INFECTION AND IMMUNITY, Dec. 1990, p. 3973-3979

Vol. 58, No. 12

0019-9567/90/123973-07$02.00/0 Copyright C 1990, American Society for Microbiology

Interleukin-1-Induced Promotion of T-Cell Differentiation in Mice Immunized with Killed Listeria monocytogenes KEN-ICHI IGARASHI,l.2* MASAO MITSUYAMA,1 KATSUMI MURAMORI,"3 HIROKI TSUKADA,1'2 AND KIKUO NOMOTO3 Department of Bacteriology' and Department of Medicine (II),2 Niigata University School of Medicine, Niigata 951, and Department of Immunology, Medical Institute for Bioregulation, Kyushu University, Fukuoka 812,3 Japan Received 17 April 1990/Accepted 11 September 1990

We studied the effects of administration of recombinant interleukin-lao (rIL-la) to mice after immunization with killed Listeria monocytogenes cells on the promotion of the functional differentiation of T cells in vivo. Mice immunized with killed L. monocytogenes were unable to express cell-mediated immunity to specific antigen in vivo, as determined by delayed-type hypersensitivity (DTH) and acquired cellular resistance (ACR), and splenic T cells obtained from such mice were unable to respond to rIL-2 and specific antigen and to produce IL-2 after antigenic restimulation in vitro. When rIL-la was given to mice after immunization with killed bacteria, T cells became capable of responding to rIL-2 and specific antigen in vitro. These functions of T cells were similar to those from mice immunized with viable listeriae. Moreover, using a local passive transfer system, it was found that effector T cells mediating DTH but not ACR to L. monocytogenes were generated in mice treated with rIL-la after immunization with killed bacteria. These T cells were able to produce macrophage chemotactic factor but not macrophage-activating factor or gamma interferon in vitro in response to stimulation with specific antigen. These results suggest that in vivo administration of rIL-la facilitates the maturation of antigen-specific T cells mediating DTH and that different effector T cells mediating DTH or ACR are involved in cell-mediated immunity to L. monocytogenes.

Listeria-specific T cells might be due to an insufficient production of IL-1 in vivo. Recombinant IL-1 (rIL-1) has become available, and it is reported that there is no difference in biological functions between IL-lac and IL-l, (10). In the present study, we investigated the effect of rIL-la administration to mice immunized with killed L. monocytogenes cells on the promotion of the functional maturation of T cells, including responsiveness to specific antigen or IL-2 in vitro and the ability to express DTH or ACR in vivo.

Protection against Listeria monocytogenes, a facultative intracellular bacterium, is mediated by cell-mediated immunity but not by humoral immunity (21, 27). Both acquired cellular resistance (ACR) and delayed-type hypersensitivity (DTH) are the manifestations of cell-mediated immunity raised against L. monocytogenes in animals (24, 25, 46). In mice, ACR against L. monocytogenes is usually measured by the capacity to eliminate challenge bacteria (23, 24, 46) and DTH is assayed by delayed footpad reaction (DFR) (25, 35). It is well-known that cell-mediated immunity, including DFR and ACR, can be induced in mice after immunization with viable L. monocytogenes but not with killed bacteria (2, 30, 31, 48). Killed bacteria are generally believed to be incapable of inducing specific cell-mediated immunity in mice. However, killed listeriae have been widely used as an effective stimulating antigen in the secondary stimulation of Listeria-specific effector T cells (6, 14, 17, 31). There is no convincing explanation as to why killed listeriae are unable to provoke DTH and ACR. Recently, we found that there was a significant difference in the ability to stimulate interleukin-1 (IL-1) production by macrophages between viable and killed cells of L. monocytogenes (29). IL-1, a cytokine produced primarily by cells of macrophage lineage, is believed to be essential in mediating several types of immune responses. In vitro, IL-1 enhances the activation and proliferation of T cells in response to mitogens (9, 37) or antigen (32). At least part of this effect may be attributed to the ability of IL-1 to induce synthesis of the cytokines active on T cells, such as IL-2 and IL-4, and to increase IL-2 receptor expression (12, 22, 43). Thus, there is a possibility that the inability of killed bacteria to generate

*

MATERIALS AND METHODS Experimental animals. C3H/He mice were raised and maintained under specific-pathogen-free conditions. Mice of both sexes were used for experiments at 7 to 12 weeks of age. Each experimental group consisted of at least five mice. Microorganism. L. monocytogenes EGD was used throughout the study. The bacteria had been maintained by serial passages in C3H/He mice, and a fresh isolate was obtained from the spleen of an infected animal. The bacteria were grown in trypticase soy broth (Difco Laboratories, Detroit, Mich.) at 37°C for 12 h, washed repeatedly, resuspended in phosphate-buffered saline, and stored at -80°C until use. The exact number of viable bacteria was determined by plating the dilutions onto nutrient agar supplemented with 0.4% glucose and counting the colonies after overnight incubation in each experiment. Heat-killed bacteria were prepared by heating viable L. monocytogenes at 74°C for 90 min. Medium. The medium consisted of RPMI 1640 (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% NU-SERUM (Collaborative Research, Inc., Waltham, Mass.), 100 U of penicillin per ml, 100 pLg of streptomycin per ml, and 5 x 10-2 mM 2-mercaptoethanol (complete RPMI medium).

Corresponding author. 3973

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IGARASHI ET AL.

Immunization. Mice were immunized intravenously with 2 x 103 viable or 108 killed L. monocytogenes cells. In some experiments, mice were injected intraperitoneally with 2 ,ug of recombinant human IL-lot (Research Laboratories, Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) 1 and 24 h after immunization with killed bacteria. Proliferative response of splenic T cells. Three or six days after immunization, Listeria-immune T cells were obtained from spleen cells by passage over a column packed with nylon wool fiber (Wako Pure Chemicals, Osaka, Japan). Control cells were taken from nonimmune mice in the same manner. Nylon wool-nonadherent T cells (105) were cultured with heat-killed listeriae (107) in the presence of various percentages of peritoneal exudate cells (PEC) as antigenpresenting cells (APC), which were obtained 3 days after intraperitoneal injection with 1.5 ml of 10% proteose peptone. In some experiments, nylon wool-nonadherent T cells (105) were stimulated with recombinant human IL-2 (TGP-3; Central Research Division, Takeda Chemical Industries, Ltd., Osaka, Japan). Cells were cultured for 3 days at 37°C in a CO2 incubator. During the last 18 h of culture, [3H]thymidine was added, and the incorporated radioactivity was counted in a liquid scintillation counter. Production of culture supernatant. Nylon wool-nonadherent spleen cells were taken from mice 3 or 6 days after immunization. Cells were cultured at 2 x 106 cells per ml in 2 ml of complete RPMI medium in the presence of APC and 2 x 108 heat-killed listeriae at 37°C in 5% CO2. After cultivation for 48 h, culture fluids were centrifuged and the supernatants were filtered through a 0.45-gLm-pore-size filter unit and kept at -20°C until use. Control culture supernatants were prepared from the spleens of nonimmune mice in the same manner. Assay of IL-2 activity. HT-2 cells, an IL-2-dependent T-cell line, were used for the assay of IL-2 activity in the supematant. HT-2 cells (104 cells per well) were cultured in the presence of a twofold-diluted supernatant in a 96-well microplate for 24 h at 37°C. During the last 6 h of culture, [3H]thymidine was added. Cells were harvested, and the incorporated radioactivity was counted in a liquid scintillation counter. Assay of MAF activity. Macrophage-activating factor (MAF) activity was assessed as the ability to induce cytotoxic activity in peritoneal macrophages against P815 mastocytoma cells, as described by Pace and Russell (40) with some modifications. P815 cells (2 x 107/ml) in medium were labeled by incubation with 100 ,Ci of Na51CrO4 (Japan Atomic Energy Research Institute, Tokyo, Japan) for 2 h at 37°C. The cells were then washed and adjusted to 2 x 105/ml with medium. The culture supernatant in a 1:1 dilution with medium was added to PEC adhering to 96-well tissue culture plates. After incubation for 12 h at 37°C, the wells were washed and 51Cr-labeled P815 cells in medium containing 25 ng of lipopolysaccharide (Escherichia coli O111:B4; Difco) per ml were added to the PEC to give an effector/target ratio of 20:1. After further incubation for 18 h, the culture plates were centrifuged for 5 min, and 100 ,ul of supernatant was taken from each well. The released radioactivity in each supernatant was measured in an automatic gamma counter, and specific 51Cr release was calculated as follows: percent specific 51Cr release = [(experimental release - spontaneous release)/(total release - spontaneous release)] x 100, where total release was the count obtained after complete lysis of the cells by 5% Triton X-100. Assay of IFN-,y by EIA. Gamma interferon (IFN--y) was quantitated by enzyme immunoassay (EIA) as described by

INFECT. IMMUN.

Firestein et al. (7) with some modifications. EIA plates were coated with 1.5 ,ug of rat anti-murine IFN--y monoclonal antibody (LEE Biomolecular Research Inc., San Diego, Calif.). After the plates were washed and blocked, serial twofold dilutions of samples and standard murine IFN--y were applied to the wells. After 90 min of incubation, the wells were washed and then incubated with 100-fold-diluted rabbit anti-IFN--y polyclonal sera for 90 min, followed by incubation with peroxidase-conjugated goat anti-rabbit immunoglobulin G (Cappel, Organon Teknika Corp., West Chester, Pa.). Finally, o-phenylenediamine in phosphatecitrate buffer with H202 was added as substrate solution, and the reaction was terminated with H2SO4. The A492 was measured, and IFN--y activity was expressed as international units per milliliter determined against a known reference standard. Murine recombinant IFN--y and rabbit anti-IFN--y polyclonal antibody were generous gifts from the Research Institute, Daiichi Seiyaku Co. Ltd., Tokyo, Japan. Assay of MCF activity. Macrophage-chemotactic factor (MCF) activity was assessed by the method of Synderman et al. (44) with some modifications. Culture supernatant in a 1:1 dilution with RPMI 1640 supplemented with 10% fetal calf serum (GIBCO) was placed in the lower wells of blind-well chemotaxis chambers. A polycarbonate membrane filter with 5-,um pores (Nuclepore Corp., Pleasanton, Calif.) was placed between the upper and lower wells. As indicator cells, PEC were collected from mice injected intraperitoneally with 1.5 ml of proteose peptone 3 days before the assay. The cells were washed three times with Hanks balanced salt solution and suspended to give 6 x 105/ml in RPMI 1640 supplemented with 10% fetal calf serum, and 500 RI of the cell suspensions was added to the upper well. After 90 min of incubation in a CO2 incubator, the filters were removed, washed, and air dried. They were stained with Giemsa solution, and the number of migrated macrophages was counted in five randomly selected high-power fields. Assessment of DTH. DTH was assessed by DFR. Six days after immunization with viable or killed L. monocytogenes, the mice were examined for DFR. For the elicitation of DFR, 108 killed bacteria in 0.05 ml of phosphate-buffered saline were injected into the left hind footpad. The thicknesses of the treated footpad and of the contralateral footpad were measured with a dial-gauge caliper 24 h after the elicitation. The difference in the thickness of the right and left footpads was expressed in 0.1-mm units as DFR. Assessment of ACR. In order to determine protection against challenge infection, immune mice were challenged by an intravenous injection of 105 viable L. monocytogenes cells. Three days after the challenge, the spleens were removed and homogenized, and the suspensions were spread on agar plates after being serially diluted. The numbers of bacteria were enumerated by counting the colonies and were expressed as log1o CFU per organ. Local transfer of DFR and ACR with lymphoid cells. Local cell transfer was carried out as described previously (31) with slight modifications. Briefly, nylon wool-nonadherent spleen cells were obtained from mice 6 days after immunization. The cells (7.5 x 106) were mixed with 7.5 x 107 heat-killed listeriae in 0.05 ml of Hanks balanced salt solution and were injected into the left hind footpads of naive recipient mice, and the degree of DFR was recorded 24 h later. Immediately after the measurement of DFR, 105 viable L. monocytogenes cells were inoculated into the same footpad. The number of bacteria in the footpad was determined 24 h after the challenge by plating the serially diluted homogenates of the foot on agar plates. Thus, ACR was

TABLE 1. Proliferative response of splenic T cells from mice immunized with viable or killed L. monocytogenes after stimulation with specific antigen or recombinant IL-2

TABLE 3. Effect of treatment of mice with recombinant IL-la on the proliferative response of splenic T cells to specific antigen or recombinant IL-2 [3H]thymidine uptake by T cells (102 cpm) in response to: rIL-2c Specific antigenb

[3H]thymidine uptake by T cells (102 cpm) in response to: o*

Days after immunization

Immunization Immuniation

Specific

antigena

None Killed bacteria Viable bacteria Killed bacteria Viable bacteria

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PROMOTION OF CELL-MEDIATED IMMUNITY BY rIL-la

VOL. 58, 1990

8.3 ± 1.1

18.1 11.0 13.3 68.3

3 3 6 6

± ± ± ±

4.0 5.6 2.8 6.9c

Immunization

Treatment with

None Killed bacteria None

-

rIL-2b

20.1 ± 26.1 ± 92.7 ± 23.3 ± 229.3 ±

2.6

4.2

6.0c 3.0 14.0c

Killed bacteria Viable bacteria

4.4 3.0 6.3 28.1 13.2

+ + -

1.2

± 0.6 ± 0.9 ± 4.5d ± 3.2e

10.2 7.2 6.9 25.9 177.0

±

2.1

± 1.3 ± 1.0 ± 3.1e ±

9.6d

Mice were intraperitoneally injected with 2 1±g of rIL-la 1 and 24 h after immunization. b T cells (106/ml) were stimulated with killed bacterial cells (108/ml) in the presence of 10%o APC. ' T cells (106/ml) were stimulated with 0.1 U of rIL-2 per ml. d p < 0.001 compared with the value for the control group. e p < 0.005 compared with the value for the control group. a

a T cells (106/ml) were stimulated with 108 killed bacterial cells per ml in the presence of 10% APC. b T cells (106/ml) were stimulated with 0.1 U of rIL-2 per ml. c P < 0.001 compared with value for the control group.

measured by the capacity to eliminate challenge bacteria at the site of cell transfer. Statistics. Results were expressed as means + standard deviations. Statistical significance of the data was determined by Student's t test. A P value of less than 0.05 was considered to be significant. RESULTS Immunization with killed or viable L. monocytogenes and development of T-cell proliferation upon stimulation with specific antigen or rIL-2. Mice were immunized with viable or killed L. monocytogenes, and proliferative responses of splenic T cells to stimulation with antigen in the presence of appropriate percentages of APC were measured 3 and 6 days after the immunization. Although spleen cells from mice immunized with viable bacteria showed the proliferative response on day 6, no proliferation was observed in spleen cells from mice immunized with killed bacteria (Table 1). When stimulated with rIL-2, spleen cells from mice immunized with viable bacteria showed a proliferative response as early as 3 days after immunization. A highly significant level of response was observed in spleen cells obtained 6 days after immunization. On the other hand, no significant level of proliferation was observed in spleen cells from mice immunized with killed bacteria even after 6 days (Table 1). IL-2 production by T cells upon stimulation with specific antigen. Since antigen-specific mature T cells are capable of secreting IL-2 after stimulation with specific antigen in the presence of APC (17), we examined the IL-2 activity in the culture supernatant of spleen cells stimulated with killed bacterial antigen. Spleen cells obtained from mice 6 days

after immunization with viable bacteria produced a high level of IL-2, while cells from killed bacteria-immune mice did not (Table 2). These results suggested that there was a difference in the stage of maturation between T cells from mice immunized with viable L. monocytogenes and those from mice immunized with heat-killed bacteria. Effect of treatment of mice with rIL-laL on T-cell functions. In a previous study, we found that there was a difference in the ability to stimulate IL-1 production by macrophages between viable and killed cells of L. monocytogenes (29). It was plausible that functional maturation of T cells, which is necessary for the expression of DTH or protection, might be arrested when immunization was done with killed bacteria, because of an insufficient production of IL-1 in vivo; therefore, we investigated whether in vivo administration of rIL-la facilitates the maturation of antigen-specific T cells. When mice were given rIL-la after immunization with killed L. monocytogenes, splenic T cells became capable of showing a high level of proliferative response against specific antigen and rIL-2 (Table 3). They could also produce IL-2 upon stimulation with specific antigen (Table 4). HT-2 cells used in this study responded to rIL-2 very well in a dosedependent manner, while they never showed any significant proliferative response to up to 200 U of rIL-4 per ml, suggesting that the activity in the supernatant could be attributed mainly to IL-2. T cells having such a responsiveness were never generated in mice after in vivo administration of rIL-la without immunization with killed bacteria. Effect of treatment of mice with rIL-la on generation of DFR and ACR to L. monocytogenes. Since spleen cells from

TABLE 2. IL-2 activity in the supernatant of immune splenic T cells stimulated with killed L. monocytogenes [3H]thymidine uptake by HT-2 cells (103 cpm) in: Immunization Immunization

Days after immunization

Culture

Medium only

supernatant diluted': 1/4

1/8

1/2

0.3 0.2 0.4 0.4 4.0c

0.9 1.1 0.9 0.5 65.9 ± 2.9c

a IL-2-dependent HT-2 cells (104) were cultured in the presence of diluted supernatant of a 2-day-old culture of splenic T cells (2 x (2 x 108/ml) and PEC (5%). b p < 0.005 compared with the value for the control group. c P < 0.001 compared with the value for the control group.

106/ml) with killed bacteria

None Killed bacteria Viable bacteria Killed bacteria Viable bacteria

3 3 6 6

3.3 2.4 1.8 2.2 2.6

± ± ± ± ±

0.1 0.2 0.4 0.2 0.5

6.2 5.2 4.5 4.1 13.1

0.7 0.5 0.7 0.7 ± 2 0b ± ± ± ±

7.3 6.6 5.7 6.2

± ± ± ±

37.6 ±

7.8 8.5 7.5 8.6

± ± ± ±

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INFECT. IMMUN.

IGARASHI ET AL. TABLE 4. Effect of treatment of mice with recombinant IL-la on antigen-induced production of IL-2 in the culture supernatant of splenic T cells Treatment rIL-1aawith

Immunization

Medium only

[3H]thymidine uptake by HT-2 cells (103 cpm) in: Supematant dilutedb:

None Killed bacteria None Killed bacteria Viable bacteria

2.2 1.5 1.3 1.2 1.8

+ + -

± ± ± ± ±

0.5 0.2 0.1 0.3 0.1

3.4 2.4 2.4 8.9 12.4

± ± ± ± ±

1/2

1/4

1/8

2.9 2.4 2.4 21.6 28.1

0.6 0.4 0.2 0.9c 1.5c

± ± ± ± ±

2.9 4.3 3.2 45.7 54.7

0.3 0.1 0.4 1.1C l.9c

± 0.1 ± 0.6

± 0.2 ± 3.6c ± 3.7c

a Mice were intraperitoneally injected with 2 ,ug of rIL-la 1 and 24 h after immunization. b IL-2-dependent HT-2 cells (104) were cultured in the presence of the diluted supernatant of a culture of nylon wool-passed T cells (2 x 106/ml) cultured for 2 days with killed bacterial cells (2 x 108/ml) and APC (5%). c P < 0.001 compared with the value for the control group.

mice treated with rIL-lao after immunization with killed L. monocytogenes acquired the ability to respond to specific antigen or rIL-2 in vitro, we next examined the effect of rIL-lot-administration on the expression of DTH and ACR in vivo. A significant degree of DFR was observed in mice immunized with viable listeriae but not in mice immunized with killed bacteria or treated only with rIL-la. On the other hand, in mice treated with rIL-la after immunization with killed listeriae, a positive reaction was observed (Table 5). In this group of mice, ACR, as determined by the elimination of challenge bacteria, was also expressed; however, it was unlikely that T cells contributing to ACR were actually generated since a similar enhancement of bacterial elimination was observed in the group given rIL-lot only. There was a possibility that the treatment with rIL-lcx resulted in the enhancement of nonspecific antibacterial resistance, as reported elsewhere (3, 39). Local transfer of DFR and local resistance. In order to determine the possible contribution of rIL-lo- to T-cell maturation at a cellular level, we employed local transfer of immune spleen cells. In this system, it was possible to assess the transferability of both DFR and local protection (31). Nonadherent immune cells were transferred into the footpads of naive recipient mice along with the eliciting antigen. When spleen cells from mice immunized with viable L. monocytogenes were transferred, significant levels of DFR and protection were detected at the site of cell transfer. Spleen cells from mice immunized with killed bacteria or from those treated only with rIL-lot never conferred any

DFR or local resistance. In contrast, when spleen cells from mice treated with rIL-lot after immunization with killed bacteria were transferred, a positive DFR was expressed (Table 6). However, bacterial elimination at the site of cell transfer was not enhanced by the same splenic T cells (Table 7). These results indicated that treatment with rIL-lot after immunization with killed L. monocytogenes generated the effector cells mediating DTH but not ACR. Lymphokine activity in culture supernatants. Among various T cell-derived lymphokines, MCF and MAF/IFN-y are of primary importance in the expression of DTH or protection against L. monocytogenes (11, 31). To determine the functional maturation of T cells with respect to lymphokine production, T cells from each group of mice were examined for the ability to produce MCF or MAF/IFN--y upon stimulation with specific antigen. Spleen cells from mice immunized with viable L. monocytogenes were capable of producing both MCF and MAF/IFN--y. Spleen cells from mice treated with rIL-lot after immunization with killed bacteria did not produce MAF/IFN--y, but these cells produced a higher level of MCF than those from mice immunized with killed bacteria alone or from mice treated only with rIL-la (Table 8).

TABLE 5. Effect of treatment of mice with recombinant IL-la on DFR and ACR to L. monocytogenes

TABLE 6. Effect of treatment of mice with recombinant IL-lot on ability of splenic T cells to transfer DFR

Immunization Immuniation

Treatment with rIL-1ca'

None Killed bacteria None Killed bacteria Viable bacteria

-

+ +

-

b

DFRmm) (0.1

2.4 2.4 3.4 4.3 6.3

Logl0 CFU in spleen'

5.85 6.18 4.45 ± 1.lf 5.67 1.5f 2.24 + 0.3 ± 0.4 ± 0.8

± 0.31

± 0.05 ± 0.21e

± 0.33 ±

0.49e

Logl0d ACR 0 -0.33 +1.40 +0.18 +3.61

a Mice were intraperitoneally injected with 2 ,ug of rIL-la 1 and 24 h after

immunization. b DFR was elicited 6 days after immunization and was measured 24 h after the elicitation. c Mice were intravenously challenged with 105 viable L. monocytogenes cells, and the numbers of CFU in the spleens were determined 3 days later. Each group consisted of four mice. d CFU in experimental group - loglo CFU in control group. e p < 0.001 compared with the value for the control group. f P < 0.005 compared with the value for the control group.

Log1o

DISCUSSION Protective immunity to facultative intracellular bacteria is mediated by specific T lymphocytes. Immunization with viable L. monocytogenes generated Listeria-specific T cells

DFR (0.1 mm)a

T cells from mice: Immunized with:

Treated with

Nothing Killed bacteria

-

Nothing Killed bacteria Viable bacteria

+ +

rIL-1ab

-

Expt 1

3.2 2.3 3.1 5.6 6.5

± ± ± ±

0.6 0.8 0.5

1.9c

± 1.4d

Expt 2

7.0 7.3 5.8 9.9 11.8

± ± ± ± ±

1.9 1.7 2.4 1.8c 3.2d

a Nylon wool-passed spleen cells (7.5 x 106) were injected into the footpads of naive recipient mice along with killed bacterial cells (7.5 x 107), and DFR was measured 20 h after the elicitation. b Mice were intraperitoneally injected with 2 p.g of rIL-la 1 and 24 h after immunization. I P < 0.05 compared with the result for mice to which control cells were transferred. d p < 0.005 compared with the value for the control group.

PROMOTION OF CELL-MEDIATED IMMUNITY BY rIL-la

VOL. 58, 1990

TABLE 7. Effect of treatment of mice with recombinant IL-la on ability of splenic T cells to transfer local resistance to L. monocytogenes T cells from mice:

Loglo CFU in footpada

Immunized

wmmunith:

Treated with

Nothing Killed bacteria Nothing Killed bacteria Viable bacteria

-

rIL-lab

Expt 1

5.53 5.61 5.58 5.52 4.84

+ + -

Expt 2

± 0.20

5.99 ± 0.12 6.12 ± 0.05 5.58 ± 0.11 5.70 ± 0.21 4.73 ± 0.19d

± 0.15

± 0.20 ± 0.19 ± 0.27c

a Viable L. monocytogenes cells (105) were locally injected into the footpads, and the numbers of CFU in footpads were determined 20 h later. b Mice were intraperitoneally injected with 2 plg of rIL-la 1 and 24 h after immunization. c P < 0.01 compared with the value for the control group. d p < 0.001 compared with the value for the control group.

capable of responding to rIL-2 and specific antigen and producing IL-2 after antigenic restimulation in vitro. On the other hand, T-cell responsiveness was never generated by immunization with killed bacteria. Recently, we found that the ability of L. monocytogenes to stimulate macrophage IL-1 production was impaired when bacteria were killed and the development of T-cell maturation was arrested at the initial stage after immunization with killed bacteria (29). These findings suggested that the insufficient production of IL-1 in vivo was responsible for the ineffectiveness of killed bacteria in inducing protective immunity. In the present study, it was shown that treatment of mice with rIL-la actually generated the proliferative response and IL-2 secretion by T cells from mice immunized with killed listeriae. Administration of rIL-la after immunization with killed bacteria could promote the functional maturation of T cells, which was never achieved without rIL-la. The in vitro functions of T cells thus obtained were similar to those of T cells from mice immunized with viable listeriae. These results implied the importance of IL-1 for genetrating Listeria-specific effector T cells in vivo. T cells from mice treated with rIL-la after immunization TABLE 8. Effect of treatment of mice with recombinant IL-la on antigen-induced production of lymphokines by splenic T cells Immunization

Treatment with rIL-loa

MCF actiVityb (no. of migrated macro-

phages)

None Killed bacteria None Killed bacteria Viable bacteria

+ +

-

72.8 70.0 105.3 117.0 128.3

± 7.3 ± 10.1 ± 6.5

± 2.2e ± 13.1e

a Mice were intraperitoneally injected with 2

immunization.

MAF acity MA sctiityc release)

5'Cr

11.2 4.9 2.6 8.8 60.5

± ± ± ±

4.0 0.7 1.2 2.1

± 7.4f

IFN_yd (IU/ml)

Interleukin-1-induced promotion of T-cell differentiation in mice immunized with killed Listeria monocytogenes.

We studied the effects of administration of recombinant interleukin-1 alpha (rIL-1 alpha) to mice after immunization with killed Listeria monocytogene...
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