Listeria monocytogenes-Induced Gamma Interferon Secretion by ...

INFECTION AND IMMUNITY, May 1993, p. 2154-2161 0019-9567/93/052154-08$02.00/0 Copyright C 1993, American Society for Microbiology

Vol. 61, No. 5

Listeria monocytogenes-Induced Gamma Interferon Secretion by Intestinal Intraepithelial y/8 T Lymphocytes SHIGEKI YAMAMOTO,t FRIEDEMANN RUSS, HENRIQUE C. TEIXEIRA, PETER CONRADT, AND STEFAN H. E. KAUFMANN* Department of Immunology, University of Ulm, Albert-Einstein-Allee 11, D-7900 Ulm, Germany Received 16 October 1992/Accepted 11 February 1993

fy/8 T cells represent a major proportion of intestinal intraepithelial lymphocytes (IEL), and it has been suggested that these IEL serve as a first immune barrier against microbial invasion and that they do so by destroying infected epithelial cells. In the present study, we confirm that both ct/Ia and -y/8 IEL from naive mice express potent cytotoxicity and produce gamma interferon (IFN-y) after T-cell receptor (TCR) engagement by specific monoclonal antibodies (MAb). Intraperitoneal administration of the anti-'y/b TCR MAb GL3 caused downregulation of the -y/8 TCR in IEL, and IEL from -y/8 TCR-modulated mice failed to express cytotoxic activity and to secrete IFN--y after fy/8 TCR engagement. In contrast, am/I3 IEL from such mice were still cytolytic and secreted IFN-y. Mice were infected orally with virulent Listeria monocytogenes at doses which caused bacterial invasion through the intestinal epithelia. Although at/,8 and -y/8 IEL from these mice expressed high cytolytic activities in antibody-redirected killer assays, target cells pulsed with listerial antigens were not lysed. In contrast, IFN-y secretion by IEL from L. monocytogenes-infected mice was induced not only by anti-TCR MAb but also by target cells pulsed with listerial antigens, whereas irrelevant antigens, including heat shock protein 60, did not induce TFN-'y secretion. Furthermore, the number of IFN-'y-secreting IEL, as assessed by the enzyme-linked immunospot technique, was increased during listeriosis. y/& TCR modulation by GL3 administration abrogated antigen-induced IFN-'y secretion by IEL from infected mice. These findings suggest that L. monocytogenes induced IFN-y secretion by fy/8 IEL from mice suffering from intestinal L. monocytogenes infection and invasion. Thus, the data provide evidence for a role of IFN--y-secreting IEL in local resistance against listeriosis and perhaps other food-borne diseases. -y/8 T cells are a minor T-cell population in the peripheral lymphoid organs of mice, in which the majority of T cells express the o1/1 T-cell receptor (TCR) and either the CD4 or the CD8 molecule. However, -y/b TCR-expressing cells are prevalent in various epithelial tissues, such as the epidermis and intestinal, respiratory, and reproductive tracts (1, 38). It has been reported that y/8 T cells recognize host-derived heat shock proteins (HSPs), which serve as ubiquitous and constant markers of stress (26), indicating that -y/1 intestinal intraepithelial lymphocytes (IEL) respond to a selected set of marker antigens on epithelial cells undergoing microbial invasion and subsequent inflammation. Although the intestinal IEL primarily express the V,,7 chain, their TCRs are

organs. However, naturally acquired listeriosis in humans is food borne, and hence invasion proceeds via the intestine (14). To date, local immunity against L. monocytogenes infection via the natural route has been greatly neglected, even though IEL may represent a first and important part of the cellular immune response against naturally acquired L. monocytogenes infection. We therefore decided to characterize the potential role of IEL in local immunity against listeriosis. We found that IEL were activated during gastrointestinal listeriosis to produce gamma interferon (IFN-,y) in situ arid after in vitro restiinulation with L. monocytogenes-pulsed stimulator cells. In vivo modulation of -y/8 IEL with monoclonal antibodies (MAb) against the -y/8 TCR abrogated this biological activity, indicating involvement of y/8 T cells in L. monocytogenesinduced IFN--y production.

quite diverse because of combination with different 8 chains and high junctional diversity (1, 2, 4, 9, 35). Thus, the potential antigen recognition repertoire of intestinal IEL is sufficiently diverse to allow recognition of various microbial antigens. Listena monocytogenes is an intracellular bacterium capable of surviving inside mononuclear phagocytes. Acquired resistance to this type of pathogen is mediated by specific T cells; antibodies are of minor, if any, importance (10). Experimental listeriosis in mice has been widely used as a model to examine the T-cell-mediated mechanisms underlying acquisition of resistance against intracellular bacteria (10). In this type of investigation, bacteria are normally administered intravenously or intraperitoneally (i.p.) and infection and immunity are assessed in central lymphoid

MATERIALS AND METHODS Mice. C57BL/6 mice of both sexes were used at 8 to 12 weeks of age. They were supplied by the animal facility of the University of Ulm and maintained under specific-pathogen-free conditions. Antigens. Live or heat-killed (HK) L. monocytogenes EGD organisms were prepared as described elsewhere (15). Lyophilized Mycobacterium tuberculosis H37Ra was obtained commercially (batch 741362; Difco, Detroit, Mich.). hsp60 and tryptic digests of hsp60 were prepared as described previously (17). Infection with L. monocytogenes. Mice were starved for 18 h before infection and then infected orally (p.o.) with various sublethal doses of bacteria, as indicated for each experiment, with a gastric tube. The 50% lethal dose of L.

* Corresponding author. t Permanent address: Department of Veterinary Public Health, Institute of Public Health, 4-6-1 Shirokanedai, Minatoku, Tokyo

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monocytogenes after p.o. infection was >5 x 109 organisms. After infection, mice were kept without food and water for 4 h. At the indicated days after infection, the killer activity of and interleukin production by IEL were examined. In some experiments, mice were injected i.p. with the anti--y/b TCR MAb GL3 at 500 jig per mouse 6 or 2 days before sacrifice or infection, respectively. The CFU of bacteria in mesenteric lymph nodes (MLN), livers, and spleens were determined by plating on tryptic soy agar plates at 1 and 5 days after infection. The detection threshold was 2.0 log1o CFU in each organ. Preparation of IEL. IEL were isolated by the method described by Taguchi et al. (34) with slight modifications. Small intestines were obtained from three to six mice, and mesenteric and connective tissues were carefully removed. The Peyer's patches were then excised from the intestinal wall. Intestines were opened longitudinally, washed three times with phosphate-buffered saline (PBS), and cut into pieces of approximately 1 cm. Pieces of tissue were placed in a bottle containing 50 ml of RPMI 1640 medium with 2% fetal calf serum (FCS), sodium bicarbonate, nonessential amino acids, L-glutamine, 100 U of penicillin per ml, 100 ,ug of streptomycin per ml, and 5 x 10' M 2-mercaptoethanol and incubated for 30 min at 37°C with gentle shaking on a rotator. Supernatants (35 ml) containing cells were collected. The remaining tissue-containing medium was transferred to a 50-ml centrifuge tube and shaken vigorously for 15 s, and the cell-containing medium was collected. This process was repeated twice with fresh medium. Cells were pooled, washed, and resuspended in medium containing 10% FCS. Cells were then passed through a 10-ml syringe column packed loosely with siliconized glass wool (150 mg; Serva Feinbiochemica GmbH & Co., Heidelberg, Germany). Discontinuous density gradients were prepared with Percoll (1.124 g/ml; Seromed GmbH & Co., Berlin, Germany) diluted in medium to obtain lymphocytes. Gradients were prepared in 10-ml centrifuge tubes by layering from the bottom 70% Percoll (1 ml) and then 40% Percoll (2 ml) containing cells. The tubes were then centrifuged at 20°C for 20 min at 600 x g. The interface between the 70 and 40% Percoll layers contained lymphocytes with >95% cell viability. Approximately 2 x 106 to 6 x 106 cells per mouse were obtained by this method. Phenotype analysis of IEL. IEL were stained with appropriate dilutions of the following MAb for surface phenotype analyses. Anti-CD3 conjugated to fluorescein isothiocyanate (FITC) (145-2C11), a hamster MAb specific for the CD3 e-chain, was a generous gift of J. A. Bluestone (22). AntiL3T4-phycoerythrin, anti-Lyt2-biotin, and anti-Lyt2-FITC were purchased from Becton Dickinson, Mountain View, Calif. Anti-a/I TCR MAb H57-597, a hamster MAb specific for a/P TCR, was a generous gift of R. Kubo (18) and was labeled with FITC and biotin. Anti--y/b TCR MAb GL-3, a hamster MAb recognizing y/8 TCR, was a kind gift of L. Lefranqois (6) and was conjugated with biotin by standard methods. Biotinylated antibodies were visualized with streptavidin-phycoerythrin (Becton Dickinson) or streptavidin-tricolour (Dianova, Hamburg, Germany) for three-color analyses. Stained cells in 104 events were analyzed by fluorescence-activated cell sorting (FACS) with a FACScan (Becton Dickinson). IEL treated with anti-y/b TCR MAb in vivo were counterstained with anti-hamster immunoglobulin G (HmIgG)-FITC (Dianova) to determine whether IEL were labeled by anti--y/b TCR MAb. Cytotoxicity assay. Cytolytic activities of IEL were measured in a standard 51Cr release assay. IEL were incubated

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with 103 51Cr-labeled P815 cells or bone marrow macrophages as target cells for 4 h at 37°C in 7% CO2 at various effector:target cell ratios. Assays were done in the presence or absence of 2 ,ug of anti-CD3, anti-a/1 TCR, anti--y/b TCR MAb, or normal HmIgG (Dianova), 108 HK listeriae, or 50 ,ug of killed M. tuberculosis. After 4 h, 100 ,ul of the supematant was removed and assayed in a gamma counter (Cobra; Canberra Packard GmbH, Frankfurt, Germany). All experiments were performed at least three times, and assays were done in triplicate. Percent specific lysis was calculated as follows: [(experimental 51Cr release - spontaneous 51Cr release)/(maximum 51Cr release - spontaneous 51Cr release)] x 100. Interleukin determination by ELISA. IEL (2 x 105) were cultured with 105 irradiated (10 Gy) P815 cells for 24 h at 37°C in 7% CO2 in the presence or absence of anti-CD3, anti-aIl TCR, anti-y/b TCR MAb, or HmIgG (2 ,ug/ml), HK listeriae (108/ml), killed M. tuberculosis (50 pg/ml), concanavalin A (ConA) (2.5 p,g/ml) (Sigma, St. Louis, Mo.), hsp60 (25 p,g/ml), or trypsin-digested hsp60 (25 ,ug/ml). All experiments were done at least twice. Culture supernatants were assayed for IFN-y, interleukin-4 (IL-4), and IL-5 by enzyme-linked immunosorbent assay (ELISA) as described recently (32). Briefly, ELISA microplates (Immunoplate, Maxi-sorb; Nunc) were coated with anti-IFN--y (R4-6A2, 6 ,ug/ml [33]), anti-IL-4 (BVD4-1D11, 1 p,g/ml), or anti-IL-5 (TRFK-4, 7 ,ug/ml [31]) MAb in PBS (pH 7.4) at 4°C overnight. The anti-IL-4 and anti-IL-5 MAb were a generous gift of R. L. Coffman. Unsaturated sites were blocked with 1% bovine serum albumin (BSA) in PBS. Plates were washed and incubated with 50-,u aliquots of culture supernatants at 37°C for 1 h. After washing, 50 IlI of biotin-labeled anti-IFN--y (AN-18.17.24, 0.5 ,ug/ml [29]), anti-IL-4 (BVD624G2.7, 0.5 ,ug/ml), or anti-IL-5 (TRFK-5, 0.5 ,g/ml [31]) MAb was added, respectively, and incubated at 37°C for 1 h. Plates were washed and incubated with streptavidin-alkaline phosphatase, the reaction was developed, and the optical density was measured in an Intermed NJ-2000 Immunoreader. Interleukin titers were determined by standard titration curves of respective recombinant interleukins. The detection threshold was three times higher than the standard deviation of the value for the medium-only control. Assay for IFN-y production by IEL. We used the modified enzyme-linked immunospot (ELISPOT) method which was described by others (24). Briefly, ELISPOT plates (Millipore Co.; STHA 09610) were coated with 1 ,ug of anti-IFN--y (R4-6A2) MAb per ml in 100 R1 of 0.05 M carbonate buffer, pH 9.6, per well at 4°C overnight. Plates were washed once with PBS and blocked with 1% BSA-PBS for 2 h at 37°C. After two washes with PBS, appropriate numbers of IEL in 10% FCS-RPMI 1640 medium were added and incubated for 16 h at 37°C in 7% CO2. The plates were washed extensively (20 times) with 0.05% Tween 20-PBS (washing solution), and then biotinylated anti-IFN-y MAb (0.25 ,ug/ml, AN18.17.24) was added. After incubation at 37°C for 2 h, the plates were washed 12 times with washing solution, and avidin-alkaline phosphatase (1:20,000 in 0.1% BSA-0.05% Tween 20-PBS) was then added. After 1 h of incubation at 37°C, they were washed eight times with washing solution and once with alkaline phosphatase buffer (pH 9.5), and 50 pl of freshly prepared substrate (nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate in 70% dimethylformamide) was added. The plates were then incubated for 15 min at 37°C in the dark, and spots were counted under a dissecting microscope.

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TABLE 1. Surface phenotypes of IEL from naive C57BL/6 mice

60-

of

50-

Phenotype

CD3+ CD4+

CD8+ CD4+ CD8+ CD4- CD8-

TCR usage

Relative±

y/8

36.0 59.1 0.1 10.3 28.9 49.9 0.4 8.9 6.8 0.3

a/03 -y/I a/P 'y/8 a/0 fy/8 a/P

+ 6.0 4.3 +0.1 ± 7.4 ± 5.6 ± 2.8 ± 0.9 ± 1.4 ± 4.7 + 0.7

-y/8 Oa/, a Relative percentages were determined from the percentage of small

co, 40o

30-

.a

a)

20-

')

100-

lymphocytes gated. Standard deviations were calculated from the results of four to five experiments.

RESULTS Characterization of phenotype and biological activities of IEL from naive mice. IEL were separated, labeled with antibodies against T-cell subsets, and analyzed by cytofluorimetry. As shown in Table 1, >90% of the small lymphocyte population was CD3+. Of these cells, approximately 60% expressed the ct,B TCR and approximately 40% expressed the -y/l TCR. The vast majority (>90%) of CD3+ cells were CD8+, including CD4- CD8+ cells and CD4+ CD8+ cells. Only a minority (80% of CD3+ y/8 TCR+ cells were CD8+. CD4 expression was restricted to a/3 T cells, with some of them (