1962. In vitro cellmigration as a model for delayed hypersensitivity. Proc.Soc. Exp. Biol. Med. 111:514-521.

11. Halliburton, B. L, and A. A. Blazkovec. 1975. Delayedhypersensitivity and acquired cellular resistance inguinea pigs infected with Listeria monocytogenes. In-fect. Immun. 11:1-7.

12. Hinsdill, R. D., and D. T. Berman. 1967. Antigens ofBrucella abortus. I. Chemical and immunoelectropho-retic characterization. J. Bacteriol. 93:544-549.

13. Mackaness, G. B. 1962. Cellular resistance to infection.J. Exp. Med. 116:381-406.

14. Mackaness, G. B. 1968. The immunology of anti-tuber-culous immunity. Am. Rev. Respir. Dis. 97:337-344.

15. Neiburger, R. G., G. P. Youmans, and A. S. Youmans.1973. Relationship between tuberculin hypersensitivityand cellular immunity to infection in mice vaccinatedwith viable attenuated mycobacterial cells or with my-cobacterial ribonucleic acid preparations. Infect. Im-mun. 8:4247.

16. Osebold, J. W., L. D. Pearson, and N. L. Medin. 1974.Relationship of antimicrobial cellular immunity to de-layed hypersensitivity in listeriosis. Infect. Immun.9:354-362.

17. Petit, J. C., and E. R. Unanue. 1974. Effects of bacterial

products on lymphocytes and macrophages: their pos-sible role in natural resistance to Listeria infection inmice. J. Immunol. 113:984-992.

18. Rocklin, R. E., R. P. MacDermott, L Chess, S. F.Schlosaman, and J. R. David. 1974. Studies on me-diator production by highly purified human T and Blymphocytes. J. Exp. Med. 140:1303-1316.

19. Rosenstreich, D. L, J. T. Blake, and A. S. Rosenthal.1971. The peritoneal exudate lymphocyte. I. Differencesin antigen responsiveness between peritoneal exudateand lymph node lymphocytes from immunized guineapigs. J. Exp. Med. 134:1170-1186.

20. Sandok, P. L., R. D. Hinsdill, and R. M. Albrecht.1975. Alterations in mouse macrophage migration: afunction of assay systems, lymphocyte activation prod-uct preparation and fractionation. Infect. Immun.11:1100-1109.

21. Soborg, M. 1968. In vitro migration of peripheral humanleucocytes in cellular hypersensitivity. Acta Med.Scand. 186:135-139.

22. Tubergen, D. G., J. D. Feldman, E. M. Pollock, andR. A. Lerner. 1972. Production of macrophage migra-tion inhibition factor by continuous cell lines. J. Exp.Med. 135:255-266.

23. Weisbart, R. H., R. Bluestone, L. S. Goldberg, and C.M. Pearson. 1974. Migration enhancement factor: anew lymphokine. Proc. Natl. Acad. Sci. U.S.A. 71:875-879.

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