Vol. 21, No. 1
INFECTION AND IMMUNITY, July 1978, p. 10-16
0019-9567/78/0021-0010$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Acquired Cellular Resistance, Delayed Hypersensitivity, and Altered Macrophage Migration in Listeria monocytogenesInfected Guinea Pigs MEHER M. DUSTOOR AND ANDREW A. BLAZKOVEC* Department of Medical Microbiology, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 16 January 1978
A Listeria monocytogenes infection in guinea pigs was used to study the interrelationship between antigen-induced macrophage migration inhibition, delayed-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 uniformly observed only on day 21 of the infection, after which a shift again to enhancement was seen. The early detection (by day 2) of migration enhancement suggested that this assay may be more sensitive than assessment of delayed type hypersensitivity in vivo, which in this system was first detectable only on day 4. Acquired cellular resistance, as measured by enhanced survival following a high dose challenge with Listeria, was present from day 7 after infection until at least day 60. By splenic clearance studies, however, acquired cellular resistance was present only until day 14 after infection, suggesting that in this system splenic clearance was not a very reliable criterion for measuring acquired cellular resistance.
The relationship between acquired cellular resistance (ACR) and delayed-type hypersensitivity (DTH) remains an area of conflict (3-5, 15-17). A Listeria monocytogenes infection in guinea pigs has been used in this laboratory to study the interrelationships between ACR and DTH (8, 11). The results obtained from in vivo studies showed that infected animals were resistant to a small challenge dose of Listeria given 2, 7, or 14 days after the primary infection. DTH, however, was not detectable until 5 or 6 days postinfection, which suggested that, for this species, ACR was not dependent on the development of DTH. In the two studies cited above, the only criterion of ACR employed was enhanced splenic clearance and, furthermore, the challenge dose used was low and hence no definite conclusions could be reached. In the present study, enhanced survival was used in addition to splenic clearance as a criterion of ACR, and a high 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 it could be used as an indicator of DTH in this system. (The research described in this paper was submitted in part to the Graduate School of the University of Wisconsin by M.M.D. in partial fulfillment of the requirements for the Ph.D. degree in Medical Microbiology.)
MATERIALS AND METHODS Animals. Randomly bred albino guinea pigs of both sexes were obtained from local suppliers. The animals weighed 500 to 700 g at the time of infection. Bacteria. The L. monocytogenes strain used and the method of cultivation have been described elsewhere (11). Immunization and challenge. For immunization, approximately one-half of a 50% lethal dose in a total volume of 2 ml was injected intracardially into each guinea pig. Ten times the 50% lethal dose of Listeria was used for challenge via the intracardial route. The exact number of viable organisms injected was determined by plate counts. Spleen assays. The number of viable Listeria in brain heart infusion broth homogenates of spleen was determined by the method of Mackaness (13). Appropriate decimal dilutions were plated in duplicate on well-dried brain heart infusion plates. Colonies were counted after 24 to 48 h of incubation at 370C. Antigen. A water-soluble extract of sonically disrupted Listeria was prepared by using a modification of the method of Hinsdill and Berman (12) as described previously (11). Before use, the lyophilized material was reconstituted and sterilized by passage through a 0.45-gm membrane filter (Millipore Corp., Bedford, Mass.). Delayed hypersensitivity tests. Guinea pigs were injected intradermally on the shaved dorsal flank with 30 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 described (11). Maximum skin reactivity was always recorded at 24 h after testing. Thus skin reactivity 10
VOL. 21, 1978
CELLULAR IMMUNITY TO L. MONOCYTOGENES
data presented in Table 2 are comprised of only the 24-h diameter of erythema. Histological examination of skin test sites at various time intervals after infection revealed a classical tuberculin-type infiltrate, thereby confirming previously published findings of this laboratory (8). PE cells. Peritoneal exudate (PE) cells were induced in guinea pigs by the intraperitoneal injection of 25 to 30 ml of sterile no. 31 heavy paraffin oil (American Oil Co., Chicago, Ill.). Three days later, the exudates were harvested with 300 ml of Eagle minimal essential medium (Grand Island Biological Co., Grand Island, N.Y.) containing 200 fig of penicillin per ml and 40 ug of streptomycin per ml. The PE cells were washed twice, and equal volumes were distributed into separate 15-ml centrifuge tubes. These were centrifuged and a 7.5% suspension of the pelleted cells was made in Eagle minimal essential medium containing 15% normal guinea pig serum and the appropriate concentration of Listeria antigen. For some experiments, purified protein derivative (Parke-Davis and Co., Detroit, Mich.) served as a specificity control. Migration inhibition assay. The 7.5% cell suspensions were drawn up to 75 mm in 100-mm capillary tubes (Kimax no. 34502, 0.8 to 1.1 by 100 mm; Kimble Products, Toledo, Ohio), which were sealed at one end with a mixture of paraffin wax and petroleum jelly. Six tubes were filled with each cell suspension. They were centrifuged at 35 x g for 4 min and cut at the cell-fluid interface. The tubes were placed in Sykes-Moore chambers (Bellco Glass, Inc., Vineland, N.J.), which were then filled with medium containing the same concentration of antigen as was used to suspend the cells and incubated for 24 h at 370C in an atmosphere of 5% CO2. The areas of cell migration were magnified with a Microprojector (Bausch and Lomb, Rochester, N.Y.), traced, cut out, and weighed. For each animal, the mean areas of migration from capillary tubes with antigen and without antigen were then used in the following formula: percentage of migration = (mean area of migration in presence of antigen/mean area of migration without antigen) x 100.
11
difference in the numbers of Listeria recovered from infected and control guinea pigs. Thus no definite conclusion could be reached regarding the presence of ACR in the animals at 2 days, since the fate of the challenge organisms alone was not followed. At 7 days after infection, splenic clearance by the infected animals was markedly enhanced over that by controls. On day 14, it appeared to be on the wane, and by 21 and 28 days the clearance by infected animals almost paralleled that by the controls. (ii) Survival. Outbred guinea pigs were infected with one-half of a 50% lethal dose of Listeria. On days 2, 4, 7, 14, 21, 28, and 60, these animals, together with a group of uninfected controls, were challenged with an intracardial injection of approximately 1Ox the 50% lethal dose of Listeria. Daily deaths were recorded, and these data are given in Table 1. There was no significant difference between the number of deaths recorded for the controls and the 2-day challenge group. After 7 days, the mortality in the infected groups was significantly reduced. From these data, ACR did not appear to be present at 2 days, but was present from day 7 through at least day 60. Macrophage migration inhibition in guinea pigs infected with Listeria. Outbred guinea pigs were infected with one-half of a 50% lethal dose of Listeria, and 2, 7, 14, 21, and 28 days later PE cells from these animals were used in the migration inhibition assay. A control group of normal animals was also used. Animals used in these studies were skin tested 24 h earlier with Listeria antigen. Migration inhibition of normal PE cells. Figure 2 shows the percentage of migration of normal cells in the presence of various concenRESULTS trations of Listeria antigen. A concentration of ACR in guinea pigs infected with ListerwL 1,000 Itg/ml was clearly toxic, inhibiting migra(i) Splenic clearance. Guinea pigs infected tion by 40%. The other four concentrations were with a sublethal dose of Listeria, together with nontoxic. In all subsequent studies, concentrauninfected controls, were challenged at 2, 7, 14, tions ranging from 0.1 to 100 tg/ml were utilized. PE cells obtained from six normal guinea pigs 21, and 28 days with an intracardial injection of lOx the 50% lethal dose of Listeria. Groups of and from six animals at 21 days after infection animals were sacrificed on days 0, 1, 2, and 3 with Listeria were incubated with 10 and 1 ,g of purified protein derivative per ml and served as following challenge. Preliminary studies utilizing a streptomycin- specificity controls. With 10 jig of purified proresistant mutant of Listeria as the challenge tein derivative, the mean migration of cells from organism have been performed. There was, how- infected and normal animals was 91.13 ± 5.66 ever, a relative lack of multiplication of the and 104.71 + 7.41%, respectively; with 1 ug of mutant organisms in outbred guinea pigs. The purified protein derivative, migration values of use of mutants in further studies was, therefore, 108.38 + 10.45 and 92.58 + 8.04%, respectively, were obtained. These findings in relationship to discontinued. At the 2-day challenge interval (Fig. 1), al- specific Listeria antigen-induced alterations of though there was no multiplication of the chal- immune PE cell migration established the speclenge organisms in the infected animals within ificity of the latter. the first 24 h, by 48 and 72 h there was no Alteration of macrophage migration
12
INFECT. IMMUN.
DUSTOOR AND BLAZKOVEC
CHALLENGE AT 28 DAYS
21 DAYS
usaa. G"
co
m
".
EL
m U
e
2
30
1
30
2
1
DAYS AFTER CHALLENGE FIG. 1. Clearance of a 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 of Listeria present in the spleen were determined. Bars indicate standard error. TABLE 1. Resistance ofguinea pigs previously infected with one-half of a 50% lethal dose of Listeria, upon challenge with lOx the 50% lethal dose of Listeria No. dead/ Time of no. chal- P value" Mortality record' challenge lenged (days) Controls 1(7), 2(9), 1(10), 2(12), 10/17C 1(13), 1(14), 1(15), 1(21)
160.0!120.0 F QC
2
1(7), 1(15), 1(17), 1(19), 1(22), 1(23)
6/13
0.2745
I 60.0 1-
7
1(8), 1(9), 1(30)
3/14
0.0402
z 60.0 I.
14
0/11
0.00001
21
0/11
0.00001
1/8
0.0378
28
1(13)
1/11 0.0104 1(23) a Number of animals (day of death). b Comparison of experimental groups with the control group made using the Fisher Exact Test. Three more animals appeared sick, but later recovered.
'a' "i
40.01F 20.0
F
60
c
after infection. The mean percentage of migration of PE cells harvested at various time intervals after infection is shown in Fig. 3. Each bar represents the group mean ± standard error based on the minimum and maximum percentage of migration for each animal. At 2 days, significant enhancement of migration (154.66 + 1.01%) over control values was obtained. On day 7, a maximum percentage of migration of 183.44 ± 17.07% was seen. At 14 days, two animals displayed significant enhancement and three others showed significant inhibition, but the val-
1000
100
10
1.0
0.1
LISTERIA ANTIGEN (pg) FIG. 2. Percentage ofmigration of normal PE cells the presence of Listeria antigen. PE cells were harvested from normal outbred guinea pigs. Each point represents the mean percentage of migration obtained with 10 animals. Bars indicate standard in
error.
ues
for the
group as a
whole did not differ
significantly from the controls. At 21 days, migration inhibition was routinely observed with all the animals. None of the animals showed any
significant enhancement. By 28 days, however, only one animal displayed inhibition, and enhancement was again observed with three animals. Preliminary experiments had indicated that
CELLULAR IMMUNITY TO L. MONOCYTOGENES
VOL. 21, 1978 VI
200.0 Oi 160.0
+ C
l,
VI
160.0
=~~~~~
140.0-
120.0 +
IL~~
+
40.0
+
20.0 .AELF92L
-
0
UNINFECTED
general, all animals showing significant migration inhibition were also skin test positive except in the case of animal no. 1 on day 2, which showed migration inhibition in the absence of a positive skin test. On day 2, in four of the five animals, significant migration enhancement was observed in the absence of a positive skin test. No correlation existed between the intensity of skin reactivity and the level of macrophage migration inhibition or enhancement. Similar observations have been reported for the BCG-rabbit system by others (9).
VI~~~~~~~~'
+ 50.0 +
100.0
60.0
13
2
7
14
21
CONTROLS
DAYS AFTER LISTERIA INFECTION FIG. 3. Alteration of PE cell migration in the presence of Listeria antigen after infection with Listeria. PE cells were harvested from outbred guinea pigs at various time intervals after a primary Listeria infection. Each bar represents the group mean based on the lowest (0) and the highest (a) percentage of migration for each animal obtained in dose-response experiments (0.1 to 1(X)pg). Groups consisted of six to seven animals. Stand&d error is indicated on each bar. The mean migration inhibition or enhancement obtained with each experimental group was compared to that of the control group by means of Student's t test. The level of significance is indicated above each bar (NS, not significant). no single antigen concentration was uniformly inhibitory or enhancing towards cells from infected donors. It was therefore necessary to employ a range of antigen concentrations. DTH and macrophage migration patterns. The onset and persistence of DTH following infection with Listeria has been reported previously by this laboratory (8, 11). Results obtained throughout the present study paralleled the previously reported data and for this reason were not detailed in the present report. Table 2 shows the DTH skin test response and the percentages of migration for individual guinea pigs at various time intervals after infection. It can be seen that after 7 days all the animals were uniformly skin test positive. On day 7, when the skin reactions were at their peak, no migration inhibition was seen and, in fact, enhancement was routinely observed. In
DISCUSSION The present experiments, as well as a study of lymphocyte blastogenesis by Fulton et al. (8), clearly establish that a state of specific cell-mediated immunity develops in guinea pigs following infection with L. monocytogenes. The results of the splenic clearance of 1Ox the 50% lethal dose of Listeria used as a challenge dose at various time intervals after an immunizing dose were essentially similar to those reported by Halliburton and Blazkovec (11). It is possible that the inactivation of the primary infection, which was seen to occur at about day 2 (11), may not have been immunological but could have been due either to an influx of inflammatory cells or to direct activation of resident macrophages by listerial products. Hence, the decline in numbers of the 2-day challenge inoculum seen over the first 24 h may have been due to such nonspecific phenomena. The fact that, after 48 h, clearance was not enhanced in the 2day challenge animals when compared with control values was in marked contrast to the clearance patterns of animals challenged at 7 and 14 days. Petit and Unanue (17) have shown that products released by Listeria are B-cell mitogens and also enhance resistance to virulent Listeria in mice. Listeria cell walls have also been shown to possess similar mitogenic and adjuvant properties (1, 2). An early nonspecific phase of resistance may, therefore, be operative prior to the onset of specific resistance. Passive transfer studies would provide more clear-cut answers to this question. The clearance studies at the other time periods suggested that at 7 and 14 days after infection sufficient numbers of sensitized lymphocytes were present for a state of immunity to be manifested. At 14 days, however, these may have been on the decline, necessitating the 24-h lag period during which time there was some multiplication of the Listeria. In addition, it also appeared that there was no generation or entry of memory cells in the spleen on day 21 and after. It should be noted that injection of 1Ox the
14
DUSTOOR AND BLAZKOVEC
INFECT. IMMUN.
TABLE 2. Comparison of Listeria antigen-specific macrophage migration and delayed hypersensitivity of guinea pigs after infection with L. monocytogenes Migration inhibition Time after infection
Guinea pig no.
(days)
Optimal antigen concna
101) (2
2
1
2 3 4 5 7
0.1 1.0
0.1
21
Optimal
a
P
24-h skin
reactin
reactivity
Percent Mc mitiml nan Percent Significance' gration' Signficancec tigen gration
(diam of erSthem in
605g/ml)
65.69 92.57 None None 80.71
12 13 14 15 16 17
0.1 0.1 1.0 0.1 1.0 100
83.86 61.28 88.22 60.51 60.77 98.09
18 19 20 21 22 23 24
0.1 1.0 10 100 10 10 0.1
79.08 69.74 47.09 24.53 60.50 76.24 70.16
mm)
0.01
100
154.65
NS
100 100 10 100
152.94 155.00 152.50 158.22
0.001 0.001 0.001 0.001 0.01
Neg
100 100 1.0 100 100 100
143.64 169.23 147.22 189.66 257.70 193.17
0.001 0.001 0.001 0.001 0.001 0.001
17.5 15.5 20 15.5 14 15.5
10
120.54 None 110.10 168.20 144.62 126.28
NS
10.5 12.5 15 13 13 17
NS
None None None None None None
6 7 8 9 10 11
14
Migration enhancement
NS 0.01 NS 0.001 0.01 NS 0.01 0.01 0.01 0.05 0.05
NS 0.05
100 100
100 10
None None None None None None None
NS 0.001 0.01 0.001
Neg Neg 4 Neg
10 14 19 16 13 14.5 7
28
25 0.1 65.84 0.001 None 13 26 None 100 202.39 0.001 6 27 10 88.08 NS 10 127.99 0.01 8 0.1 28 99.21 NS 1.0 115.55 0.02 11 0.1 29 95.30 NS 10 122.80 NS 10.5 30 None 10 157.16 0.01 15 31 1.0 78.03 NS 100 161.55 0.01 16.5 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 animals of Listeria results in an acute short-term infecdid not lead either to progressive growth of tion in guinea pigs, it is very likely that the high organisms in the spleen or to enhanced multi- level of resistance to reinfection seen on days 21, plication. Over the first 72 h at least, the growth 28, and 60 was due to the persistence of memory pattern appeared to be independent of the dose cells which, presumably, rapidly generated a used. It thus appeared that, for the guinea pig state of immunity. This, however, was not remodel, splenic counts did not constitute a reflec- flected in the 21- and 28-day splenic clearance tion of the degree of resistance. results. The length of time that these memory The results of survival experiments also sup- cells persisted is not known, since survival studported this concept. Using the survival criterion, ies were not extended beyond 60 days. In all resistance was present when measured on day 7 studies carried out in this laboratory to date, and persisted until at least day 60, at which time titrations of sera by the passive hemagglutinathe last assessment was made. Because injection tion method have shown that little or no anti-
VOL. 21, 1978
CELLULAR IMMUNITY TO L. MONOCYTOGENES
15 Results reported by Fulton et al. (8) derived from the use of PE cells for the study of lymphocyte blastogenesis during the course of a Listeria infection in guinea pigs revealed that significant blastogenesis was first measurable on day 7 in about 40% of the animals. Skin tests became positive on day 4. The results of the present study showed that enhancement of macrophage migration was evident as early as day 2, suggesting that this assay may be more sensitive than assessment of levels of DTH in vivo. All animals that were DTH positive also tested positive 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 correlation exists between ACR, DTH, and migration inhibition/enhancement. Passive transfer studies, together with dissection of the in vitro response by assaying purified lymphocyte supernatant fractions, are warranted to clarify these interrelationships further.
body can be demonstrated after either a primary or a secondary infection of guinea pigs with L. monocytogenes (8). It is, therefore, unlikely that antibodies play a significant role in terms of protection against Listeria in this species. In this system, peripheral DTH has been shown to be present from day 4 onwards, peaking at 7 days and persisting for almost 1 year (11). The results of the present study are in general agreement with these findings, although skin testing was not carried out beyond 84 days. These results, in conjunction with those obtained from the survival studies, suggest that DTH precedes the expression of ACR. Lymphocyte-derived mediators such as migration inhibition factor have been implicated in the expression of cellular resistance to infection. Although cell types other than T cells have been reported to produce migration inhibition factor (18, 22), the use in these studies of PE cell populations, which represent a rich source of T cells (19), has made it possible to assess the inhibition factor-producing capacity of T cells ACKNOWLEDGMENTS primarily. This investigation was supported by grant no. 5-R22AIThe results in Table 2 and Fig. 3 make it from the United States-Japan Cooperative Medical evident that the macrophages were being sub- 08608 Science administered by the U.S. Department of jected to opposing influences. Many reports have Health, Program, Education and Welfare; by funds from American documented the enhancement of macrophage Cancer Society Institution Grant no. IN-35022; by funds from Health Service General Research Support Grant no. migration and the secretion of a distinct migra- Public G-474-17 to the University of Wisconsin Medical School from tion enhancement factor by antigen-stimulated the Division of Research Facilities and Resources, National lymphocyte cultures (7, 20, 21, 23). In a few Institutes of Health; by funds from the University of Wisconsin instances, the existence of migration enhance- Graduate School Research Committee; and by funds from ment factor was demonstrable only after migra- Public Health Service training grant no. AI-00451 from the tion inhibition factor activity was first removed. National Institute of Allergy and Infectious Diseases. It is possible that the enhancement observed in LITERATURE CITED the present study was also due to the presence 1. Campbell, P. A., C. Schuffler, and G. E. Rodriguez. of such a factor. This phenomenon is immuno1976. Listeria cell wall fraction: a B cell adjuvant. J. logical, since enhancement was observed only Immunol. 116:590-594. 2. Cohen, J. J., G. E. Rodriguez, P. D. Kind, and P. A. when using cells from hypersensitive donors. Campbell. 1975. Listeria cell wall fraction: a B cell It is not known whether the same or different mitogen. J. Immunol. 114:1132-1134. cell types secrete migration inhibition and en- 3. Collins, F. M. 1971. Mechanisms in antimicrobial immuhancement factors. In the present study, the nity. RES J. Reticuloendothel. Soc. 10:58-99. cells harvested from 2 animals at 14 days after 4. Collins, F. M., and G. B. Mackaness. 1970. The relationship of delayed hypersensitivity to acquired antiinfection were shown to be both enhanced and tuberculous immunity. I. Tuberculin sensitivity and inhibited, depending on the antigen concentraresistance to reinfection in BCG vaccinated mice. Cell. tion employed, suggesting that both activities Immunol. 1:253-265. can be released in measurable quantities by a 5. Collins, F. M., and G. B. Mackaness. 1970. The relationship of delayed hypersensitivity to acquired antigiven cell population. Early after sensitization, tuberculous immunity. II. Effect of adjuvant on the enhancing activity was predominant, with high allergenicity and immunogenicity of heat-killed tubercle levels of enhancement demonstrable on days 2 bacilli. Cell. Immunol. 1:266-275. and 7. It was not clear what the significance of 6. David, J. R. 1975. Macrophage activation by lymphocyte Fed. Proc. 34:1730-1736. the shift to migration inhibition and the return 7. mediators. Fox, R. A., D. S. Gregory, and J. D. Feldman. 1974. to migration enhancement at 28 days was. The Migration inhibition factor (MIF) and migration stimpresence of inhibition factor demonstrable at 21 ulation factor (MSF) in fetal calf serum. J. Immunol. 112:1861-1866. days constituted a real phenomenon, since all seven of the animals assayed were positive and 8. Fulton, A. M., M. M. Dustoor, J. E. Kasinski, and A. A. Blazkovec. 1975. Blastogenesis as an in vitro corenhancement of cell migration was never obrelate of delayed hypersensitivity in guinea pigs infected served at any of the antigen concentrations emwith Listeria monocytogenes. Infect. Immun. 12: 647-655. ployed.
16
DUSTOOR AND BLAZKOVEC
9. Galindo, B., and Q. N. Myrvik. 1970. Migratory response of granulomatous alveolar cells from BCG-sensitized rabbits. Infect. Immun. 1:227-237. 10. George, M., and J. H. Vaughan. 1962. In vitro cell migration as a model for delayed hypersensitivity. Proc. Soc. Exp. Biol. Med. 111:514-521. 11. Halliburton, B. L, and A. A. Blazkovec. 1975. Delayed hypersensitivity and acquired cellular resistance in guinea pigs infected with Listeria monocytogenes. Infect. Immun. 11:1-7. 12. Hinsdill, R. D., and D. T. Berman. 1967. Antigens of Brucella abortus. I. Chemical and immunoelectrophoretic 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-tuberculous immunity. Am. Rev. Respir. Dis. 97:337-344. 15. Neiburger, R. G., G. P. Youmans, and A. S. Youmans. 1973. Relationship between tuberculin hypersensitivity and cellular immunity to infection in mice vaccinated with viable attenuated mycobacterial cells or with mycobacterial ribonucleic acid preparations. Infect. Immun. 8:4247. 16. Osebold, J. W., L. D. Pearson, and N. L. Medin. 1974. Relationship of antimicrobial cellular immunity to delayed hypersensitivity in listeriosis. Infect. Immun. 9:354-362. 17. Petit, J. C., and E. R. Unanue. 1974. Effects of bacterial
INFECT. IMMUN.
18.
19.
20.
21. 22.
23.
products on lymphocytes and macrophages: their possible role in natural resistance to Listeria infection in mice. J. Immunol. 113:984-992. Rocklin, R. E., R. P. MacDermott, L Chess, S. F. Schlosaman, and J. R. David. 1974. Studies on mediator production by highly purified human T and B lymphocytes. J. Exp. Med. 140:1303-1316. Rosenstreich, D. L, J. T. Blake, and A. S. Rosenthal. 1971. The peritoneal exudate lymphocyte. I. Differences in antigen responsiveness between peritoneal exudate and lymph node lymphocytes from immunized guinea pigs. J. Exp. Med. 134:1170-1186. Sandok, P. L., R. D. Hinsdill, and R. M. Albrecht. 1975. Alterations in mouse macrophage migration: a function of assay systems, lymphocyte activation product preparation and fractionation. Infect. Immun. 11:1100-1109. Soborg, M. 1968. In vitro migration of peripheral human leucocytes in cellular hypersensitivity. Acta Med. Scand. 186:135-139. Tubergen, D. G., J. D. Feldman, E. M. Pollock, and R. A. Lerner. 1972. Production of macrophage migration inhibition factor by continuous cell lines. J. Exp. Med. 135:255-266. Weisbart, R. H., R. Bluestone, L. S. Goldberg, and C. M. Pearson. 1974. Migration enhancement factor: a new lymphokine. Proc. Natl. Acad. Sci. U.S.A. 71: 875-879.
INFECTION AND IMMUNITY, July 1978, p. 10-16
0019-9567/78/0021-0010$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Acquired Cellular Resistance, Delayed Hypersensitivity, and Altered Macrophage Migration in Listeria monocytogenesInfected Guinea Pigs MEHER M. DUSTOOR AND ANDREW A. BLAZKOVEC* Department of Medical Microbiology, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 16 January 1978
A Listeria monocytogenes infection in guinea pigs was used to study the interrelationship between antigen-induced macrophage migration inhibition, delayed-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 uniformly observed only on day 21 of the infection, after which a shift again to enhancement was seen. The early detection (by day 2) of migration enhancement suggested that this assay may be more sensitive than assessment of delayed type hypersensitivity in vivo, which in this system was first detectable only on day 4. Acquired cellular resistance, as measured by enhanced survival following a high dose challenge with Listeria, was present from day 7 after infection until at least day 60. By splenic clearance studies, however, acquired cellular resistance was present only until day 14 after infection, suggesting that in this system splenic clearance was not a very reliable criterion for measuring acquired cellular resistance.
The relationship between acquired cellular resistance (ACR) and delayed-type hypersensitivity (DTH) remains an area of conflict (3-5, 15-17). A Listeria monocytogenes infection in guinea pigs has been used in this laboratory to study the interrelationships between ACR and DTH (8, 11). The results obtained from in vivo studies showed that infected animals were resistant to a small challenge dose of Listeria given 2, 7, or 14 days after the primary infection. DTH, however, was not detectable until 5 or 6 days postinfection, which suggested that, for this species, ACR was not dependent on the development of DTH. In the two studies cited above, the only criterion of ACR employed was enhanced splenic clearance and, furthermore, the challenge dose used was low and hence no definite conclusions could be reached. In the present study, enhanced survival was used in addition to splenic clearance as a criterion of ACR, and a high 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 it could be used as an indicator of DTH in this system. (The research described in this paper was submitted in part to the Graduate School of the University of Wisconsin by M.M.D. in partial fulfillment of the requirements for the Ph.D. degree in Medical Microbiology.)
MATERIALS AND METHODS Animals. Randomly bred albino guinea pigs of both sexes were obtained from local suppliers. The animals weighed 500 to 700 g at the time of infection. Bacteria. The L. monocytogenes strain used and the method of cultivation have been described elsewhere (11). Immunization and challenge. For immunization, approximately one-half of a 50% lethal dose in a total volume of 2 ml was injected intracardially into each guinea pig. Ten times the 50% lethal dose of Listeria was used for challenge via the intracardial route. The exact number of viable organisms injected was determined by plate counts. Spleen assays. The number of viable Listeria in brain heart infusion broth homogenates of spleen was determined by the method of Mackaness (13). Appropriate decimal dilutions were plated in duplicate on well-dried brain heart infusion plates. Colonies were counted after 24 to 48 h of incubation at 370C. Antigen. A water-soluble extract of sonically disrupted Listeria was prepared by using a modification of the method of Hinsdill and Berman (12) as described previously (11). Before use, the lyophilized material was reconstituted and sterilized by passage through a 0.45-gm membrane filter (Millipore Corp., Bedford, Mass.). Delayed hypersensitivity tests. Guinea pigs were injected intradermally on the shaved dorsal flank with 30 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 described (11). Maximum skin reactivity was always recorded at 24 h after testing. Thus skin reactivity 10
VOL. 21, 1978
CELLULAR IMMUNITY TO L. MONOCYTOGENES
data presented in Table 2 are comprised of only the 24-h diameter of erythema. Histological examination of skin test sites at various time intervals after infection revealed a classical tuberculin-type infiltrate, thereby confirming previously published findings of this laboratory (8). PE cells. Peritoneal exudate (PE) cells were induced in guinea pigs by the intraperitoneal injection of 25 to 30 ml of sterile no. 31 heavy paraffin oil (American Oil Co., Chicago, Ill.). Three days later, the exudates were harvested with 300 ml of Eagle minimal essential medium (Grand Island Biological Co., Grand Island, N.Y.) containing 200 fig of penicillin per ml and 40 ug of streptomycin per ml. The PE cells were washed twice, and equal volumes were distributed into separate 15-ml centrifuge tubes. These were centrifuged and a 7.5% suspension of the pelleted cells was made in Eagle minimal essential medium containing 15% normal guinea pig serum and the appropriate concentration of Listeria antigen. For some experiments, purified protein derivative (Parke-Davis and Co., Detroit, Mich.) served as a specificity control. Migration inhibition assay. The 7.5% cell suspensions were drawn up to 75 mm in 100-mm capillary tubes (Kimax no. 34502, 0.8 to 1.1 by 100 mm; Kimble Products, Toledo, Ohio), which were sealed at one end with a mixture of paraffin wax and petroleum jelly. Six tubes were filled with each cell suspension. They were centrifuged at 35 x g for 4 min and cut at the cell-fluid interface. The tubes were placed in Sykes-Moore chambers (Bellco Glass, Inc., Vineland, N.J.), which were then filled with medium containing the same concentration of antigen as was used to suspend the cells and incubated for 24 h at 370C in an atmosphere of 5% CO2. The areas of cell migration were magnified with a Microprojector (Bausch and Lomb, Rochester, N.Y.), traced, cut out, and weighed. For each animal, the mean areas of migration from capillary tubes with antigen and without antigen were then used in the following formula: percentage of migration = (mean area of migration in presence of antigen/mean area of migration without antigen) x 100.
11
difference in the numbers of Listeria recovered from infected and control guinea pigs. Thus no definite conclusion could be reached regarding the presence of ACR in the animals at 2 days, since the fate of the challenge organisms alone was not followed. At 7 days after infection, splenic clearance by the infected animals was markedly enhanced over that by controls. On day 14, it appeared to be on the wane, and by 21 and 28 days the clearance by infected animals almost paralleled that by the controls. (ii) Survival. Outbred guinea pigs were infected with one-half of a 50% lethal dose of Listeria. On days 2, 4, 7, 14, 21, 28, and 60, these animals, together with a group of uninfected controls, were challenged with an intracardial injection of approximately 1Ox the 50% lethal dose of Listeria. Daily deaths were recorded, and these data are given in Table 1. There was no significant difference between the number of deaths recorded for the controls and the 2-day challenge group. After 7 days, the mortality in the infected groups was significantly reduced. From these data, ACR did not appear to be present at 2 days, but was present from day 7 through at least day 60. Macrophage migration inhibition in guinea pigs infected with Listeria. Outbred guinea pigs were infected with one-half of a 50% lethal dose of Listeria, and 2, 7, 14, 21, and 28 days later PE cells from these animals were used in the migration inhibition assay. A control group of normal animals was also used. Animals used in these studies were skin tested 24 h earlier with Listeria antigen. Migration inhibition of normal PE cells. Figure 2 shows the percentage of migration of normal cells in the presence of various concenRESULTS trations of Listeria antigen. A concentration of ACR in guinea pigs infected with ListerwL 1,000 Itg/ml was clearly toxic, inhibiting migra(i) Splenic clearance. Guinea pigs infected tion by 40%. The other four concentrations were with a sublethal dose of Listeria, together with nontoxic. In all subsequent studies, concentrauninfected controls, were challenged at 2, 7, 14, tions ranging from 0.1 to 100 tg/ml were utilized. PE cells obtained from six normal guinea pigs 21, and 28 days with an intracardial injection of lOx the 50% lethal dose of Listeria. Groups of and from six animals at 21 days after infection animals were sacrificed on days 0, 1, 2, and 3 with Listeria were incubated with 10 and 1 ,g of purified protein derivative per ml and served as following challenge. Preliminary studies utilizing a streptomycin- specificity controls. With 10 jig of purified proresistant mutant of Listeria as the challenge tein derivative, the mean migration of cells from organism have been performed. There was, how- infected and normal animals was 91.13 ± 5.66 ever, a relative lack of multiplication of the and 104.71 + 7.41%, respectively; with 1 ug of mutant organisms in outbred guinea pigs. The purified protein derivative, migration values of use of mutants in further studies was, therefore, 108.38 + 10.45 and 92.58 + 8.04%, respectively, were obtained. These findings in relationship to discontinued. At the 2-day challenge interval (Fig. 1), al- specific Listeria antigen-induced alterations of though there was no multiplication of the chal- immune PE cell migration established the speclenge organisms in the infected animals within ificity of the latter. the first 24 h, by 48 and 72 h there was no Alteration of macrophage migration
12
INFECT. IMMUN.
DUSTOOR AND BLAZKOVEC
CHALLENGE AT 28 DAYS
21 DAYS
usaa. G"
co
m
".
EL
m U
e
2
30
1
30
2
1
DAYS AFTER CHALLENGE FIG. 1. Clearance of a 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 of Listeria present in the spleen were determined. Bars indicate standard error. TABLE 1. Resistance ofguinea pigs previously infected with one-half of a 50% lethal dose of Listeria, upon challenge with lOx the 50% lethal dose of Listeria No. dead/ Time of no. chal- P value" Mortality record' challenge lenged (days) Controls 1(7), 2(9), 1(10), 2(12), 10/17C 1(13), 1(14), 1(15), 1(21)
160.0!120.0 F QC
2
1(7), 1(15), 1(17), 1(19), 1(22), 1(23)
6/13
0.2745
I 60.0 1-
7
1(8), 1(9), 1(30)
3/14
0.0402
z 60.0 I.
14
0/11
0.00001
21
0/11
0.00001
1/8
0.0378
28
1(13)
1/11 0.0104 1(23) a Number of animals (day of death). b Comparison of experimental groups with the control group made using the Fisher Exact Test. Three more animals appeared sick, but later recovered.
'a' "i
40.01F 20.0
F
60
c
after infection. The mean percentage of migration of PE cells harvested at various time intervals after infection is shown in Fig. 3. Each bar represents the group mean ± standard error based on the minimum and maximum percentage of migration for each animal. At 2 days, significant enhancement of migration (154.66 + 1.01%) over control values was obtained. On day 7, a maximum percentage of migration of 183.44 ± 17.07% was seen. At 14 days, two animals displayed significant enhancement and three others showed significant inhibition, but the val-
1000
100
10
1.0
0.1
LISTERIA ANTIGEN (pg) FIG. 2. Percentage ofmigration of normal PE cells the presence of Listeria antigen. PE cells were harvested from normal outbred guinea pigs. Each point represents the mean percentage of migration obtained with 10 animals. Bars indicate standard in
error.
ues
for the
group as a
whole did not differ
significantly from the controls. At 21 days, migration inhibition was routinely observed with all the animals. None of the animals showed any
significant enhancement. By 28 days, however, only one animal displayed inhibition, and enhancement was again observed with three animals. Preliminary experiments had indicated that
CELLULAR IMMUNITY TO L. MONOCYTOGENES
VOL. 21, 1978 VI
200.0 Oi 160.0
+ C
l,
VI
160.0
=~~~~~
140.0-
120.0 +
IL~~
+
40.0
+
20.0 .AELF92L
-
0
UNINFECTED
general, all animals showing significant migration inhibition were also skin test positive except in the case of animal no. 1 on day 2, which showed migration inhibition in the absence of a positive skin test. On day 2, in four of the five animals, significant migration enhancement was observed in the absence of a positive skin test. No correlation existed between the intensity of skin reactivity and the level of macrophage migration inhibition or enhancement. Similar observations have been reported for the BCG-rabbit system by others (9).
VI~~~~~~~~'
+ 50.0 +
100.0
60.0
13
2
7
14
21
CONTROLS
DAYS AFTER LISTERIA INFECTION FIG. 3. Alteration of PE cell migration in the presence of Listeria antigen after infection with Listeria. PE cells were harvested from outbred guinea pigs at various time intervals after a primary Listeria infection. Each bar represents the group mean based on the lowest (0) and the highest (a) percentage of migration for each animal obtained in dose-response experiments (0.1 to 1(X)pg). Groups consisted of six to seven animals. Stand&d error is indicated on each bar. The mean migration inhibition or enhancement obtained with each experimental group was compared to that of the control group by means of Student's t test. The level of significance is indicated above each bar (NS, not significant). no single antigen concentration was uniformly inhibitory or enhancing towards cells from infected donors. It was therefore necessary to employ a range of antigen concentrations. DTH and macrophage migration patterns. The onset and persistence of DTH following infection with Listeria has been reported previously by this laboratory (8, 11). Results obtained throughout the present study paralleled the previously reported data and for this reason were not detailed in the present report. Table 2 shows the DTH skin test response and the percentages of migration for individual guinea pigs at various time intervals after infection. It can be seen that after 7 days all the animals were uniformly skin test positive. On day 7, when the skin reactions were at their peak, no migration inhibition was seen and, in fact, enhancement was routinely observed. In
DISCUSSION The present experiments, as well as a study of lymphocyte blastogenesis by Fulton et al. (8), clearly establish that a state of specific cell-mediated immunity develops in guinea pigs following infection with L. monocytogenes. The results of the splenic clearance of 1Ox the 50% lethal dose of Listeria used as a challenge dose at various time intervals after an immunizing dose were essentially similar to those reported by Halliburton and Blazkovec (11). It is possible that the inactivation of the primary infection, which was seen to occur at about day 2 (11), may not have been immunological but could have been due either to an influx of inflammatory cells or to direct activation of resident macrophages by listerial products. Hence, the decline in numbers of the 2-day challenge inoculum seen over the first 24 h may have been due to such nonspecific phenomena. The fact that, after 48 h, clearance was not enhanced in the 2day challenge animals when compared with control values was in marked contrast to the clearance patterns of animals challenged at 7 and 14 days. Petit and Unanue (17) have shown that products released by Listeria are B-cell mitogens and also enhance resistance to virulent Listeria in mice. Listeria cell walls have also been shown to possess similar mitogenic and adjuvant properties (1, 2). An early nonspecific phase of resistance may, therefore, be operative prior to the onset of specific resistance. Passive transfer studies would provide more clear-cut answers to this question. The clearance studies at the other time periods suggested that at 7 and 14 days after infection sufficient numbers of sensitized lymphocytes were present for a state of immunity to be manifested. At 14 days, however, these may have been on the decline, necessitating the 24-h lag period during which time there was some multiplication of the Listeria. In addition, it also appeared that there was no generation or entry of memory cells in the spleen on day 21 and after. It should be noted that injection of 1Ox the
14
DUSTOOR AND BLAZKOVEC
INFECT. IMMUN.
TABLE 2. Comparison of Listeria antigen-specific macrophage migration and delayed hypersensitivity of guinea pigs after infection with L. monocytogenes Migration inhibition Time after infection
Guinea pig no.
(days)
Optimal antigen concna
101) (2
2
1
2 3 4 5 7
0.1 1.0
0.1
21
Optimal
a
P
24-h skin
reactin
reactivity
Percent Mc mitiml nan Percent Significance' gration' Signficancec tigen gration
(diam of erSthem in
605g/ml)
65.69 92.57 None None 80.71
12 13 14 15 16 17
0.1 0.1 1.0 0.1 1.0 100
83.86 61.28 88.22 60.51 60.77 98.09
18 19 20 21 22 23 24
0.1 1.0 10 100 10 10 0.1
79.08 69.74 47.09 24.53 60.50 76.24 70.16
mm)
0.01
100
154.65
NS
100 100 10 100
152.94 155.00 152.50 158.22
0.001 0.001 0.001 0.001 0.01
Neg
100 100 1.0 100 100 100
143.64 169.23 147.22 189.66 257.70 193.17
0.001 0.001 0.001 0.001 0.001 0.001
17.5 15.5 20 15.5 14 15.5
10
120.54 None 110.10 168.20 144.62 126.28
NS
10.5 12.5 15 13 13 17
NS
None None None None None None
6 7 8 9 10 11
14
Migration enhancement
NS 0.01 NS 0.001 0.01 NS 0.01 0.01 0.01 0.05 0.05
NS 0.05
100 100
100 10
None None None None None None None
NS 0.001 0.01 0.001
Neg Neg 4 Neg
10 14 19 16 13 14.5 7
28
25 0.1 65.84 0.001 None 13 26 None 100 202.39 0.001 6 27 10 88.08 NS 10 127.99 0.01 8 0.1 28 99.21 NS 1.0 115.55 0.02 11 0.1 29 95.30 NS 10 122.80 NS 10.5 30 None 10 157.16 0.01 15 31 1.0 78.03 NS 100 161.55 0.01 16.5 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 animals of Listeria results in an acute short-term infecdid not lead either to progressive growth of tion in guinea pigs, it is very likely that the high organisms in the spleen or to enhanced multi- level of resistance to reinfection seen on days 21, plication. Over the first 72 h at least, the growth 28, and 60 was due to the persistence of memory pattern appeared to be independent of the dose cells which, presumably, rapidly generated a used. It thus appeared that, for the guinea pig state of immunity. This, however, was not remodel, splenic counts did not constitute a reflec- flected in the 21- and 28-day splenic clearance tion of the degree of resistance. results. The length of time that these memory The results of survival experiments also sup- cells persisted is not known, since survival studported this concept. Using the survival criterion, ies were not extended beyond 60 days. In all resistance was present when measured on day 7 studies carried out in this laboratory to date, and persisted until at least day 60, at which time titrations of sera by the passive hemagglutinathe last assessment was made. Because injection tion method have shown that little or no anti-
VOL. 21, 1978
CELLULAR IMMUNITY TO L. MONOCYTOGENES
15 Results reported by Fulton et al. (8) derived from the use of PE cells for the study of lymphocyte blastogenesis during the course of a Listeria infection in guinea pigs revealed that significant blastogenesis was first measurable on day 7 in about 40% of the animals. Skin tests became positive on day 4. The results of the present study showed that enhancement of macrophage migration was evident as early as day 2, suggesting that this assay may be more sensitive than assessment of levels of DTH in vivo. All animals that were DTH positive also tested positive 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 correlation exists between ACR, DTH, and migration inhibition/enhancement. Passive transfer studies, together with dissection of the in vitro response by assaying purified lymphocyte supernatant fractions, are warranted to clarify these interrelationships further.
body can be demonstrated after either a primary or a secondary infection of guinea pigs with L. monocytogenes (8). It is, therefore, unlikely that antibodies play a significant role in terms of protection against Listeria in this species. In this system, peripheral DTH has been shown to be present from day 4 onwards, peaking at 7 days and persisting for almost 1 year (11). The results of the present study are in general agreement with these findings, although skin testing was not carried out beyond 84 days. These results, in conjunction with those obtained from the survival studies, suggest that DTH precedes the expression of ACR. Lymphocyte-derived mediators such as migration inhibition factor have been implicated in the expression of cellular resistance to infection. Although cell types other than T cells have been reported to produce migration inhibition factor (18, 22), the use in these studies of PE cell populations, which represent a rich source of T cells (19), has made it possible to assess the inhibition factor-producing capacity of T cells ACKNOWLEDGMENTS primarily. This investigation was supported by grant no. 5-R22AIThe results in Table 2 and Fig. 3 make it from the United States-Japan Cooperative Medical evident that the macrophages were being sub- 08608 Science administered by the U.S. Department of jected to opposing influences. Many reports have Health, Program, Education and Welfare; by funds from American documented the enhancement of macrophage Cancer Society Institution Grant no. IN-35022; by funds from Health Service General Research Support Grant no. migration and the secretion of a distinct migra- Public G-474-17 to the University of Wisconsin Medical School from tion enhancement factor by antigen-stimulated the Division of Research Facilities and Resources, National lymphocyte cultures (7, 20, 21, 23). In a few Institutes of Health; by funds from the University of Wisconsin instances, the existence of migration enhance- Graduate School Research Committee; and by funds from ment factor was demonstrable only after migra- Public Health Service training grant no. AI-00451 from the tion inhibition factor activity was first removed. National Institute of Allergy and Infectious Diseases. It is possible that the enhancement observed in LITERATURE CITED the present study was also due to the presence 1. Campbell, P. A., C. Schuffler, and G. E. Rodriguez. of such a factor. This phenomenon is immuno1976. Listeria cell wall fraction: a B cell adjuvant. J. logical, since enhancement was observed only Immunol. 116:590-594. 2. Cohen, J. J., G. E. Rodriguez, P. D. Kind, and P. A. when using cells from hypersensitive donors. Campbell. 1975. Listeria cell wall fraction: a B cell It is not known whether the same or different mitogen. J. Immunol. 114:1132-1134. cell types secrete migration inhibition and en- 3. Collins, F. M. 1971. Mechanisms in antimicrobial immuhancement factors. In the present study, the nity. RES J. Reticuloendothel. Soc. 10:58-99. cells harvested from 2 animals at 14 days after 4. Collins, F. M., and G. B. Mackaness. 1970. The relationship of delayed hypersensitivity to acquired antiinfection were shown to be both enhanced and tuberculous immunity. I. Tuberculin sensitivity and inhibited, depending on the antigen concentraresistance to reinfection in BCG vaccinated mice. Cell. tion employed, suggesting that both activities Immunol. 1:253-265. can be released in measurable quantities by a 5. Collins, F. M., and G. B. Mackaness. 1970. The relationship of delayed hypersensitivity to acquired antigiven cell population. Early after sensitization, tuberculous immunity. II. Effect of adjuvant on the enhancing activity was predominant, with high allergenicity and immunogenicity of heat-killed tubercle levels of enhancement demonstrable on days 2 bacilli. Cell. Immunol. 1:266-275. and 7. It was not clear what the significance of 6. David, J. R. 1975. Macrophage activation by lymphocyte Fed. Proc. 34:1730-1736. the shift to migration inhibition and the return 7. mediators. Fox, R. A., D. S. Gregory, and J. D. Feldman. 1974. to migration enhancement at 28 days was. The Migration inhibition factor (MIF) and migration stimpresence of inhibition factor demonstrable at 21 ulation factor (MSF) in fetal calf serum. J. Immunol. 112:1861-1866. days constituted a real phenomenon, since all seven of the animals assayed were positive and 8. Fulton, A. M., M. M. Dustoor, J. E. Kasinski, and A. A. Blazkovec. 1975. Blastogenesis as an in vitro corenhancement of cell migration was never obrelate of delayed hypersensitivity in guinea pigs infected served at any of the antigen concentrations emwith Listeria monocytogenes. Infect. Immun. 12: 647-655. ployed.
16
DUSTOOR AND BLAZKOVEC
9. Galindo, B., and Q. N. Myrvik. 1970. Migratory response of granulomatous alveolar cells from BCG-sensitized rabbits. Infect. Immun. 1:227-237. 10. George, M., and J. H. Vaughan. 1962. In vitro cell migration as a model for delayed hypersensitivity. Proc. Soc. Exp. Biol. Med. 111:514-521. 11. Halliburton, B. L, and A. A. Blazkovec. 1975. Delayed hypersensitivity and acquired cellular resistance in guinea pigs infected with Listeria monocytogenes. Infect. Immun. 11:1-7. 12. Hinsdill, R. D., and D. T. Berman. 1967. Antigens of Brucella abortus. I. Chemical and immunoelectrophoretic 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-tuberculous immunity. Am. Rev. Respir. Dis. 97:337-344. 15. Neiburger, R. G., G. P. Youmans, and A. S. Youmans. 1973. Relationship between tuberculin hypersensitivity and cellular immunity to infection in mice vaccinated with viable attenuated mycobacterial cells or with mycobacterial ribonucleic acid preparations. Infect. Immun. 8:4247. 16. Osebold, J. W., L. D. Pearson, and N. L. Medin. 1974. Relationship of antimicrobial cellular immunity to delayed hypersensitivity in listeriosis. Infect. Immun. 9:354-362. 17. Petit, J. C., and E. R. Unanue. 1974. Effects of bacterial
INFECT. IMMUN.
18.
19.
20.
21. 22.
23.
products on lymphocytes and macrophages: their possible role in natural resistance to Listeria infection in mice. J. Immunol. 113:984-992. Rocklin, R. E., R. P. MacDermott, L Chess, S. F. Schlosaman, and J. R. David. 1974. Studies on mediator production by highly purified human T and B lymphocytes. J. Exp. Med. 140:1303-1316. Rosenstreich, D. L, J. T. Blake, and A. S. Rosenthal. 1971. The peritoneal exudate lymphocyte. I. Differences in antigen responsiveness between peritoneal exudate and lymph node lymphocytes from immunized guinea pigs. J. Exp. Med. 134:1170-1186. Sandok, P. L., R. D. Hinsdill, and R. M. Albrecht. 1975. Alterations in mouse macrophage migration: a function of assay systems, lymphocyte activation product preparation and fractionation. Infect. Immun. 11:1100-1109. Soborg, M. 1968. In vitro migration of peripheral human leucocytes in cellular hypersensitivity. Acta Med. Scand. 186:135-139. Tubergen, D. G., J. D. Feldman, E. M. Pollock, and R. A. Lerner. 1972. Production of macrophage migration inhibition factor by continuous cell lines. J. Exp. Med. 135:255-266. Weisbart, R. H., R. Bluestone, L. S. Goldberg, and C. M. Pearson. 1974. Migration enhancement factor: a new lymphokine. Proc. Natl. Acad. Sci. U.S.A. 71: 875-879.
