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beta interferons and gamma interferon produced in mice infected with Listeria monocytogenes
CELLULAR
IMMUNOLOGY
88,29-40
(1984)
The Significance of Alpha/Beta lnterferons and Gamma Interferon Produced in Mice Infected with Listeria monocytogenes AKIO NAKANE AND TOMONORI MINAGAWA Department of Microbiology, Hokkaido University School ojh4edicine, Kita 15 Nishi 7, Kita-Ku, Sapporo 060, Japan Received January 30. 1984; accepted April 25, 1984 Interferon (IFN)-a//B was induced in the circulation of mice infected intravenously with Lisferia monocytogenes 24 to 72 hr aher infection, but was not induced by the administration of heat-
killed Listeria, listerial cell wall fraction (LCWF), or iisterial soluble fraction. Appearance of IFN-a/p showed a pattern similar to that of the growth of bacteria in the spleen and the liver of mice. IFN-@I production was abrogated by pretreatment of mice with anti-asialo GM1 antibody, antithymocyte serum, or hydrocortisone, but not with cyclophosphamideor carrageenan. Such treatments which suppressedIFN-o/p production did not influence bacterial growth in the organs of mice in the early stage of Listeria infection. Administration of IFN-(u//3 exogenously also did not. After 5 days of infection when the specific resistanceagainst reinfection with Lisferia was established,IFN-+r but not IFN-(r//3 was induced in the circulation 3 to 6 hr after stimulation with LCWF or reinfection with Listeria. IFN--r production was abrogated completely by cyclophosphamide and antithymocyte serum, and partially by hydrocortisone and carrageenan, but not by anti-asialo GM1 antibody in Listeria-infected mice treated with these agents before induction of IFN-7 by LCWF. Presumably, IFN-a/b might be produced by asialo GM 1-bearing cells but IFN-y might not. However, IFN-7 production was suppressedin Listeria-infected mice, when IFN-c&3 production had been inhibited by treatment with anti-asialo GM1 antibody or when the IFN produced had been neutralized with anti-mouse IFN-a/P antibody. Therefore, it is conceivable that IFN-a/6 might be essential for the generation or the expression of antigenspecific T cells involving EN-7 production and acquired resistanceduring Listeria infection. In fact, the bacterial growth in the organs of mice in the early stage of infection was normal in IFN-culfidepleted mice but it resulted in the delay of T-celldependent elimination of bacteria from the organs of mice in the late stage.
INTRODUCTION The major host resistance to facultative intracellular bacteria, such as Listeriu monocytogenes,Salmonella typhimurium, and Mycobacterium bovis BCG, is mediated by cellular factors but not by the serum antibody (l-3). Among these, only Listeria has been studied in detail. Listeria infection is eliminated from the tissues by macrophages acting in two steps:during the first 2 or 3 days after infection, mononuclear phagocytes, which are activated by a T-cell-independent mechanism other than complement, protect the murine host against explosive multiplication of bacteria (4-7). Thereafter, virtual elimination of Listeria is performed by macrophages which are activated by some mediators released from sensitized T cells over the next 4 to 5 days (8- 12). Recently, our understanding of the defensemechanisms against Listeria 29 0008~8749184$3.00 Copyright @ 1984 by Academic FXSS, Inc. All rights of reproduction in any form reserved.
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NAKANE
AND MINAGAWA
infection was augmented by the information that natural killer (NK) activity in murine peritoneal exudate cells (PEC) increased Days 2 through 6 after infection (13). Therefore, it is conceivable that macrophages, T cells, and NK cells may form the framework of the major cellular defensemechanisms and these cells may produce some mediators which activate each other in order to eliminate Listeria from the tissues. Actually, it was demonstrated that PEC from Listeria-infected mice could produce interleukin 1 (IL-l), interleukin 2 (IL-2), and interferon (IFN)-y by stimulation with the specific antigen (14-16). There are three antigenic types of IFN, referred to as a, fl, and y in both human and murine systems (17). In the murine system, IFN-cy//3 production is induced nonspecifically not only by viral infections but also by other microbial infections involving bacteria ( 18), Mycoplasma ( 19), Rickettsia (20), Chlamydia (21), or Protozoa (22). In contrast, IFN-7 can be produced by stimulation with specific antigen in the immunized mice (23) and is classified as one of the lymphokines. Hence, the host’s immunological status can be estimated by determining which type of IFN is induced. However, neither the IFN-(Y/P nor the IFNy production mechanism has been studied independently in viva In the present study, we demonstrate that IFN-a//3 and IFN-7 can be produced alternatively in the circulation and that the speciesproduced reflectsthe immunological status of the murine host during Listeriu infection. IFN-(u/p is produced in the primary infection by the cells sensitive to anti-asialo GM1 antibody, while induction of IFN-y, by stimulation with specific antigen or by reinfection, is abrogated completely by pretreatment of mice with antithymocyte serum or cyclophosphamide. We also demonstrate that IFN-(u//3 produced in the early stage of Listeriu infection may not participate in T-cell-independent elimination of bacteria in the early stage but that suppression of IFN-(u/p production by pretreatment of mice with anti-asialo GM1 antibody or neutralization of the IFN-(u/b produced endogenously by anti-IFN-c*l/& antibody administration results in both suppression of IFN-7 production and the delay of T-cell-dependent elimination of bacteria from the organs of mice in the late stage of infection. MATERIALS AND METHODS Mice. Female ddY mice 5 to 7 weeks of age were purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Hamamatsu, Shizuoka, Japan). Infection with bacteria. Listeria monocytogenes lb strain 1684, kindly provided by Dr. T. Nagai of the Department of Microbiology, Sapporo Medical College Hospital, Sapporo, were prepared as previously reported (24). The concentration of washed cells was adjusted spectrophotometricahy (at 550 nm). Mice were infected intravenously with 0.2 ml of 1 X lo4 colony-forming units (CFU) of viable Listeria in 0.01 M phosphate-buffered saline (PBS, pH 7.4). The numbers of viable organisms in the spleen and the liver of infected animals was established by plating serial tenfold dilutions of the organ homogenate in PBS on trypticase soy agar (BBL, Cockeysville, Md.) containing 0.3% glucose. The colony counts were routinely performed 18 to 24 hr later. Preparation of heat-killed Listeria and listerial cell components. Heat-killed Listeria (HK-LM) and Listeriu soluble fraction (LSF) were prepared as previously reported (24). Listeriu cell wall fraction (LCWF) was prepared as follows: the precipitate of
INTERFERONS
AND
Listeria INFECTION
31
the sonically disrupted organisms obtained by differential centrifugation (24) was washed three times with sterile, nonpyrogenic, double-distilled water. The LCWF obtained was lyophilized, weighed, reconstituted with sterile, nonpyrogenic 0.85% saline to a concentration of 2 mg/ml, and autoclaved. All preparations were carried out under aseptic conditions. HK-LM, LCWF, and LSF were stored at -70°C. When used for injections, these preparations were diluted with 0.01 M PBS. Drug treatment. Mice were injected intraperitoneally with single doses of 240 mg/kg of cyclophosphamide (Sigma Chemical Co., St. Louis, MO.) (25), 200 mg/kg of hydrocortisone acetate (Wako Pure Chemical Ind., Osaka, Japan) (26), or 240 (Wako) (27) in 0.5 ml of 0.0 1 M PBS 48 hr before infection mg/kg of K-Carrageenan with Listeria or stimulation with LCWF. Antithymocyte serum treatment. Rabbit anti-mouse thymocyte serum (0.25 ml; M. A. Bioproducts, Walkersville, Md.) was injected into mice intraperitoneally twice (48 and 24 hr before Listeria infection or stimulation with LCWF) (26). Normal rabbit serum was injected into the other group of animals as a control in the same manner. Anti-asialo GM1 antibody treatment. GM1 was isolated from porcine brain by a combination of anion-exchange chromatography and high-performance adsorption chromatography (28, 29). Asialo GM1 was prepared from GM1 by treatment with 1 N formic acid at 100°C for 1 hr (30) and then purified by chromatography on diethylaminoethyl (DEAE)-Sephadex A-25 (Pharmacia Fine Chemicals, Uppsala, Sweden) and Iatrobeads 6RS8060 (Iatron Laboratory, Inc., Tokyo, Japan). New Zealand rabbits were immunized with asialo GM1 dispersed in 0.85% saline containing methylated bovine serum albumin (Sigma) and emulsified with complete Freund’s adjuvant asdescribedbefore (3 1). The titer of anti-a&lo GM 1 antibody was determined by enzyme-linked immunosorbent assay (ELISA) according to the method reported elsewhere(32). A globulin portion of the antibody was obtained by precipitation with 50% saturated ammonium sulfate. Anti-asialo GM 1 globulin (0.2 ml; diluted 1:3) was injected intravenously 48 hr before Listeria infection or stimulation with LCWF (33). In the control animals, rabbit mock globulin obtained from rabbits immunized with only methylated bovine serum albumin emulsified with complete Freund’s adjuvant was injected instead of the antibody. Anti-mouseIFN-cx/@antibody treatment.The globulin portion of anti-mouse L-cell IFN-(r//3 antibody (Catalog No. G-024-501-568) was kindly provided by Dr. G. J. Galasso of The Microbiology and Infectious Disease Program, National Institute of Allergy and Infectious Diseases.Anti-mouse IFN-(r//3 sheep globulin (0.2 ml) that had been adjusted to the IFN-neutralizing titer of 10,000 U/ml with serum-free RPM1 1640 medium (GIBCO, Grand Island, N.Y.) was injected into the tail vein of mice after Listeria infection. One neutralizing unit of the antibody can neutralize 8 to 10 international interferon units (IU) of mouse IFN-(r//3. Control animals were treated the same amount of sheep globulin to mock L-cell IFN-a/P (Catalogue No. G-025501-568) in the same manner. Administration of mouseIFN-(Y/P.Partially purified Newcastle diseasevirus-induced mouse L-cell IFN-(Y/P (sp act: 9 X lo6 IU/mg) was kindly provided by Dr. S. Kobayashi of Torey Basic Institute, Kamakura. Mice were given intravenously 0.5 ml of mouse IFN-(u//3 containing 1 X IO5 IU. Determination of protein. Protein contents were determined by the method of Lowry et al. (34).
NAKANE
32
AND MINAGAWA
Assays and characterization of ZFN. The IFN samples were pooled sera obtained from at least four mice in each group. The IFN assay was carried out by the dyebinding method (35) using mouse L-929 cells and vesicular stomatitis virus (Indiana strain) as previously reported (36). Neutralization tests using anti-mouse IFN-a//3 antibody and acid stability of IFN samples were carried out as reported elsewhere (37). RESULTS ZFN Response in the Circulation of Mice by the Primary Infection of Listeria After mice were infected intravenously with 1 X lo4 CFU of Listeria, the antiviral activities of the pooled sera obtained from four or five mice of each group were followed at various times. As shown in Fig. la, no antiviral activity was detected in the sera until at least 16 hr after infection. The antiviral activity appeared at 24 hr and was maintained until 72 hr after infection. All antiviral activities were completely neutralized with anti-mouse IFN-a/p antibody (Fig. la), and were not reduced by pH 2.0 treatment (data not shown). On the other hand, the growth of Listeria in the spleens and the livers reached a maximum at 48 hr and then declined (Fig. lb). We also studied the possibility that IFN could be produced in the circulation of mice injected with killed Listeria (HK-LM) or listerial cell components (LCWF and LSF). No antiviral activity was demonstrated when mice were injected with HK-LM, LCWF, or LSF at any period tested (Table 1).
111,
36916
II
2432
I
I
I
1
48
72
96
120
HOURS AFTER LISTERIA INFECTION
FIG. 1. Kinetics of IFN production in the circulation and bacterial growth in spleensand livers following Listeriu infection. Mice weTe infected intravenously with 1 X 104CFU of viable Listeriu, and sera were
collected at various times. IFN titer before (0) and after (0) treatment with anti-IFN-ol/fi antibody (a). At the same time, the numbers of bacteria in spleens (0) and livers (0) were estimated (b).
33
INTERFERONS AND Listeriu INFECTION TABLE I IFN Response in the Circulation of Mice Injected with Different Preparations of Lisferiu IFN titer (IU/ml) Hours after injection”
Viable LM
HK-LM
LSF
LCWF
3 6 24 48 12
<4 <4 40 50 20
<4 <4 <4 <4 <4
14 <4 <4 <4 <4
<4 <4 <4 <4 <4
’ The sera were collected at the specified times after mice were injected intravenously with 1 X lo4 CFU of viable Listeria, 5 X lo6 cells of HK-LM, 1.9 mg protein of LSF, or 75 pg of LCWF.
Efects of Immunosuppressive Agents on IFN Response to the Primary Infection of Listeria The results of the IFN response to the primary infection of bacteria in mice which had been treated with various immunosuppressive agents are summarized in Table 2. IFN-a/(3 production was completely abrogated in mice pretreated with hydrocortisone. Similarly, anti-asialo GM1 antibody rather than antithymocyte serum sup TABLE 2 Effects of Various Immunosuppressive Agents on the IFN Response to the Primary Infection of Listeria IFN titer (IU/ml)b Expt
Induced
After neutralization with anti-IFN-a/p
None Cyclophosphamide Hydrocortisone Carrageenan
120 210 <4 100
10 10 ND‘ <4
None Normal rabbit serum Antithymocyte serum
95 110 40
<4 <4 <4
None Mock anti-asialo GM 1 globulin Anti-a&lo GM I globulin
130 130 35
15 10 <4
Treatment’
’ Mice were injected intraperitoneally with single doses of each drug: 240 mg/kg of cyclophosphamide, 200 mg/kg of hydrocortisone, or 240 mg/kg of catrageenan. Also other groups of mice were injected intravenously with 0.2 ml of rabbit anti-asialo GM1 globulin (diluted 1:3, approximately 800 units in ELISA) or the same amount of rabbit mock anti-asialo GM1 globulin. Thesemice were infected intravenously with 1 X lo4 CFU of viable Lisferiu 48 hr later. Antithymocyte serum or normal rabbit serum (0.25 ml) was injected intraperitoneally 48 and 24 hr before bacteriaI infection. b Sera were collected 48 hr after Listerin infection, and then IFN activities and the antigenicity of the specimens were determined. ’ Not done.
34
NAKANE
AND MINAGAWA
pressed IFN induced by List&a infection. However, IFN-a/B production was not affected by treatment with cyclophosphamide or carrageenan. IFN-y Production in Listeria-Infected Mice Stimulated with the Specific Antigen or Reinfected with Bacteria Though no IFN activity was induced when uninfected mice were injected with LCWF, we assumed that the IFN response to the specific antigen would occur in mice after the specific immunity was established. LCWF (50 pg) was injected intravenously into mice on Day 6 of Listeria infection, and IFN activity in the pooled sera was determined at various times (Fig. 2). IFN activity appeared 3 hr and peaked 6 hr after stimulation. Thereafter the activity decreasedand disappeared 10 hr after injection with LCWF. The neutralization test was carried out using anti-mouse IFN-cr//3 antibody, and IFN activities produced at any period were not affected by the treatment (Fig. 2). Treatment with pH 2.0 buffer resulted in complete inactivation of the IFN activity (Fig. 2). We examined which type of IFN could be induced by reinfection with viable Listeria. On Day 6 of infection, mice were reinfected intravenously with 1 X lo5 CFU of Listeria, and the sera were collected 3, 6, 24, and 48 hr later. As shown in Table 3, IFN activity was demonstrated in the circulation 3 hr after reinfection and peaked at 6 hr. The IFN produced in the sera by reinfection was of the y type as was that produced in mice injected with LCWF. No antiviral activity was demonstrated 24 or 48 hr after reinfection with Listeria different from the primary infection. The IFN response to the specific antigen occurred in mice on Day 5 of infection, and the maximum response was demonstrated on Day 7 (Fig. 3). Thereafter, the IFN response gradually decreased, and no antiviral activity was seen in the sera when mice were stimulated with LCWF on Day 21 of infection. IFN activities induced by LCWF on Days 5 to 14 of infection were not neutralized with anti-mouse IFN-a/@ antibody (Fig. 3) and were labile at pH 2.0 (data not shown). When mice were sensitized with various dosesof HK-LM instead of viable Listeria, no IFN responsewas observed in the circulation of mice on Day 6 of administration of any dose of HK-LM (data not shown).
HOURS AFTER INJECTION WITH LCWF FIG. 2. Kinetics of IFN production in the circulation following injection with LCWF in List&z-infected mice. LCWF (50 pg) was injected intravenously into mice on Day 6 of Listeriu infection, and sexawere collected at various times. IFN titer before (0) and after (0) treatment with anti-IFN-43 antibody, and after pH 2.0 treatment (0).
35
INTERFERONS AND Listeria INFECTION TABLE 3 The IFN Response in the Circulation of Mice after Reinfection with Listeria IFN titer (IU/ml) Hours after stimulation
Stimulus” Infected mice Viable LM
LCWF
Uninfected mice Viable LM
Induced
After neutralization with anti-IFN-cY/Bb
3 6 24 48
55 80 <4 <4
50 80 ND’ ND
3 6 24 48
240 5100 <4 <4
240 5100 ND ND
3 6 24 48
<4 <4 40 50
ND ND <4 14
’ Viable Listeria (1 X 10’ CFU) or LCWF (50 ~~39) was injected intravenously into mice on Day 6 of Listeriu infection. Uninfected mice were injected with 1 X IO4CFU of viable Listeriu as a control. bThe neutralization test was carried out as described in the text. ’ Not done.
Efects of Various Immunosuppressive IFN-7
Agents on Antigen-SpeciJic
Induction
of
Mice were infected with Listeria and treated with various immunosuppressive agents. The animals were stimulated with LCWF on Day 6 of infection, and IFN titers in the circulation were determined 6 hr later (Table 4). Both cyclophosphamide
1600 t ‘3 x >
800
; 400 c” z 200 h -
100
-i/‘; f
3 5
7 9 II DAYS AFTER LISTERIA
A 14 21 INFECTION
RG. 3. IFN response to stimulation with specific antigen in the circulation of mice during Listeria infection. After mice were infected intravenously with 1 X lo4 CFU of Listerin, 50 pg of LCWF was injected intravenously into mice on different days after Listeriu infection. The sera were collected 6 hr later. IFN titer before (0) and after (0) treatment with anti-IFN-cY/@antibody.
36
NAKANE
AND MINAGAWA TABLE 4
Effects of Various Immunosuppressive Agents on Antigen-Specific Induction of IFN in Wisteria-Infected Mice IFN titer (IU/ml)” Expt
Treatment”
Induced
After neutralization with anti-IFN-a/b
I
None Cyclophosphamide Hydrocortisone Carrageenan
3200 <16 450 630
3200 NDC 450 630
2
None Normal rabbit serum Antithymocyte serum
2470 2520 <16
2450 2520 ND
3
None Mock anti-asialo GM I globulin Anti-asialo GM I globulin
2600 2530 2300
2600 2530 2300
’ Mice were infected intravenously with 1 X 10’ CFU of viable Listeriu. The animals were treated with cyclophosphamide, hydrocortisone, carrageenan,anti-asialo GM I globulin, or rabbit mock anti-asialo GM I globulin on Day 4 of infection, or with antithymocyte serum or normal rabbit serum on Days 4 and 5 of infection, as described in Table 2. These mice were stimulated with 50 a of LCWF on Day 6 of infection. ’ Sera were collected 6 hr after injection with LCWF, and then IFN activities and the antigenicity of the specimens were determined. ’ Not done.
and antithymocyte serum completely suppressed IFN-y production. The IFN-y responsewas also suppressedin hydrocortisone- or carrageenan-treated mice. However, a normal IFN-7 responseto LCWF was observed in anti-asialo GM 1 antibody-treated animals.
Signijcance of Early EndogenousIFN-CY/@ on the Expressionof Late IFN-y Response in Listeria-Infected Mice The above results demonstrated that IFN-a/B production by the primary infection of Listeria was suppressed in asialo GM1 antibody-treated mice (Table 2) but IFN-7 production was not affected when mice were treated with the antibody before stimulation with LCWF (Table 4). We investigated the IF%y response in mice in which IFN-cr//3 production had been suppressed (Table 5). Mice were treated with anti-a&lo GM1 antibody 2 days before Listeria infection, and IFN-y production stimulated with the specific antigen was estimated on Day 6 of infection. IFN-y production was significantly suppressedin the antibody-treated animals. Furthermore, IFNy production was completely suppressed in Listeria-infected mice in which IFN-(u/p produced endogenously had been depleted by administration of anti-mouse IFN-cu//3antibody (Table 5). No suppression of IFN-y production was observed when anti-mouse IFN-(Y/B antibody was injected soon after stimulation with the specific antigen (data not shown).
Role of IFN+3 on Elimination of Bacteriafrom the Organsof Listeria-InfectedMice It was demonstrated that IFN-(u//l produced in the early stageof Listeria infection would be necessary for the establishment or the expression of the EN-y-producing
INTERFERONS AND Lisferiu INFECTION
37
TABLE 5 The IFN-7 Response to Specific Antigen in Lisle&Infected IFN-o//3 Production Was Suppressed
Mice in Which
IFN titer (RI/ml) Induced
After neutralization with anti-IFN-a//3
None
2200
2200
Mock anti-a&lo GM I globulin Anti-asialo GM 1 globulin
3000 400
3000 400
Mock anti-IFN globulin Anti-IFN-a/B globulin
2400
2400
Treatment a
’ Mice were injected intravenously with 0.2 ml of anti-asialo GM1 globulin (diluted 1:3) or the same amount of rabbit mock anti-asialo GM 1 globulin 2 daysbefore Lisferiu infection. Also the animals were injected intravenously with 0.2 ml of anti-mouse IFI%/@ globulin involving 2000 neutralizing units or the same amount of mock anti-IFN globulin 8 hr after Lisferiu infection. b Mice were stimulated with LCWF on Day 6 of Listeriu infection, and the sera were collected 6 hr later.
system (Table 5). Hence, it was investigated whether IFN-a!//3 might play a role in both nonspecific and specific elimination of bacteria from the organs of mice infected with Listeria. The numbers of bacteria in the spleen and the liver of mice on Day 2 of infection in which IFN-(u//3 production had been suppressed by pretreatment with anti-a&lo GM 1 antibody or IFN-(Y//~produced had been neutralized with antimouse EN-m//3 antibody was determined (Table 6). No difference was observed between the numbers of bacteria in the organs of the control group and the IFN+ bdepleted mice. Furthermore, no fluctuation was seen in the numbers of bacteria in the organs of mice which had been administered 1 X lo5 IU of partially purified mouse IFN-(u//3 exogenously (data not shown). However, the efficiency of the elimination of bacteria decreasedin IFN-alpdepleted mice on Day 5 of infection while antigen-specific elimination of bacteria progressed(Table 6). TABLE 6 Growth of Listeriu in the Organs of Mice in Which IFI%/@ Production Was Suppressed Log bacteria/organ b Day 2 Treatment’
Day 5
Spleen
Liver
Spleen
Liver
None
6.25 + 0.08
5.32 + 0.40
3.72 + 0.55
3.05 + 0.04
Mock anti-r&lo GM 1 globulin Anti-asialo GM 1 globulin
6.18 + 0.12 7.08 + 0.09
5.42 + 0.16 6.25 f 0.08
3.58 k 0.42 5.12 f 0.14
3.16 + 0.34 5.06 f 0.97
Mock anti-IFN globulin Anti-IFN-a/@ globulin
6.20 f 0.28 6.03 3~0.32
5.23 + 0.30 5.12 f 0.31
3.53 + 0.47 4.82 f 1.02
2.98 2 0.27 4.19 f 0.77
’ Mice were treated with those antibodies as described in Table 6. b The numbers of viable Listeriu in the spleens and the livers of infected animals were established by plating out tenfold dilutions of the organ homogenates in PBS on trypticase soy agar.
38
NAKANE
AND MINAGAWA
DISCUSSION For the first time, we have demonstrated the alternative induction of IFN-ar//3 or IFN-y in the same system in viva IFN-cr//3 was nonspecifically induced in the early stage of Listeriu infection by asialo GM 1-bearing cells, which may be equivalent to NK cells in mice (3 1, 33). IFN-a//3 production by these cells was evidenced by our previous in vitro study using heat-killed Listeriu as a nonspecific IFN inducer (24, 37). When almost all of IFN-cu//3inducers involving bacterial lipopolysaccharide (38), synthetic polynucleotides (39), and viruses (40) are injected intravenously into mice, IFN-(u//3 is releasedinto the circulation within several hours after injection and thereafter rapidly disappears. Here, however, IFN-a//3 appeared in the circulation 24 hr after infection and persisted until 72 hr. This result shows that injection of bacteria may not directly induce IFN but multiplication of Listeriu in the tissues may be necessaryfor IFN induction, although it is not obvious which component(s) of Listeriu is a direct IFN-(u//3 inducer. Heat-killed Listeriu or bacterial cell components involving LCWF and LSF could not induce antiviral activity in the circulation. Therefore, it is unlikely that bacteriophages endogenously infecting bacteria (4 1) or an endotoxinlike material (42) induced IFN-(u/p in our experiments. On the other hand, IFN-7 production was induced by the specific antigen and by bacterial reinfection; this result is consistent in the kinetics of the expression of acquired cellular resistance (ACR) and delayed hypersensitivity (8-10). IFNy production was abrogated in cyclophosphamide-, hydrocortisone-, antithymocyte serum-, or carrageenan-treated mice. Among these, cyclophosphamide and antithymocyte serum completely inhibited IFN-7 production. It has been reported that cyclophosphamide can eliminate B-cell population (43) and that the drug inhibits functions of suppressor T cells (25). Antithymocyte serum can abrogate lymphoid cells involving T cells. In an in vitro study, it was demonstrated that T cells collaborating with macrophages were responsible for IFN-7 production induced by Listeriu in mouse spleen cell cultures (37). From our study of the effectsof those agents,it may be postulated that T cells and macrohages mainly participate in IFN-y production in viva Furthermore, we have presumed that suppressor/cytotoxic T cells among T-cell subpopulations participate in IFN-7 production as demonstrated by studies on the cellular origins of IFN-7 production in vitro (44, 45). Though participation of B cells cannot be eliminated as demonstrated by the in vivo study of IFN-y production in BCG-sensitized mice (46), NK cells can be excluded becauseanti-a&lo GM 1 antibody does not suppress IFN-y production. Recently, studies using recombinant IFNs produced by gene technology have distinguished between the biological activities of IFN-a/P and IFN-y. IFN-y can enhance the expression of major histocompatibility complex (MHC) classII antigen (Ia antigen) and Fc, receptor of macrophages, but IFN-a//3 cannot (47,48). Furthermore, it may be suggestedthat macrophage-activating factor (MAF) and IF%y are the same molecule (49, 50). Actually, the lymphokine, which activates macrophages to kill intracellular pathogens including Chlumydiu psittuci, Leishmunia donoviani, and Toxoplasma gondii, has been identified as IFNy (5 l-53). It is postulated that IFN-y produced by sensitized T cells may activate macrophages and that the virtual elimination of bacteria from the organs may be performed by the activated phagocytic mononuclear cells. This hypothesis is supported by the increase in the proportion of Ia-positive macrophages in PEC during Listeria infection (54) and by the fact that IFN-7 is produced by T cells from Listeriu-immune mice (16, 55). We could not detect antiviral activity in serum specimens of mice after 4 days of Listeriu infection.
INTERFERONS AND Listeriu INFECTION
39
However, it is likely that sensitized T cells in lymph organs produce IFN-y locally but not systemically. In fact, our study demonstrated that amplified IFNy production occurred at the systemic level in Listeriu-immune mice by stimulation with the specific antigen or by bacterial reinfection. We were interested in the role of IFN-a/B induced in the early stage of Listeria infection. It is known that IFN-a/,!? plays a key role in the defense mechanism of viral infections (56, 57). The IFN+/@ produced activates NK cells, and macrophages and virus-infected cells are killed by these cells. Therefore, we hypothesized that IFN-(Y/P might play an important role in nonspecific resistance in the early stage of Listeria infection. However, our hypothesis was disproven: the numbers of bacteria in the organs in the early stageof infection in IFN-o/B-depleted mice were not affected by treatment with anti-mouse IFN-(Y/P antibody or anti-asialo GM1 antibody, or in IFN-a/p-administered animals. On the contrary, the present study suggestsan unexpected action for IFN-(Y/B. Depletion of IFN-a/8 resulted in both suppression of IFNy production and delay of T-cell-dependent elimination of bacteria from the organs of mice in the late stage of infection. It may be assumed that IFN-a/P plays an important role in the establishment or the expression of both the UN-y-producing system and ACR. After Listeria infection, the pathogen multiplied in the organs, and NK cells stimulated by the organisms and/or interacting with bacteria-infected macrophages may have produced IFN-(u/p. The IFN-a/P produced may act as a “messenger” to aid in the establishment or the expression of T-cell-dependent events including IFN-7 production and ACR. Our concept may apply to the interpretation of lymphocytic choriomeningitis virus (LCMV)-induced encephalitis. It is known that LCMV diseaseis attributed to the development of virus-specific cytotoxic T cells (58). However, no death is observed in anti-IFN-cr/@ antibody-treated mice (59). It may be postulated that IFN-a/@ induced by LCMV infection would aid the generation of virus-specific cytotoxic T cells and the suppression of its generation by elimination of IFN-(Y/P results in the escapefrom death due to LCMV infection. Attempts are now being made to elucidate the role of IFN-a/p as a “messenger” in more detail both in vivo and in vitro. ACKNOWLEDGMENTS We thank Dr. H. Iida for his advice and encouragement and Ms. C. Matsumoto for her help in the assay of titers of anti-asialo GM 1 antibody.
REFERENCES 1. Mackaness, G. B., .I. Exp. Med. 116, 1962. 2. Blanden, R. V., Ma&mess, G. B., and Collins, F. M., J. Exp. Med. 124, 585, 1966. 3. Blanden, R. V., L&ford, M. J., and Mackaness, G. B., J. Exp. Med. 129, 1079, 1969. 4. Chan, C., Kongshaven, P. A. L., and Skamene, E., Immunology 32, 529, 1977. 5. Takeya, K., Shimototi, S., Taniguchi, T., and Nomoto, K., J. Gen. Microbial. 100, 373, 1977. 6. Newbor& M. F., and North, R. J., J. Immunol. 124, 571, 1980. 7. Petit, J., Infect. Immun. 21, 6 I, 1980. 8. Mackaness, G. B., J. Exp. Med. 129, 973, 1969. 9. Lane, F. C., and Unanue, E. R., J. Exp. Med. 135, 1104, 1972. 10. North, R. J., J. Exp. Med. 138, 342, 1973. 11. Kaufmann, S. H. E., Simon, M. M., and Hahn, H., J. Exp. Med. 150, 1033, 1979. 12. Kaufmann, S. H. E., and Hahn, H., J. Exp. Med. 155, 1754, 1982. 13. Holmberg, L. A., Springer, T. A., and Ault, K. A., J. Immunol. 127, 1792, 198I. 14. Farr, A. G., Dorf, M. E., and Unanue, E. R., Proc. Natl. Acad. Sci. USA 74, 3542, 1977. 15. Finke, J. H., Sharma, S. D., and Scott, J. W., J. Immunol. 127, 2354, 1981.
NAKANE
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16. Hsu, L., Finke, J. H., Scott, J. W., and Proffitt, M. R., J. Interferon Rex 1, 271, 1981. 17. Stewart, W. E., Blalock, J. E., Burke, D. C., Chang, C., Dunnic, J. C., Falcoff, E., Friedman, R. M., GaIasso, G. J., JokIick, W. K., Vilcek, J. T., Youngner, J. S., and Zoon, K. C., Nature @ondon) 286, 110, 1980. 18. Stinebring, W. R., and Youngner, J. S., Nature (London) 204, 712, 1964. 19. Sokhey, J., Soloviev, A. I., and Vasilieva, V. I., Acta Viral. (Engl. Ed.) 21, 485, 1977. 20. Kazt, J., Acta Viroi. 10, 227, 1966. 21. Merigan, T. C., and Hanna, L., Proc. Sot. Exp. Biol. Med. 122,421, 1966. 22. Friedman, R. M., Merigan, T. C., Remington, J. S., and Brownlee, I. E., Proc. Sot. Exp. Biol. Med. 123, 862, 1966.
23. Youngner, J. S., and Salvin, S. B., J. Immunol. 111, 1914, 1973. 24. Nakane, A., and Minagawa, T., J. Immunol. 126, 2139, 198 1. 25. Riillinghof, M., Staminski-Prowitz, A., Phxenheimer, K., and Wagner, H., J. Exp. Med. 145, 455, 1977. 26. Virelier, J. L., Transplantation 27, 353, 1979. 27. Catanxaro, P. J., Schwartz, H. J., and Graham, R. C., Amer. J. Pathol. 64, 387, 1971. 28. Momoi, T., Ando, S., and Nagai, Y., B&him. Biophys. Acta 441,488, 1976. 29. Iwamori, M., and Nagai, Y., B&him. Biophys. Acta 528,257, 1978. 30. Svennerholm, L., Mansson, J. E., and Li, Y.-T., J. Biol. Chem. 248,740, 1973. 31. Kasai, M., Iwamori, M., Nagai, Y., Okumura, K., and Tada, T., Eur. J. Immunol. 10, 175, 1980. 32. Sanai, Y., Ogawa, J., and Nagai, Y., Japan J. Exp. Med. 51, 309, 1981. 33. Kasai, M., Yoneda, T., Habu, S., Maruyama, Y., Okumura, K., and Tokunaga, T., Nature (London) 291, 334, 1981. 34. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265, 1951. 35. Armstrong, J. A., Appl. Microbial. 21, 723, 1971. 36. Nakane, A., and Minagawa, T., Infect. Immun. 36, 966, 1982. 37. Nakane, A., and Minagawa, T., Cell. Immunol. 75, 283, 1983. 38. Youngner, J. S., and Stinebring, W. R., Nature (London) 208, 456, 1965. 39. Sharpe, T. J., Birch, P. J., and Planterose, D. W., J. Gen. Viral. 12, 331, 1971. 40. Baron, S., and Buckler, C. E., Science (Washington, D.C.) 141, 1061, 1963. 4 1. Sword, C. P., and Pickett, M. J., J. Gen. Microbial. 25, 241, 1961. 42. Wexler, H., and Oppenheim, J. D,, In&t. Immun. 23, 845, 1979. 43. Turk, J. L., and Poulter, L. W., Clin. Exp. Immunol. 10, 283, 1972. 44. Torres, B. A., Farrar, W., and Johnson, H. M., J. Immunol. 128,2271, 1982. 45. Sonnenfeld, G., Salvin, S. B., and Youngner, J. S., Infect. Immun. 18, 283, 1977. 46. Sonnenfeld, G., Mandel, A. D., and Merigan, T. C., Immunology 36,883, 1979. 47. Steeg, P. A., Moore, R. N., Johnson, H. M., and Oppenheim, J. J., J. Exp. Med. 156, 1780, 1982. 48. Guyre, P. M., Morganelli, P. M., and Miller, R., J. Clin. Invest. 72, 393, 1983. 49. Zlotnik, A., Roberts, W. K., Vasil, A., Blumenthal, E., Larosa, F., Leibson, H. J., Enders, R. O., Graham, S. M., Jr., White, J., Hill, J., Henson, P., Klein, J. R., Bevan, M. J., Marrack, P., and Kappler, J. W., J. Immunol. 131, 794, 1983. 50. Schreiber, R. D., Pace,J. L., Russell, S. W., Altman, A., and Katz, D. H., J. Immunol. 131,826, 1983. 51. Rothermel, C. D., Rubin, B. Y., and Murray, H. W., J. Immunol. 131, 2542, 1983. 52. Murray, H. W., Rubin, B. Y., and Rothermel, C. D., J. Clin. Invest. 72, 1506, 1983. 53. Nathan, C. F., Murray, H. W., Wiebe, M. E., and Rubin, B. Y., J. Exp. Med. 158, 670, 1983. 54. Beller, D. I., Kiely, J.-M., and Unanue, E. R., J. Immunol. 124, 1426, 1980. 55. Kaufmann, H. E., Hahn, H., Berger, R., and Kirchner, H., Eur. J. Immunol. 13, 265, 1983. 56. Gresser, I., Tovey, M. G., Bandu, M.-T., Maury, C., and Brouty-Boy&, D., J. Exp. Med. 144, 1305, 1976.
57. Gresser, I., Tovey, M. G., Maury, C., and Bandu, M.-T., J. Exp. Med. 144, 13 16, 1976. 58. Cole, G. A., Nathanson, N., and Prendergast, R. A., Nature (London) 238, 335, 1972. 59. Pfau, C. J., Gresser, I., and Hunt, K. D., J. Gen. Virol. 64, 1827, 1983.
IMMUNOLOGY
88,29-40
(1984)
The Significance of Alpha/Beta lnterferons and Gamma Interferon Produced in Mice Infected with Listeria monocytogenes AKIO NAKANE AND TOMONORI MINAGAWA Department of Microbiology, Hokkaido University School ojh4edicine, Kita 15 Nishi 7, Kita-Ku, Sapporo 060, Japan Received January 30. 1984; accepted April 25, 1984 Interferon (IFN)-a//B was induced in the circulation of mice infected intravenously with Lisferia monocytogenes 24 to 72 hr aher infection, but was not induced by the administration of heat-
killed Listeria, listerial cell wall fraction (LCWF), or iisterial soluble fraction. Appearance of IFN-a/p showed a pattern similar to that of the growth of bacteria in the spleen and the liver of mice. IFN-@I production was abrogated by pretreatment of mice with anti-asialo GM1 antibody, antithymocyte serum, or hydrocortisone, but not with cyclophosphamideor carrageenan. Such treatments which suppressedIFN-o/p production did not influence bacterial growth in the organs of mice in the early stage of Listeria infection. Administration of IFN-(u//3 exogenously also did not. After 5 days of infection when the specific resistanceagainst reinfection with Lisferia was established,IFN-+r but not IFN-(r//3 was induced in the circulation 3 to 6 hr after stimulation with LCWF or reinfection with Listeria. IFN--r production was abrogated completely by cyclophosphamide and antithymocyte serum, and partially by hydrocortisone and carrageenan, but not by anti-asialo GM1 antibody in Listeria-infected mice treated with these agents before induction of IFN-7 by LCWF. Presumably, IFN-a/b might be produced by asialo GM 1-bearing cells but IFN-y might not. However, IFN-7 production was suppressedin Listeria-infected mice, when IFN-c&3 production had been inhibited by treatment with anti-asialo GM1 antibody or when the IFN produced had been neutralized with anti-mouse IFN-a/P antibody. Therefore, it is conceivable that IFN-a/6 might be essential for the generation or the expression of antigenspecific T cells involving EN-7 production and acquired resistanceduring Listeria infection. In fact, the bacterial growth in the organs of mice in the early stage of infection was normal in IFN-culfidepleted mice but it resulted in the delay of T-celldependent elimination of bacteria from the organs of mice in the late stage.
INTRODUCTION The major host resistance to facultative intracellular bacteria, such as Listeriu monocytogenes,Salmonella typhimurium, and Mycobacterium bovis BCG, is mediated by cellular factors but not by the serum antibody (l-3). Among these, only Listeria has been studied in detail. Listeria infection is eliminated from the tissues by macrophages acting in two steps:during the first 2 or 3 days after infection, mononuclear phagocytes, which are activated by a T-cell-independent mechanism other than complement, protect the murine host against explosive multiplication of bacteria (4-7). Thereafter, virtual elimination of Listeria is performed by macrophages which are activated by some mediators released from sensitized T cells over the next 4 to 5 days (8- 12). Recently, our understanding of the defensemechanisms against Listeria 29 0008~8749184$3.00 Copyright @ 1984 by Academic FXSS, Inc. All rights of reproduction in any form reserved.
30
NAKANE
AND MINAGAWA
infection was augmented by the information that natural killer (NK) activity in murine peritoneal exudate cells (PEC) increased Days 2 through 6 after infection (13). Therefore, it is conceivable that macrophages, T cells, and NK cells may form the framework of the major cellular defensemechanisms and these cells may produce some mediators which activate each other in order to eliminate Listeria from the tissues. Actually, it was demonstrated that PEC from Listeria-infected mice could produce interleukin 1 (IL-l), interleukin 2 (IL-2), and interferon (IFN)-y by stimulation with the specific antigen (14-16). There are three antigenic types of IFN, referred to as a, fl, and y in both human and murine systems (17). In the murine system, IFN-cy//3 production is induced nonspecifically not only by viral infections but also by other microbial infections involving bacteria ( 18), Mycoplasma ( 19), Rickettsia (20), Chlamydia (21), or Protozoa (22). In contrast, IFN-7 can be produced by stimulation with specific antigen in the immunized mice (23) and is classified as one of the lymphokines. Hence, the host’s immunological status can be estimated by determining which type of IFN is induced. However, neither the IFN-(Y/P nor the IFNy production mechanism has been studied independently in viva In the present study, we demonstrate that IFN-a//3 and IFN-7 can be produced alternatively in the circulation and that the speciesproduced reflectsthe immunological status of the murine host during Listeriu infection. IFN-(u/p is produced in the primary infection by the cells sensitive to anti-asialo GM1 antibody, while induction of IFN-y, by stimulation with specific antigen or by reinfection, is abrogated completely by pretreatment of mice with antithymocyte serum or cyclophosphamide. We also demonstrate that IFN-(u//3 produced in the early stage of Listeriu infection may not participate in T-cell-independent elimination of bacteria in the early stage but that suppression of IFN-(u/p production by pretreatment of mice with anti-asialo GM1 antibody or neutralization of the IFN-(u/b produced endogenously by anti-IFN-c*l/& antibody administration results in both suppression of IFN-7 production and the delay of T-cell-dependent elimination of bacteria from the organs of mice in the late stage of infection. MATERIALS AND METHODS Mice. Female ddY mice 5 to 7 weeks of age were purchased from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Hamamatsu, Shizuoka, Japan). Infection with bacteria. Listeria monocytogenes lb strain 1684, kindly provided by Dr. T. Nagai of the Department of Microbiology, Sapporo Medical College Hospital, Sapporo, were prepared as previously reported (24). The concentration of washed cells was adjusted spectrophotometricahy (at 550 nm). Mice were infected intravenously with 0.2 ml of 1 X lo4 colony-forming units (CFU) of viable Listeria in 0.01 M phosphate-buffered saline (PBS, pH 7.4). The numbers of viable organisms in the spleen and the liver of infected animals was established by plating serial tenfold dilutions of the organ homogenate in PBS on trypticase soy agar (BBL, Cockeysville, Md.) containing 0.3% glucose. The colony counts were routinely performed 18 to 24 hr later. Preparation of heat-killed Listeria and listerial cell components. Heat-killed Listeria (HK-LM) and Listeriu soluble fraction (LSF) were prepared as previously reported (24). Listeriu cell wall fraction (LCWF) was prepared as follows: the precipitate of
INTERFERONS
AND
Listeria INFECTION
31
the sonically disrupted organisms obtained by differential centrifugation (24) was washed three times with sterile, nonpyrogenic, double-distilled water. The LCWF obtained was lyophilized, weighed, reconstituted with sterile, nonpyrogenic 0.85% saline to a concentration of 2 mg/ml, and autoclaved. All preparations were carried out under aseptic conditions. HK-LM, LCWF, and LSF were stored at -70°C. When used for injections, these preparations were diluted with 0.01 M PBS. Drug treatment. Mice were injected intraperitoneally with single doses of 240 mg/kg of cyclophosphamide (Sigma Chemical Co., St. Louis, MO.) (25), 200 mg/kg of hydrocortisone acetate (Wako Pure Chemical Ind., Osaka, Japan) (26), or 240 (Wako) (27) in 0.5 ml of 0.0 1 M PBS 48 hr before infection mg/kg of K-Carrageenan with Listeria or stimulation with LCWF. Antithymocyte serum treatment. Rabbit anti-mouse thymocyte serum (0.25 ml; M. A. Bioproducts, Walkersville, Md.) was injected into mice intraperitoneally twice (48 and 24 hr before Listeria infection or stimulation with LCWF) (26). Normal rabbit serum was injected into the other group of animals as a control in the same manner. Anti-asialo GM1 antibody treatment. GM1 was isolated from porcine brain by a combination of anion-exchange chromatography and high-performance adsorption chromatography (28, 29). Asialo GM1 was prepared from GM1 by treatment with 1 N formic acid at 100°C for 1 hr (30) and then purified by chromatography on diethylaminoethyl (DEAE)-Sephadex A-25 (Pharmacia Fine Chemicals, Uppsala, Sweden) and Iatrobeads 6RS8060 (Iatron Laboratory, Inc., Tokyo, Japan). New Zealand rabbits were immunized with asialo GM1 dispersed in 0.85% saline containing methylated bovine serum albumin (Sigma) and emulsified with complete Freund’s adjuvant asdescribedbefore (3 1). The titer of anti-a&lo GM 1 antibody was determined by enzyme-linked immunosorbent assay (ELISA) according to the method reported elsewhere(32). A globulin portion of the antibody was obtained by precipitation with 50% saturated ammonium sulfate. Anti-asialo GM 1 globulin (0.2 ml; diluted 1:3) was injected intravenously 48 hr before Listeria infection or stimulation with LCWF (33). In the control animals, rabbit mock globulin obtained from rabbits immunized with only methylated bovine serum albumin emulsified with complete Freund’s adjuvant was injected instead of the antibody. Anti-mouseIFN-cx/@antibody treatment.The globulin portion of anti-mouse L-cell IFN-(r//3 antibody (Catalog No. G-024-501-568) was kindly provided by Dr. G. J. Galasso of The Microbiology and Infectious Disease Program, National Institute of Allergy and Infectious Diseases.Anti-mouse IFN-(r//3 sheep globulin (0.2 ml) that had been adjusted to the IFN-neutralizing titer of 10,000 U/ml with serum-free RPM1 1640 medium (GIBCO, Grand Island, N.Y.) was injected into the tail vein of mice after Listeria infection. One neutralizing unit of the antibody can neutralize 8 to 10 international interferon units (IU) of mouse IFN-(r//3. Control animals were treated the same amount of sheep globulin to mock L-cell IFN-a/P (Catalogue No. G-025501-568) in the same manner. Administration of mouseIFN-(Y/P.Partially purified Newcastle diseasevirus-induced mouse L-cell IFN-(Y/P (sp act: 9 X lo6 IU/mg) was kindly provided by Dr. S. Kobayashi of Torey Basic Institute, Kamakura. Mice were given intravenously 0.5 ml of mouse IFN-(u//3 containing 1 X IO5 IU. Determination of protein. Protein contents were determined by the method of Lowry et al. (34).
NAKANE
32
AND MINAGAWA
Assays and characterization of ZFN. The IFN samples were pooled sera obtained from at least four mice in each group. The IFN assay was carried out by the dyebinding method (35) using mouse L-929 cells and vesicular stomatitis virus (Indiana strain) as previously reported (36). Neutralization tests using anti-mouse IFN-a//3 antibody and acid stability of IFN samples were carried out as reported elsewhere (37). RESULTS ZFN Response in the Circulation of Mice by the Primary Infection of Listeria After mice were infected intravenously with 1 X lo4 CFU of Listeria, the antiviral activities of the pooled sera obtained from four or five mice of each group were followed at various times. As shown in Fig. la, no antiviral activity was detected in the sera until at least 16 hr after infection. The antiviral activity appeared at 24 hr and was maintained until 72 hr after infection. All antiviral activities were completely neutralized with anti-mouse IFN-a/p antibody (Fig. la), and were not reduced by pH 2.0 treatment (data not shown). On the other hand, the growth of Listeria in the spleens and the livers reached a maximum at 48 hr and then declined (Fig. lb). We also studied the possibility that IFN could be produced in the circulation of mice injected with killed Listeria (HK-LM) or listerial cell components (LCWF and LSF). No antiviral activity was demonstrated when mice were injected with HK-LM, LCWF, or LSF at any period tested (Table 1).
111,
36916
II
2432
I
I
I
1
48
72
96
120
HOURS AFTER LISTERIA INFECTION
FIG. 1. Kinetics of IFN production in the circulation and bacterial growth in spleensand livers following Listeriu infection. Mice weTe infected intravenously with 1 X 104CFU of viable Listeriu, and sera were
collected at various times. IFN titer before (0) and after (0) treatment with anti-IFN-ol/fi antibody (a). At the same time, the numbers of bacteria in spleens (0) and livers (0) were estimated (b).
33
INTERFERONS AND Listeriu INFECTION TABLE I IFN Response in the Circulation of Mice Injected with Different Preparations of Lisferiu IFN titer (IU/ml) Hours after injection”
Viable LM
HK-LM
LSF
LCWF
3 6 24 48 12
<4 <4 40 50 20
<4 <4 <4 <4 <4
14 <4 <4 <4 <4
<4 <4 <4 <4 <4
’ The sera were collected at the specified times after mice were injected intravenously with 1 X lo4 CFU of viable Listeria, 5 X lo6 cells of HK-LM, 1.9 mg protein of LSF, or 75 pg of LCWF.
Efects of Immunosuppressive Agents on IFN Response to the Primary Infection of Listeria The results of the IFN response to the primary infection of bacteria in mice which had been treated with various immunosuppressive agents are summarized in Table 2. IFN-a/(3 production was completely abrogated in mice pretreated with hydrocortisone. Similarly, anti-asialo GM1 antibody rather than antithymocyte serum sup TABLE 2 Effects of Various Immunosuppressive Agents on the IFN Response to the Primary Infection of Listeria IFN titer (IU/ml)b Expt
Induced
After neutralization with anti-IFN-a/p
None Cyclophosphamide Hydrocortisone Carrageenan
120 210 <4 100
10 10 ND‘ <4
None Normal rabbit serum Antithymocyte serum
95 110 40
<4 <4 <4
None Mock anti-asialo GM 1 globulin Anti-a&lo GM I globulin
130 130 35
15 10 <4
Treatment’
’ Mice were injected intraperitoneally with single doses of each drug: 240 mg/kg of cyclophosphamide, 200 mg/kg of hydrocortisone, or 240 mg/kg of catrageenan. Also other groups of mice were injected intravenously with 0.2 ml of rabbit anti-asialo GM1 globulin (diluted 1:3, approximately 800 units in ELISA) or the same amount of rabbit mock anti-asialo GM1 globulin. Thesemice were infected intravenously with 1 X lo4 CFU of viable Lisferiu 48 hr later. Antithymocyte serum or normal rabbit serum (0.25 ml) was injected intraperitoneally 48 and 24 hr before bacteriaI infection. b Sera were collected 48 hr after Listerin infection, and then IFN activities and the antigenicity of the specimens were determined. ’ Not done.
34
NAKANE
AND MINAGAWA
pressed IFN induced by List&a infection. However, IFN-a/B production was not affected by treatment with cyclophosphamide or carrageenan. IFN-y Production in Listeria-Infected Mice Stimulated with the Specific Antigen or Reinfected with Bacteria Though no IFN activity was induced when uninfected mice were injected with LCWF, we assumed that the IFN response to the specific antigen would occur in mice after the specific immunity was established. LCWF (50 pg) was injected intravenously into mice on Day 6 of Listeria infection, and IFN activity in the pooled sera was determined at various times (Fig. 2). IFN activity appeared 3 hr and peaked 6 hr after stimulation. Thereafter the activity decreasedand disappeared 10 hr after injection with LCWF. The neutralization test was carried out using anti-mouse IFN-cr//3 antibody, and IFN activities produced at any period were not affected by the treatment (Fig. 2). Treatment with pH 2.0 buffer resulted in complete inactivation of the IFN activity (Fig. 2). We examined which type of IFN could be induced by reinfection with viable Listeria. On Day 6 of infection, mice were reinfected intravenously with 1 X lo5 CFU of Listeria, and the sera were collected 3, 6, 24, and 48 hr later. As shown in Table 3, IFN activity was demonstrated in the circulation 3 hr after reinfection and peaked at 6 hr. The IFN produced in the sera by reinfection was of the y type as was that produced in mice injected with LCWF. No antiviral activity was demonstrated 24 or 48 hr after reinfection with Listeria different from the primary infection. The IFN response to the specific antigen occurred in mice on Day 5 of infection, and the maximum response was demonstrated on Day 7 (Fig. 3). Thereafter, the IFN response gradually decreased, and no antiviral activity was seen in the sera when mice were stimulated with LCWF on Day 21 of infection. IFN activities induced by LCWF on Days 5 to 14 of infection were not neutralized with anti-mouse IFN-a/@ antibody (Fig. 3) and were labile at pH 2.0 (data not shown). When mice were sensitized with various dosesof HK-LM instead of viable Listeria, no IFN responsewas observed in the circulation of mice on Day 6 of administration of any dose of HK-LM (data not shown).
HOURS AFTER INJECTION WITH LCWF FIG. 2. Kinetics of IFN production in the circulation following injection with LCWF in List&z-infected mice. LCWF (50 pg) was injected intravenously into mice on Day 6 of Listeriu infection, and sexawere collected at various times. IFN titer before (0) and after (0) treatment with anti-IFN-43 antibody, and after pH 2.0 treatment (0).
35
INTERFERONS AND Listeria INFECTION TABLE 3 The IFN Response in the Circulation of Mice after Reinfection with Listeria IFN titer (IU/ml) Hours after stimulation
Stimulus” Infected mice Viable LM
LCWF
Uninfected mice Viable LM
Induced
After neutralization with anti-IFN-cY/Bb
3 6 24 48
55 80 <4 <4
50 80 ND’ ND
3 6 24 48
240 5100 <4 <4
240 5100 ND ND
3 6 24 48
<4 <4 40 50
ND ND <4 14
’ Viable Listeria (1 X 10’ CFU) or LCWF (50 ~~39) was injected intravenously into mice on Day 6 of Listeriu infection. Uninfected mice were injected with 1 X IO4CFU of viable Listeriu as a control. bThe neutralization test was carried out as described in the text. ’ Not done.
Efects of Various Immunosuppressive IFN-7
Agents on Antigen-SpeciJic
Induction
of
Mice were infected with Listeria and treated with various immunosuppressive agents. The animals were stimulated with LCWF on Day 6 of infection, and IFN titers in the circulation were determined 6 hr later (Table 4). Both cyclophosphamide
1600 t ‘3 x >
800
; 400 c” z 200 h -
100
-i/‘; f
3 5
7 9 II DAYS AFTER LISTERIA
A 14 21 INFECTION
RG. 3. IFN response to stimulation with specific antigen in the circulation of mice during Listeria infection. After mice were infected intravenously with 1 X lo4 CFU of Listerin, 50 pg of LCWF was injected intravenously into mice on different days after Listeriu infection. The sera were collected 6 hr later. IFN titer before (0) and after (0) treatment with anti-IFN-cY/@antibody.
36
NAKANE
AND MINAGAWA TABLE 4
Effects of Various Immunosuppressive Agents on Antigen-Specific Induction of IFN in Wisteria-Infected Mice IFN titer (IU/ml)” Expt
Treatment”
Induced
After neutralization with anti-IFN-a/b
I
None Cyclophosphamide Hydrocortisone Carrageenan
3200 <16 450 630
3200 NDC 450 630
2
None Normal rabbit serum Antithymocyte serum
2470 2520 <16
2450 2520 ND
3
None Mock anti-asialo GM I globulin Anti-asialo GM I globulin
2600 2530 2300
2600 2530 2300
’ Mice were infected intravenously with 1 X 10’ CFU of viable Listeriu. The animals were treated with cyclophosphamide, hydrocortisone, carrageenan,anti-asialo GM I globulin, or rabbit mock anti-asialo GM I globulin on Day 4 of infection, or with antithymocyte serum or normal rabbit serum on Days 4 and 5 of infection, as described in Table 2. These mice were stimulated with 50 a of LCWF on Day 6 of infection. ’ Sera were collected 6 hr after injection with LCWF, and then IFN activities and the antigenicity of the specimens were determined. ’ Not done.
and antithymocyte serum completely suppressed IFN-y production. The IFN-y responsewas also suppressedin hydrocortisone- or carrageenan-treated mice. However, a normal IFN-7 responseto LCWF was observed in anti-asialo GM 1 antibody-treated animals.
Signijcance of Early EndogenousIFN-CY/@ on the Expressionof Late IFN-y Response in Listeria-Infected Mice The above results demonstrated that IFN-a/B production by the primary infection of Listeria was suppressed in asialo GM1 antibody-treated mice (Table 2) but IFN-7 production was not affected when mice were treated with the antibody before stimulation with LCWF (Table 4). We investigated the IF%y response in mice in which IFN-cr//3 production had been suppressed (Table 5). Mice were treated with anti-a&lo GM1 antibody 2 days before Listeria infection, and IFN-y production stimulated with the specific antigen was estimated on Day 6 of infection. IFN-y production was significantly suppressedin the antibody-treated animals. Furthermore, IFNy production was completely suppressed in Listeria-infected mice in which IFN-(u/p produced endogenously had been depleted by administration of anti-mouse IFN-cu//3antibody (Table 5). No suppression of IFN-y production was observed when anti-mouse IFN-(Y/B antibody was injected soon after stimulation with the specific antigen (data not shown).
Role of IFN+3 on Elimination of Bacteriafrom the Organsof Listeria-InfectedMice It was demonstrated that IFN-(u//l produced in the early stageof Listeria infection would be necessary for the establishment or the expression of the EN-y-producing
INTERFERONS AND Lisferiu INFECTION
37
TABLE 5 The IFN-7 Response to Specific Antigen in Lisle&Infected IFN-o//3 Production Was Suppressed
Mice in Which
IFN titer (RI/ml) Induced
After neutralization with anti-IFN-a//3
None
2200
2200
Mock anti-a&lo GM I globulin Anti-asialo GM 1 globulin
3000 400
3000 400
Mock anti-IFN globulin Anti-IFN-a/B globulin
2400
2400
Treatment a
’ Mice were injected intravenously with 0.2 ml of anti-asialo GM1 globulin (diluted 1:3) or the same amount of rabbit mock anti-asialo GM 1 globulin 2 daysbefore Lisferiu infection. Also the animals were injected intravenously with 0.2 ml of anti-mouse IFI%/@ globulin involving 2000 neutralizing units or the same amount of mock anti-IFN globulin 8 hr after Lisferiu infection. b Mice were stimulated with LCWF on Day 6 of Listeriu infection, and the sera were collected 6 hr later.
system (Table 5). Hence, it was investigated whether IFN-a!//3 might play a role in both nonspecific and specific elimination of bacteria from the organs of mice infected with Listeria. The numbers of bacteria in the spleen and the liver of mice on Day 2 of infection in which IFN-(u//3 production had been suppressed by pretreatment with anti-a&lo GM 1 antibody or IFN-(Y//~produced had been neutralized with antimouse EN-m//3 antibody was determined (Table 6). No difference was observed between the numbers of bacteria in the organs of the control group and the IFN+ bdepleted mice. Furthermore, no fluctuation was seen in the numbers of bacteria in the organs of mice which had been administered 1 X lo5 IU of partially purified mouse IFN-(u//3 exogenously (data not shown). However, the efficiency of the elimination of bacteria decreasedin IFN-alpdepleted mice on Day 5 of infection while antigen-specific elimination of bacteria progressed(Table 6). TABLE 6 Growth of Listeriu in the Organs of Mice in Which IFI%/@ Production Was Suppressed Log bacteria/organ b Day 2 Treatment’
Day 5
Spleen
Liver
Spleen
Liver
None
6.25 + 0.08
5.32 + 0.40
3.72 + 0.55
3.05 + 0.04
Mock anti-r&lo GM 1 globulin Anti-asialo GM 1 globulin
6.18 + 0.12 7.08 + 0.09
5.42 + 0.16 6.25 f 0.08
3.58 k 0.42 5.12 f 0.14
3.16 + 0.34 5.06 f 0.97
Mock anti-IFN globulin Anti-IFN-a/@ globulin
6.20 f 0.28 6.03 3~0.32
5.23 + 0.30 5.12 f 0.31
3.53 + 0.47 4.82 f 1.02
2.98 2 0.27 4.19 f 0.77
’ Mice were treated with those antibodies as described in Table 6. b The numbers of viable Listeriu in the spleens and the livers of infected animals were established by plating out tenfold dilutions of the organ homogenates in PBS on trypticase soy agar.
38
NAKANE
AND MINAGAWA
DISCUSSION For the first time, we have demonstrated the alternative induction of IFN-ar//3 or IFN-y in the same system in viva IFN-cr//3 was nonspecifically induced in the early stage of Listeriu infection by asialo GM 1-bearing cells, which may be equivalent to NK cells in mice (3 1, 33). IFN-a//3 production by these cells was evidenced by our previous in vitro study using heat-killed Listeriu as a nonspecific IFN inducer (24, 37). When almost all of IFN-cu//3inducers involving bacterial lipopolysaccharide (38), synthetic polynucleotides (39), and viruses (40) are injected intravenously into mice, IFN-(u//3 is releasedinto the circulation within several hours after injection and thereafter rapidly disappears. Here, however, IFN-a//3 appeared in the circulation 24 hr after infection and persisted until 72 hr. This result shows that injection of bacteria may not directly induce IFN but multiplication of Listeriu in the tissues may be necessaryfor IFN induction, although it is not obvious which component(s) of Listeriu is a direct IFN-(u//3 inducer. Heat-killed Listeriu or bacterial cell components involving LCWF and LSF could not induce antiviral activity in the circulation. Therefore, it is unlikely that bacteriophages endogenously infecting bacteria (4 1) or an endotoxinlike material (42) induced IFN-(u/p in our experiments. On the other hand, IFN-7 production was induced by the specific antigen and by bacterial reinfection; this result is consistent in the kinetics of the expression of acquired cellular resistance (ACR) and delayed hypersensitivity (8-10). IFNy production was abrogated in cyclophosphamide-, hydrocortisone-, antithymocyte serum-, or carrageenan-treated mice. Among these, cyclophosphamide and antithymocyte serum completely inhibited IFN-7 production. It has been reported that cyclophosphamide can eliminate B-cell population (43) and that the drug inhibits functions of suppressor T cells (25). Antithymocyte serum can abrogate lymphoid cells involving T cells. In an in vitro study, it was demonstrated that T cells collaborating with macrophages were responsible for IFN-7 production induced by Listeriu in mouse spleen cell cultures (37). From our study of the effectsof those agents,it may be postulated that T cells and macrohages mainly participate in IFN-y production in viva Furthermore, we have presumed that suppressor/cytotoxic T cells among T-cell subpopulations participate in IFN-7 production as demonstrated by studies on the cellular origins of IFN-7 production in vitro (44, 45). Though participation of B cells cannot be eliminated as demonstrated by the in vivo study of IFN-y production in BCG-sensitized mice (46), NK cells can be excluded becauseanti-a&lo GM 1 antibody does not suppress IFN-y production. Recently, studies using recombinant IFNs produced by gene technology have distinguished between the biological activities of IFN-a/P and IFN-y. IFN-y can enhance the expression of major histocompatibility complex (MHC) classII antigen (Ia antigen) and Fc, receptor of macrophages, but IFN-a//3 cannot (47,48). Furthermore, it may be suggestedthat macrophage-activating factor (MAF) and IF%y are the same molecule (49, 50). Actually, the lymphokine, which activates macrophages to kill intracellular pathogens including Chlumydiu psittuci, Leishmunia donoviani, and Toxoplasma gondii, has been identified as IFNy (5 l-53). It is postulated that IFN-y produced by sensitized T cells may activate macrophages and that the virtual elimination of bacteria from the organs may be performed by the activated phagocytic mononuclear cells. This hypothesis is supported by the increase in the proportion of Ia-positive macrophages in PEC during Listeria infection (54) and by the fact that IFN-7 is produced by T cells from Listeriu-immune mice (16, 55). We could not detect antiviral activity in serum specimens of mice after 4 days of Listeriu infection.
INTERFERONS AND Listeriu INFECTION
39
However, it is likely that sensitized T cells in lymph organs produce IFN-y locally but not systemically. In fact, our study demonstrated that amplified IFNy production occurred at the systemic level in Listeriu-immune mice by stimulation with the specific antigen or by bacterial reinfection. We were interested in the role of IFN-a/B induced in the early stage of Listeria infection. It is known that IFN-a/,!? plays a key role in the defense mechanism of viral infections (56, 57). The IFN+/@ produced activates NK cells, and macrophages and virus-infected cells are killed by these cells. Therefore, we hypothesized that IFN-(Y/P might play an important role in nonspecific resistance in the early stage of Listeria infection. However, our hypothesis was disproven: the numbers of bacteria in the organs in the early stageof infection in IFN-o/B-depleted mice were not affected by treatment with anti-mouse IFN-(Y/P antibody or anti-asialo GM1 antibody, or in IFN-a/p-administered animals. On the contrary, the present study suggestsan unexpected action for IFN-(Y/B. Depletion of IFN-a/8 resulted in both suppression of IFNy production and delay of T-cell-dependent elimination of bacteria from the organs of mice in the late stage of infection. It may be assumed that IFN-a/P plays an important role in the establishment or the expression of both the UN-y-producing system and ACR. After Listeria infection, the pathogen multiplied in the organs, and NK cells stimulated by the organisms and/or interacting with bacteria-infected macrophages may have produced IFN-(u/p. The IFN-a/P produced may act as a “messenger” to aid in the establishment or the expression of T-cell-dependent events including IFN-7 production and ACR. Our concept may apply to the interpretation of lymphocytic choriomeningitis virus (LCMV)-induced encephalitis. It is known that LCMV diseaseis attributed to the development of virus-specific cytotoxic T cells (58). However, no death is observed in anti-IFN-cr/@ antibody-treated mice (59). It may be postulated that IFN-a/@ induced by LCMV infection would aid the generation of virus-specific cytotoxic T cells and the suppression of its generation by elimination of IFN-(Y/P results in the escapefrom death due to LCMV infection. Attempts are now being made to elucidate the role of IFN-a/p as a “messenger” in more detail both in vivo and in vitro. ACKNOWLEDGMENTS We thank Dr. H. Iida for his advice and encouragement and Ms. C. Matsumoto for her help in the assay of titers of anti-asialo GM 1 antibody.
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