International Immunology, Vol. 4, No. 10, pp
© 1992 Oxford University Press
1129-1136
The appearance and role of yd T cells in the peritoneal cavity and liver during primary infection with Listeria monocytogenes in rats Takashi Hasegawa, Toshiyuki Tanaka1, and Yasunobu Yoshikai
Key words: yd T cells, bacterial infection, Listeria monocytogenes, protective immunity
Abstract We have previously reported that 76 T cells play important roles In protection during the early stage of infection with Listeria monocytogenes in mice. To generalize the protective roles of 76 T cells in Material Infection to different species, we examined the appearance of 76 T cells during infection with L. monocytogenes In Fisher F344 rats. The numbers of bacteria In the peritoneal cavity and liver increased to a maximum level on day 3 and then decreased to an undetectable level by day 10 after an intraperitoneal infection with a sublethal dose (1 x 10s) of viable L. monocytogenes in rats. CD3+a/3~ T cells In the peritoneal cavity and liver began to Increase on day 3, reached a maximum level on day 6, and thereafter decreased gradually by day 10 after infection. Northern blot analysis confirmed that the CD3 + a0- T cells expressed TCR 6 and 7 gene messages. In vivo treatment with anti-TCRa/3 mAb, which suppressed most of the a/3 T cells in the periphery and impaired resistance during the late stage of Material Infection, did not affect the host defense by day 6 after infection. A significantly Increased number of 76 T cells was detected in the peritoneal cavity of the TCRa/3-suppressed rats on day 6 after infection. These results suggest that the early appearing y& T cells may contribute to the host defense at a relatively early stage during listeriosis In rats. Introduction T lymphocytes represent the major mediators and coordinators of the host defense against intracellular bacteria such as Listeria monocytogenes and Mycobacteria tuberculosis (1 - 8). T lymphocytes can be divided into several subsets on the basis of accessory molecules and TCR. CD4+CD8~ T cells recognize the bacterial antigens in the context of self-MHC class II antigens and secrete various cytokines to activate macrophages and help B cells for antibody production, while CD4~CD8+ T cells recognize the antigens in the context of self-MHC class I antigens and exhibit cytotoxic activity against the infected cells. Most of these T cells in the peripheral lymphoid tissues express TCRa/S but a minor population of T cells are known to use TCR76. TCR-yS represents the first CD3-associated TCR in ontogeny (9) and displays a more limited diversity than TCRa0 (10). Furthermore 76 T cells are in resident populations in epidermis, intestine, and reproductive organs (11-19). There are several lines of evidence
that 76 T cells recognize non-polymorphic MHC class l-like molecules such as CD1c and thymus leukemic (TL) antigens (20,21). However, antigen-unselected 76 T cell hybridomas from mouse new born thymus have been reported to respond to PPD (derived from M. tuberculosis) recombinant Mycobacterium bovis 65 kDa heat shock protein (HSP), and an epitope spanning a highly conserved reigon of the 65 kDa HSP (4,22). Rajasekar et al. have reported that a subset of murine 76 T cells in lung react to antigens on self-cells in which a heat shock response is induced (23). Human 76 T cells in peripheral blood have also been shown to react to M. bovis 65 kDa HSP (6). Thus, a significant fraction of yS T cells in mouse and human are specialized to recognize phylogenically conserved HSP. The roles of the HSP- specific 76 T cells in host defense against bacterial infection and autoimmunity remain to be elucidated. We have previously reported that 76 T cells precede a/3 T cells
Correspondence to: Y. Yoshikai Transmitting editor: S. H E. Kaufmann
Received 16 March 1992, accepted 22 June 1992
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Laboratory of Germfree Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466, Japan 'Department of Immunology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
1130
yS T cells during listeriosis in rats
in appearance during infection with L. monocytogenes in mice and the early appearing 75 T cells play an important role in the host defense at an early stage of listerial infection (24,25). To generalize our earlier findings to different species, we examined the appearance of 76 T cells during listeriosis in rats. Our results indicated that, as in the case of mice, significant levels of y8 T cells were detected in the infection sites at a relatively early stage of infection with L. monocytogenes in rats.
procedures (26). Anti-TCRa/3 mAbs were obtained from supernatants of hybridoma cells R73 [kindly provided by Dr T. Hunig (27)] growing in serum-free SFM-101 medium (Nissui Laboratories). The antibodies were concentrated and then confirmed by SDS - PAGE. The concentration of antibodies was determined by Lowry methods. mAbs diluted 750 /*g/ml in PBS were stored at - 70°C until use. Two millihters (1.5 mg) of mAb was injected into the peritoneal cavity 3 days prior to infection with L. monocytogenes.
Methods
Flow cytometry (FCM)
Animals
Bacteria LJstena monocytogenes strain EGD was grown in tryptic soy broth (Nissui Laboratories, Tokyo, Japan) after two passages of BALB/c mice. Bacteria isolates were resuspended in PBS and stored at - 7 0 ° C in 1 ml aliquots until use for experiments. Individual rats were infected intrapentoneally (i.p.) with various doses of L. monocytogenes. The LDso of L. monocytogenes inoculated i.p. was 3 x 108 in F344 rats. The rats were killed at various times after infection by cardiac puncture under ether anesthetic. After bleeding, 25 ml of PBS was injected into their peritoneal cavities and harvested after gentle massage. Their spleens and livers were removed and separated into the sterile Teflon homogenizers containing 10 ml of cold PBS in the case of the spleen or 25 ml of cold PBS in the case of the liver. After each organ was homogenized thoroughly, the homogenates were established by plating serial 10-fold dilutions in sterile distilled water on tryptic soy agar (Nissui Laboratories). Colonies were counted 2 4 - 4 8 h later after incubation at 37°C.
Northern blot analysis Using the acid guanidium thiocyanate - phenol - chloroform extraction method (28), total RNA was extracted from the nonadherent cells from PECs and liver lymphocytes of normal rats or of rats infected with L. monocytogenes 6 days previously. The total RNA (10 ng) was electrophoresed on 0.8% agarose in a 10 mM sodium phosphate buffer, pH 7.0, and transferred to GeneScreen Plus (Biotechnology Systems NEN® Research Products, Boston, MA), then hybridized with ^P-labeled mouse VT2-JT2-CT2 cDNA probes (NG6) (29), mouse V j 6 - D j 1 - D j 2 - J 6 1 - C 4 cDNA (RD11V), and mouse Cff1 cDNA (NB2) (30). Following 16 h at 60°C in 1 M NaCI, 10% dextran sulfate and 100/ig/ml heat denatured salmon sperm DNA, the filters were washed for 30 min in 2 x SSC, 1 % SDS
Preparation of lymphocytes Rats were injected i.p. with 1 x 108 of viable L. monocytogenes on day 0. The peritoneal exudate cells (PECs) were harvested after gentle massage on days 3, 6, and 10 after infection. For liver lymphocytes, fresh liver was immediately perfused with sterile PBS through large branches of the portal vein to wash out all remaining peripheral blood and then mashed using a stainless steel mesh. After the coarse pieces were removed by centrifugation at 500 r.p.m. for 1 min, the cell suspensions were again centrifuged, re-suspended in 8 ml of 44% Percoll (Sigma), and layered on 5 ml of 67.5% Percoll. The gradients were centnfuged at 600 g at 20°C for 20 min. Lymphocytes at the interface were harvested and washed twice with Hank's balanced salt solution (HBSS). The spleen was homogenized with HBSS using a glass slide and filtered by cotton gauze. Antibodies Fluorescein isothiacyanate (FITC)-conjugated anti-TCRa/3 mAb, FITC-conjugated anti-CD8 mAb, phycoerythrin (PE)-conjugated anti-CD8 mAb, and PE-conjugated anti-CD4 mAb were purchased from Serotec (Oxford, UK). Biotin-conjugated anti-rat CD3 mAbs (1F4) were conjugated according to standard
Days altar
Infection
Fig. 1. Bacteria) growth in the peritoneal cavity, liver, and spleen of F344 rats inoculated i.p. with 1 x 10s L. monocytogenes. Male F344 rats were injected i.p. with 1 x 108 viable L. monocytogenes on day 0. The number of Listeria in the peritoneal cavity ( O ), liver (•- • -), and spleen (••••A--) of infected rats on the indicated days were determined by colony formation assay. Each point indicated the means of five rats ± SD
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Male Fisher F344/N (F344) rats were purchased from Japan SLC (Shizuoka, Japan). Rats aged 8 - 1 0 weeks were used for the experiments. Athymic nude rats (F344/N nulnu) were obtained from Institute for Laboratory Animal Research, Nagoya University School of Medicine.
For two-color FCM analysis, cells were stained with FITCconjugated mAb and biotin-conjugated mAb followed by streptoavidin - PE or FITC-conjugated mAb and PE-conjguated mAb. For three-color FCM analysis, cells were stained with FITCconjugated mAb, PE-conjugated mAb, and biotin-conjugated mAb followed by streptoavidin - RED613. Streptoavidin - PE was purchased from Becton-Dickinson (Mountain View, CA) and streptoavidin-RED613 was purchased from Gibco BRL (Gaithersburg, MD).
T cells during listeriosis in rats 1131
PEC
395
4.4
Spleen 18
Normal
3d
56 3
6d
0.9
lOd
Days after infection Fig. 2. Two-color FCM analysis for the expression of TCRa/3 and CD3 on non-adherent PECs, liver lymphocytes, and spleen lymphocytes of F344 rats inoculated i.p with 1 x 108 with L. monocytogenes. Non-adherent PECs, liver lymphocytes and spleen cells on days 3, 6, and 10 after an i.p injection of viable L. monocytogenes were stained with FITC-conjugated anti-TCRajS and biotin-conjugated CD3 (1F4) mAb, then with streptoavidin - PE.
at 60°C, and exposed to X-ray film at - 7 0 ° C with intensifying screens. The probe was removed by washing for 15 mm in boiling water.
Results Kinetics of bacterial growth in the organs after i.p. infection with L. monocytogenes As shown in Fig. 1, the numbers of bacteria in the peritoneal cavity reached a peak on day 3 after infection with a sublethal dose (1 x 108) of viable L. monocytogenes and then decreased to an undetectable level by day 10. The kinetics of bacterial growth in the liver were much the same as in the peritoneal cavity. In the spleen, the number of bacteria increased to a maximum level and remained at the relatively higher level on day 6, followed by a rapid decrease from day 6 to day 10 after infection. Appearance of CD3* TCRa/3' T cells in the peritoneal cavity and the liver in rats infected with L. monocytogenes We examined the kinetics of T cells subsets in the peritoneal cavity, liver, and spleen after an i.p. infection with L. monocytogenes using FCM analysis. A typical result from three different experiments is shown in Fig. 2. Only a few CD3+TCRa/3" T cells were detected in the resident PECs from normal F344 rats. The proportion of CD3+TCRa/S~ T cells in the PECs increased from 9.5% on day 3 to 17.1% on day 6 after
infection, and thereafter decreased on day 10 after infection. The absolute number of non-adherent cells only slightly increased from 8.0 x 1 C^/rat in normal rats to 1.2 x IC^/rat on day 6, and 1.7 x lO^rat on day 10 after infection. The number of liver lymphocytes remained at a stable level ( - 2 x 1 C^/rat) from day 3 to day 10 after infection. Similar to that seen in the PECs, the proportion of CD3+TCRa/3" T cells in the liver increased to 12.0% on day 6 and then decreased to normal levels on day 10 after infection. In addition, a significant increase in the proportion of CD3+TCRa/3+ T cells was evident in the liver on day 3 after infection. Thus, the absolute number of CD3+TCRa/3" T cells in the peritoneal cavity and liver temporarily increased on day 6 after infection. However, an increase in the proportion of the CD3+TCRaj3- T cells was not evident in the spleen (Fig. 2) and mesenteric lymph node (data not shown) at any stage after listerial infection. Next, we examined expression of CD3 and CD8 on the T cells appearing in the peritoneal cavity, liver, and spleen during listerial infection. As shown in Fig. 3, which represents in three different experiments, the proportion of CD3 + CD8 + T cells in the peritoneal cavity was significantly increased from 20.5% in normal rats to 36.1 % in rats on day 6 after infection. Similar to that seen in the peritoneal cavity, the liver showed a significant increase in the proportion of CD3 + CD8 + T cells on day 6 after infection, whereas such an increase was not evident in the spleen at this stage. Notably, an appreciable level of CD3~CD8+ cells was detected in all tissues that we examined (Fig. 3). This population
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yS T cells during listeriosis in rats
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24.7
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3.7
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Days after infection Fig. 3. Two-color FCM analysis of the expression of CD8 and CD3 on non-adherent PECs, liver lymphocytes, and spleen lymphocytes of F344 rats inoculated i.p. with 1 x 108 L monocytogenes. Non-adherent PECs, liver lymphocytes, and spleen ceOs on days 3, 6, and 10 after i.p. injection viable L. monocytogenes were stained with FITC-conjugated anti-CD8 and txotin-conjugated CD3 (1F4) mAb, then with streptoavidm-PE.
is thought to belong to natural killer cells in rats (31). The percentage of CD3~CD8 + cells in the peritoneal cavity decreased on day 6 after infection. The profile of expression of CD4 and CD8 on CD3 + T cells in the peritoneal cavity, liver, and spleen on day 6 after infection is shown in Fig. 4. Most CD3 + T cells in the peritoneal cavity expressed either CD4 or CD8, and only a few CD4" CD8~ cells were detected in the CD3 + T cells. Similar to those in the peritoneal cavity, liver and spleen contained very few CD4~CD8~ T cells at any stage after infection. In contrast to mouse listeriosis, CD3 + CD4"CD8- T cells were not induced during listeriosis in rats. We further determined the expression of CD4 and CD8 on CD3+TCRa/3~ T cells in the peritoneal cavity and liver on day 6 afer infection. The results of these experiments are summarized in Table 1. More than 70% of CD3+ TCRa/3" cells were found to express CD8 on their surface.
a relatively higher proportion of CD3 + TCRa/3" T cells (Fig. 5). These results suggest that the early appearing CD3 + TCRa/3 T cells in the peritoneal cavity and liver of rats during listeriosis may express TCR76 on the their surface. Effects of pretreatment with anti-TCRa/3 mAb in vivo on the growth of bacteria in rats infected with L. monocytogenes
Our results indicated that the CD3 + TCRa/9- T cells bearing CD8 and presumably TCR76 appeared temporarily at the inflamed sites such as the peritoneal cavity and liver at day 6 after i.p. infection with L. monocytogenes. To further investigate the protective role of the 76 T cells appearing in the peritoneal cavity during listeriois, TCRa0+ T cell-supressed rats were prepared by an i.p. injection of anti-rat TCRa/3 mAb. As shown in Fig. 6, CD3 + TCRa/3+ T cells were hardly detected in the peritoneal cavity, liver, and spleen until day 6 after treatment wrth 1.5 mg R73. CD3 + T cells in the peritoneal cavity were almost completely deleted on day 6 after treatment, while the liver and Expression of TCR y and S genes in peritoneal non-adherent cells spleen on day 3 and on day 6 after the treatment contained a and liver lymphocytes significant number of CD3 + TCRa/3" T cells, which might + comprise both TCR76 T cells and T cells modulated in expression To confirm that the CD3 TCRa/?~ T cells appearing in the of TCRa/3 by the treatment with the mAb. An appreciable level inflamed sites during listeriosis expressed TCR76, we examined of CD3 + TCRa/3+ T cells was detected in these organs on day the expression of TCR 7 and 6 genes of the lymphocytes in the 10 after treatment. The number of bacteria was counted on days peritoneal cavity and liver of rats inoculated i.p. with 3, 6, and 10 after i.p. infection with a sublethal dose of viable L. monocytogenes 6 days previously by Northern Wot analysis L. monocytogenes (1 x 107). As shown in Fig. 7, the number using murine TCR 7, 5, and /3 chain cDNA probes. The abundant of bacteria in the peritoneal cavity of the control rats was expression of TCR y and 5 genes was detected in the non4.3 x 10" c.f.u. on day 3 and 1.6 x 104 c.f.u. on day 6 after adherent PECs and liver lymphocytes on day 6, which contained
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191
yS T cells during listeriosis in rats infection, and then decreased to 3.0 x 102 c.f.u. by day 10. The numbers of L. monocytogenes in the peritoneal cavity of TCRa0 T cell-suppressed rats were much the same as those of the control rats on day 3 and day 6 after infection, while the numbers of bacteria were significantly increased in the peritoneal
3.6
4.4
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Discussion 48.8
Spleen
CD8 Fig. 4. Three-color FCM analysis for expression of CD3, CD4, and CD8 on non-adherent PECs, liver lymphocytes, and spleen cells on day 6 after infection with 1 x 108 L. monocytogenes. The non-adherent PECs, liver lymphocytes, and spleen cells were stained with FITC-conjugated a/3. PE-conjugated CD4, and biotin-conjugated anti-CD3 (1F4), then with streptoavidin-RED613. The profile of CD4 and CD8 expression was displayed after gating on the CD3 + cells.
We obtained the first evidence that yd T cells appeared in the peritoneal cavity and liver at an early stage after listerial infection in rats, as in the case of mice (25,26). The a/3 T cell-suppressed rats showed much the same resistance at the early stage after infection as that seen in control rats. At this stage, a significantly increased number of y8 T cells were detected in the a/3 T cellsuppressed rats. During listeriois, the early appearing yd T cells may participate in the host defense at an early stage of primary infection with L. monocytogenes among various species. L. monocytogenes is a facultative intracellular Gram-positive bacterium that evokes a strong T cell-mediated immune responses in mice (1,32-36). The kinetics of bacterial growth in rats infected with L. monocytogenes showed much the same pattern as that seen in mice, although the LD 50 was considerably higher in rats than in mice. The number of bacteria in the liver and spleen increased to a maximum by day 3 after i.p. infection with L. monocytogenes and then gradually decreased to an undetectable level by day 10 after infection. At
Table 1. The proportion of CD4 + T cells, CD8 + T cells, or CD4-CD8" T cells in CD3 + a/3" or CD3 + a/3 + cells in non-adherent PECs and liver lymphocytes from rats inoculated i.p. with 1 x 108 L. monocytogenes 6 days previously* CD3+a0Normal rat CD4 + CD8 + CD4-CD8" Non-adherent PECs Liver lymphocytes
11.1 kb R g . 5. Expression of TCR 7 and 5 genes in non-adherent PECs and liver lymphocytes of F344 rats inoculated i.p. with 1 x 108 L. monocytogenes 6 days previously. Total RNA (10 ^g) was electrophoresed and transferred to GeneScreen Plus, then hybridized with murine V 2 - J 2 - C 2 cDNA probes (NG6) or murine V ^ - D j i - D S 2 - J 5 1 - C 6 (RD11V) and munne C^I cDNA (NB2)
>, PEC
Liver
40.8
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45.4
14.4
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255
24.7
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25.2
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3d
6d
10d
Days after R73 treatment Fig. 6. Two-color FCM analysis of the expression of TCRa/3 and CD3 on non-adherent PECs, Dver lymphocytes, and spleen cells of F344 rats treated i.p. with anti-ral TCRa£ mAb. F344 rats were injected i.p. with 1.5 mg of anti-rat TCRa/3 mAb. Two-color FCM analysis of the expression of TCRa/3 and CD3 was performed on days 3, 6, and 10 after infection.
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o
this stage, a strong delayed footpad reaction could be observed (data not shown), indicating that cell-mediated immunity against L. monocytogenes developed on day 10 after infection. Our present data clearly show that, similar to mouse listeriosis, the y8 T cells appeared in the inflamed sites at the early stages after infection with L. monocytogends in rats. The early appearing yd T cells may cover the gap between the very early phase protection mediated by non-specific inflammatory phagocytes and the late phase protection mediated by /./sfena-specific immunity. Anti-TCR a/3 mAbs can be used in vivo depletion/modulation of a/3 T cells in the peripheral lymphoid organs. Our present results showed that i.p. injection with antiTCR a/3 mAbs depletes/modulates the a/3 T cells in the peritoneal cavity, liver, and spleen for at least 6 days after administration. Strikingly, the anti-listeria resistance on day 10 after infection was severely impaired by pretreatment with anti-TCRa/3 mAb, confirming that the a/3 T cells play a crucial role in protection against listenal infection at the late stage of infection. However, the numbers of bacteria in the TCRa/3+ T cell-suppressed rats on days 3 and 6 afer infection were much the same as seen in control rats; however, athymic nude rats showed significantly increased number of bacteria in the PECs and liver on day 6 after infection. At these stages after infection, components other than the a/3 T cells may contribute to the resistance against listerial infection. L. monocytogenes usually escape from the host phagosome and exploit the cytoskeletal machinery, and then grow in the resident macrophages (37). However, the bacteria are easily killed by macrophages activated by interferon-y (38).
yd T cells during listeriosis in rats 1135
a)
Jd
6d
ICkl
Days after birccuon
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Fig. 7. Bactena growth in the per'itoneaJ cavity of anti-TCRa/3 mAb (R73)-treated F344 rats inoculated i p. with 1 x 107 L. monocytogenes 6 days previously. F344 rats were injected i p with 1.5 mg of R73 on day - 3 Rats were inoculated i.p with 1 x 107 L monocytogenes on day 0. (a) The number of Listeria from peritoneal cavities of infected rats on days 3, 6, and 10 was determined by colony formation assays. Each point indicates the mean of three rats, (b) Two-color FCM analysis of the expression of TCRa/3 and CD3 on non-adherent PECs which passed through nylon wood column. Rats were inoculated i.p. with 1 x 10' L. monocytogenes on day 0 and thei non-ai non-adherent PECs passed through nylon wool columns on day 6 after infection were stained with FITC-conjugated TCRa/3 and CD3.
NK cells may play an important role in protection through production of a significant level of interferon-7 at the very early stage of listerial infection in mice (39). However, in the case of rats in our experiments, any increase in the number of CD3~CD8+ cells, presumably NK cells, was not evident at day 6 after infection. On the contrary, a significantly increased number of CD3+TCRa/3~ T cells was detected in the peritoneal cavity in the TCRa/3+ T cell-suppressed rats at the early stage after infection, suggesting that the early appearing yd T cells may play a protective role in listerial infection at this stage, just before the appearance of Listeria TCRa/3+ T cells. It is another notable finding that the number of TCRa/3+ T cells in the liver was significantly increased on day 3 after listerial infection. Recently, Abo et a!. (40) have reported the predominant appearance of unique T cells with an intermediate intensity of TCRa/3 in the liver of mice after bacterial stimulation, which are thought to differentiate extrathymically in the sinusoid of the liver. Although no difference was observed in the intensity of the TCR/CD3 between liver T cells and spleen T cells in the case of rats, it is possible that the early increase in TCRa/3+ T cells in the liver after listerial infection may be due to an increase in T cells differentiating extrathymically in the liver. So far the specificity of the early appearing 76 T cells in rats during listeriois remains to be elucidated. In the case of mice, the early appearing 76 T cells respond to PPD, mycobacterial 65 kDa HSP, and oligopeptides spanning amino acids 180 - 1 9 6
of the 65 kDa HSP, and produce a significant level of interferon-7 (26). We speculate that the 76 T cells appearing at the early stage afer listerial infection in rats may also show specificity for exogenous and/or endogenous 65 kDa HSP and produce interferon-7 in response to the HSP, and consequently contribute to the host defense against listerial infection in rats. Ongoing analysis of cloning the yd T cells at the early stage of listerial infection in rats and the consecutive functional analysis may provide more information not only on elucidating the protective mechanism against the bacteria but also on clarifying the role of TCR yd recognition in the first lines of defense in rats. Acknowledgements We thank Dr T. Hunig for proving anti-TCRa0 mAb (R73). This work was supported in part (to Y.Y.) from the Ministry of Education, Science and Culture, the Ministry of Heafth and Welfare, The Nitto Foundation, The Ishida Foundation, The Arima Memorial Foundation, Japanese Foundation for Multidisciplinary Treatment of Cancer, and Special Coordination Funds of the Science and Technology Agency of the Japanese Government
Abbreviations FCM HBSS HSP MCF
flow cytometry Hank's balanced salt solution heat shock protein macrophage chemotacttc factor
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b)
t
1136
yS T cells during listeriosis in rats
NK PE PEC TL
natural killer phycoerythrin peritoneal exudate cell thymus leukemic
References
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1 Lane, F. C. and Unanue, E.R. 1972 Requirement of thymus (T) lymphocytes for resistance to listeriosis. J. Exp. Med. 135-1104. 2 Hahn, H and Kaufman, S. H. E. 1981. The role of cell-mediated immunity on bacterial infections. Rev. Infect. Dis 3'1221. 3 Modlin, R. L, Pirmez. C , Hofman, F. M., Torigian, V., Uyemura, K., Rea, T. H., Bloom, B R., and Brenner, M. B. 1989. Lymphocytes bearing antigen-specific yS T cell receptors accumulate in human infectious disease lesions. Nature 339.544. 4 O'Brien, R. L., Happ, M. P , Dallas, A., Palmer, E., Kubo, R , and Born, W. K 1989. Stimulation of a major subset of lymphocytes expressing T cell receptor 76 by an antigen derived from Mycobacterium tuberculosis. Cell 57:667. 5 Janis, E. M., Kaufmann, S. H. E., Schwartz, R H., and Pardoll.D. M. 1989. Activation of 76 T cells in the primary immune response to Mycobacterium tuberculosis. Science 244:713. 6 Haregewoin, A , Soman, G., Horn, R. C , and Finberg, R. W. 1989. Human y&* T cells respond to mycobacterial heat-shock protein. Nature 340.309. 7 Augustin, A., Kubo, R. T., and Sim, G. K. 1989. Resident pulmonary lymphocytes expressing the gamma/delta T-cell receptor Nature 340:239. 8 Holoshitz, J., Konnmg, F., Colligan, J. E., Bruyn, J D , and Strober, S. 1989. Isolation of CD4~CD8~ mycobacteria-reactive T lymphocyte clones from rheumatoid arthritis synovia! fluid. Nature 339:226. 9 Fowlkes, B. J and Pardoll, D. M. 1989. Molecular and cellular events of T cell development. Adv. Immunol. 44:207 10 Brenner, M. B., Strominger, J. L., and Krangel, M. S. 1988 The 76 T cell receptor. Adv. Immunol 43 132 11 Konig, F., Stingl, S., Yokoyama, W. M., Yamada, H., Tschachler, E , Scjvach, E. M., Coligan, J. E., and Maloy, W L 1987. Identification of a T3-asscciated 76 T cell receptor on Thy-1 + epidermal cell line. Science 236834. 12 Stingl, G , Konig, F., Yamada, H , Yokoyama, W. M., Tschachler, E , Bluestone, J. A., Steiner, G., Samelson, L. E., Lew, A M., Coligan, J. E , and Schvach, E. M. 1987 Thy-1 + dendritic epidermal cells express T3 antigen and the T-cell receptor 76 chain Proc. NatlAcad. Sci. USA 84:4586 13 Goodman, T. and Lefrancois, L. 1988. Expression of the 76-T cell receptor on intestinal CD8 + intraepithelial lymphocytes. Nature 333:855. 14 Bonneville, M C , Janeway, J C A , Ito, K , Haser, W., Ishida, I., Nakanishi, N., and Tonegawa, S. 1988. Intestinal intraepithelial lymphocytes are a distinct set of 76 T cells. Nature 336:479. 15 Lafaille, J. J., DeCloux, A., Bonneville, M., Takagaki, Y, and Tonegawa, S. 1989. Junctional sequences of T ceS receptor 76 genes implications for 76 T cell lineages and for a novel intermediate of V - ( D ) - J joining. Cell 59859. 16 Asarnow, D. M., Kuziet, W. A., Bonyhadi, M., Tigelaar, R. E., Tucker, P. W., and Allison, J P. 1988. Limited cfiversrty of gamma delta antigen receptor genes of Thy-1 + dendritic epidermal cells. Cell 55:837. 17 Bucy, R P., Chen, C.-L H., Cihak, J., Losch, U., and Cooper, M. D 1988. Avian T cells expressing gamma delta receptors localize in the splenic sinnsoids and the intestinal epithelium. J. Immunol. 141:2200. 18 Groh, V., Porcelli, S., Fabbi, M., Lanier, L, Piker, L J., Anderson, T., Warnke, R. A., Bahn, A. K., Strominger, J. L., and Brenner, M. B. 1989. Human lymphocytes beanng T cell receptor gamma/delta are phenotypicaBy diverse and evenly distributed throughout the tymphoid system. J. Exp. Med. 169:1277. 19.ltohara, S., Farr, A. G., Lafaille, J J., Bonneville, M., Takagaki, Y., Haas, W., and Tonegawa, S. 1990, Homing of a gamma delta thymocyte subset with homogeneous T-cell receptors to mucosal eptthelia. Nature 343:754.
20 Le francois, L. 1991. Extrathymic differentiation of intraepithelial lymphocytes: generation of a separate and unequal T-cell repertoire? Immunol Today 12436. 21 Kaer, L V., Wu, M. Ichikawa, Y., Ito, K , Bonneville, M., OstrandRosenberg, S., Murphy, K., and Tonegawa, S. 1991. Recognition of MHC TL gene products by 75 T cells. Immunol. Rev 12089. 22 Born, W., Hall, L, Dallas, A., Boyme), J., Shinnick, T. Young, D., Brennan, P., and O'Brien, R. 1990. Recognition of a peptide antigen by heat-shock-reactive 76 T lymphocytes. Science 249.67. 23 Rajasekar, R , Sim, G , and Augustin, A 1990 Self heat shock and 76 T cell reactivity. Proc. Nat! Acad. Sci. USA 87.1767. 24 Ohga, S , Yoshikai, Y., Takeda, Y., Hiromatsu, K., and Nomoto, K. 1990. Sequential appearance of 7*- and a/3-beanng T cells in the peritoneal cavity during an intraperitoneal infection with Listeria monocytogenes. Eur. J. Immunol 20:533. 25 Hiromatsu, K., Yoshikai, Y , Matsuzaki, G., Ohga, S., Muramori, K., Matsumoto.K., Jeffrey, A. B , and Nomoto, K. 1992. A protective role of 7* T cells in primary infection with Listena monocytogenes. J. Exp Med 175:49-56. 26 Tanaka, T., Masuko, T., Yagita, H., Tamura, T., and Hasimoto, Y. 1989. Characterization of a CD3-like rat T cell surface antigen recognized by a monoclonal antibody. J. Immunol. 142-2791 27 Huntg, T., Wallny, H.-J , Hartley, J. K., Lawetzky, A., and Tiefenthaler, G. 1989 A monoclonal antibody to a constant determinant of the rat T cell antigen receptor that induces T cell activation. Differential reactivity with subsets if immature and mature T lymohocytes. J Exp. Med. 16973. 28 Chomczynski, P. and Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156. 29 Yoshikai, Y , Reis, M., and Mak, T. W. 1986. Athymic express a high level of functional 7-chain but greatly reduced levels of a- and 0-chain T-cell receptor messages. Nature 324:482. 30 Kishihara, K , Yoshikai, Y., Matsuzaki, G., Tomooka, S., and Nomoto, K. 1988. 'Radioresistant' intrathymic T cell percursors express T cell receptor C gamma 4- and C delta-specific gene messages. Eur J. Immunol. 18:841. 31 Bouwens, L and Wisse, E. 1987. Immuno-electron microscopic characterization of large granular lymphocytes (natural killer cells) from rat liver. Eur J Immunol 17:1423. 32 Mitsuyama, M , Takeya, K., Nomoto, K., and Shimoton, S 1978 Three phases of phagocyte contribution to resistance against Listeria monocytogenes. J. Gen. Microbiol., 106:165. 33 Kaufmann, S H E , Simon, M. M., and Hahn, H. 1979. Specific Lyt 123 T cells are involved in protection against Listeria monocytogenes and in delayed-type hypersensitivity to listerial antigens. J. Exp. Med. 150:1033 34 Kaufman, S. H. E., Hug, E., Vath, U , and Muller, I. 1985. Effective protection against Listeria monocytogenes and delayed-type hypersensitivity to listenal antigens depend on cooperation between specific L3T4 + and Lyt2 + T cells. Infect. Immun 48:263. 35 Stolpmann, R. M , Naher, H , Osawa, H., Herrmann, T., Hahn, H., and Diamantstein, T. 1985. Production of Listera-specific rat T-cell clones and role of interieukin-2 receptors in regulation of Listeriadependent T-cell done growth in vitro. Infect. Immun 47:822 36. Melissa, C. W. and Douglas, D. M. 1984 The mediators of acquired resistance to Listeria monocytogenes are contained within a population of cytotoxic T. cells. Cell. Immunol. 87:538. 37 Hahn, H and Kaufman, S. H. E. 1981. The role of cell-mediated immunity in bacterial infections. Rev. Infect. Dis. 3:122 1. 38 Buchmaier, N. A. and Schreiber, R. D. 1985. Requirement of endogenous interferon-7 production for resolution of Listena monocytogenes infection. Proc Natl Acad Sci. USA 82:7404. 39 Dunn, P. L. and North, R. J. 1991. Early gamma interferon production by natural kHIer ceHs is important in defense against murine fisteriosis. Infect. Immun. 59:2892. 40 Abo, T., Ohteki, T., Seki, S., Koyamada.N., Yoshikai, Y., Matsuda, T., Rikiishi, H., and Kumagai, K. 1991. The appearance of T cells bearing self-reactive T cell receptor in the livers of mice injected with bacteria J. Exp. Med. 174:417.
© 1992 Oxford University Press
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The appearance and role of yd T cells in the peritoneal cavity and liver during primary infection with Listeria monocytogenes in rats Takashi Hasegawa, Toshiyuki Tanaka1, and Yasunobu Yoshikai
Key words: yd T cells, bacterial infection, Listeria monocytogenes, protective immunity
Abstract We have previously reported that 76 T cells play important roles In protection during the early stage of infection with Listeria monocytogenes in mice. To generalize the protective roles of 76 T cells in Material Infection to different species, we examined the appearance of 76 T cells during infection with L. monocytogenes In Fisher F344 rats. The numbers of bacteria In the peritoneal cavity and liver increased to a maximum level on day 3 and then decreased to an undetectable level by day 10 after an intraperitoneal infection with a sublethal dose (1 x 10s) of viable L. monocytogenes in rats. CD3+a/3~ T cells In the peritoneal cavity and liver began to Increase on day 3, reached a maximum level on day 6, and thereafter decreased gradually by day 10 after infection. Northern blot analysis confirmed that the CD3 + a0- T cells expressed TCR 6 and 7 gene messages. In vivo treatment with anti-TCRa/3 mAb, which suppressed most of the a/3 T cells in the periphery and impaired resistance during the late stage of Material Infection, did not affect the host defense by day 6 after infection. A significantly Increased number of 76 T cells was detected in the peritoneal cavity of the TCRa/3-suppressed rats on day 6 after infection. These results suggest that the early appearing y& T cells may contribute to the host defense at a relatively early stage during listeriosis In rats. Introduction T lymphocytes represent the major mediators and coordinators of the host defense against intracellular bacteria such as Listeria monocytogenes and Mycobacteria tuberculosis (1 - 8). T lymphocytes can be divided into several subsets on the basis of accessory molecules and TCR. CD4+CD8~ T cells recognize the bacterial antigens in the context of self-MHC class II antigens and secrete various cytokines to activate macrophages and help B cells for antibody production, while CD4~CD8+ T cells recognize the antigens in the context of self-MHC class I antigens and exhibit cytotoxic activity against the infected cells. Most of these T cells in the peripheral lymphoid tissues express TCRa/S but a minor population of T cells are known to use TCR76. TCR-yS represents the first CD3-associated TCR in ontogeny (9) and displays a more limited diversity than TCRa0 (10). Furthermore 76 T cells are in resident populations in epidermis, intestine, and reproductive organs (11-19). There are several lines of evidence
that 76 T cells recognize non-polymorphic MHC class l-like molecules such as CD1c and thymus leukemic (TL) antigens (20,21). However, antigen-unselected 76 T cell hybridomas from mouse new born thymus have been reported to respond to PPD (derived from M. tuberculosis) recombinant Mycobacterium bovis 65 kDa heat shock protein (HSP), and an epitope spanning a highly conserved reigon of the 65 kDa HSP (4,22). Rajasekar et al. have reported that a subset of murine 76 T cells in lung react to antigens on self-cells in which a heat shock response is induced (23). Human 76 T cells in peripheral blood have also been shown to react to M. bovis 65 kDa HSP (6). Thus, a significant fraction of yS T cells in mouse and human are specialized to recognize phylogenically conserved HSP. The roles of the HSP- specific 76 T cells in host defense against bacterial infection and autoimmunity remain to be elucidated. We have previously reported that 76 T cells precede a/3 T cells
Correspondence to: Y. Yoshikai Transmitting editor: S. H E. Kaufmann
Received 16 March 1992, accepted 22 June 1992
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Laboratory of Germfree Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466, Japan 'Department of Immunology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
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yS T cells during listeriosis in rats
in appearance during infection with L. monocytogenes in mice and the early appearing 75 T cells play an important role in the host defense at an early stage of listerial infection (24,25). To generalize our earlier findings to different species, we examined the appearance of 76 T cells during listeriosis in rats. Our results indicated that, as in the case of mice, significant levels of y8 T cells were detected in the infection sites at a relatively early stage of infection with L. monocytogenes in rats.
procedures (26). Anti-TCRa/3 mAbs were obtained from supernatants of hybridoma cells R73 [kindly provided by Dr T. Hunig (27)] growing in serum-free SFM-101 medium (Nissui Laboratories). The antibodies were concentrated and then confirmed by SDS - PAGE. The concentration of antibodies was determined by Lowry methods. mAbs diluted 750 /*g/ml in PBS were stored at - 70°C until use. Two millihters (1.5 mg) of mAb was injected into the peritoneal cavity 3 days prior to infection with L. monocytogenes.
Methods
Flow cytometry (FCM)
Animals
Bacteria LJstena monocytogenes strain EGD was grown in tryptic soy broth (Nissui Laboratories, Tokyo, Japan) after two passages of BALB/c mice. Bacteria isolates were resuspended in PBS and stored at - 7 0 ° C in 1 ml aliquots until use for experiments. Individual rats were infected intrapentoneally (i.p.) with various doses of L. monocytogenes. The LDso of L. monocytogenes inoculated i.p. was 3 x 108 in F344 rats. The rats were killed at various times after infection by cardiac puncture under ether anesthetic. After bleeding, 25 ml of PBS was injected into their peritoneal cavities and harvested after gentle massage. Their spleens and livers were removed and separated into the sterile Teflon homogenizers containing 10 ml of cold PBS in the case of the spleen or 25 ml of cold PBS in the case of the liver. After each organ was homogenized thoroughly, the homogenates were established by plating serial 10-fold dilutions in sterile distilled water on tryptic soy agar (Nissui Laboratories). Colonies were counted 2 4 - 4 8 h later after incubation at 37°C.
Northern blot analysis Using the acid guanidium thiocyanate - phenol - chloroform extraction method (28), total RNA was extracted from the nonadherent cells from PECs and liver lymphocytes of normal rats or of rats infected with L. monocytogenes 6 days previously. The total RNA (10 ng) was electrophoresed on 0.8% agarose in a 10 mM sodium phosphate buffer, pH 7.0, and transferred to GeneScreen Plus (Biotechnology Systems NEN® Research Products, Boston, MA), then hybridized with ^P-labeled mouse VT2-JT2-CT2 cDNA probes (NG6) (29), mouse V j 6 - D j 1 - D j 2 - J 6 1 - C 4 cDNA (RD11V), and mouse Cff1 cDNA (NB2) (30). Following 16 h at 60°C in 1 M NaCI, 10% dextran sulfate and 100/ig/ml heat denatured salmon sperm DNA, the filters were washed for 30 min in 2 x SSC, 1 % SDS
Preparation of lymphocytes Rats were injected i.p. with 1 x 108 of viable L. monocytogenes on day 0. The peritoneal exudate cells (PECs) were harvested after gentle massage on days 3, 6, and 10 after infection. For liver lymphocytes, fresh liver was immediately perfused with sterile PBS through large branches of the portal vein to wash out all remaining peripheral blood and then mashed using a stainless steel mesh. After the coarse pieces were removed by centrifugation at 500 r.p.m. for 1 min, the cell suspensions were again centrifuged, re-suspended in 8 ml of 44% Percoll (Sigma), and layered on 5 ml of 67.5% Percoll. The gradients were centnfuged at 600 g at 20°C for 20 min. Lymphocytes at the interface were harvested and washed twice with Hank's balanced salt solution (HBSS). The spleen was homogenized with HBSS using a glass slide and filtered by cotton gauze. Antibodies Fluorescein isothiacyanate (FITC)-conjugated anti-TCRa/3 mAb, FITC-conjugated anti-CD8 mAb, phycoerythrin (PE)-conjugated anti-CD8 mAb, and PE-conjugated anti-CD4 mAb were purchased from Serotec (Oxford, UK). Biotin-conjugated anti-rat CD3 mAbs (1F4) were conjugated according to standard
Days altar
Infection
Fig. 1. Bacteria) growth in the peritoneal cavity, liver, and spleen of F344 rats inoculated i.p. with 1 x 10s L. monocytogenes. Male F344 rats were injected i.p. with 1 x 108 viable L. monocytogenes on day 0. The number of Listeria in the peritoneal cavity ( O ), liver (•- • -), and spleen (••••A--) of infected rats on the indicated days were determined by colony formation assay. Each point indicated the means of five rats ± SD
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Male Fisher F344/N (F344) rats were purchased from Japan SLC (Shizuoka, Japan). Rats aged 8 - 1 0 weeks were used for the experiments. Athymic nude rats (F344/N nulnu) were obtained from Institute for Laboratory Animal Research, Nagoya University School of Medicine.
For two-color FCM analysis, cells were stained with FITCconjugated mAb and biotin-conjugated mAb followed by streptoavidin - PE or FITC-conjugated mAb and PE-conjguated mAb. For three-color FCM analysis, cells were stained with FITCconjugated mAb, PE-conjugated mAb, and biotin-conjugated mAb followed by streptoavidin - RED613. Streptoavidin - PE was purchased from Becton-Dickinson (Mountain View, CA) and streptoavidin-RED613 was purchased from Gibco BRL (Gaithersburg, MD).
T cells during listeriosis in rats 1131
PEC
395
4.4
Spleen 18
Normal
3d
56 3
6d
0.9
lOd
Days after infection Fig. 2. Two-color FCM analysis for the expression of TCRa/3 and CD3 on non-adherent PECs, liver lymphocytes, and spleen lymphocytes of F344 rats inoculated i.p with 1 x 108 with L. monocytogenes. Non-adherent PECs, liver lymphocytes and spleen cells on days 3, 6, and 10 after an i.p injection of viable L. monocytogenes were stained with FITC-conjugated anti-TCRajS and biotin-conjugated CD3 (1F4) mAb, then with streptoavidin - PE.
at 60°C, and exposed to X-ray film at - 7 0 ° C with intensifying screens. The probe was removed by washing for 15 mm in boiling water.
Results Kinetics of bacterial growth in the organs after i.p. infection with L. monocytogenes As shown in Fig. 1, the numbers of bacteria in the peritoneal cavity reached a peak on day 3 after infection with a sublethal dose (1 x 108) of viable L. monocytogenes and then decreased to an undetectable level by day 10. The kinetics of bacterial growth in the liver were much the same as in the peritoneal cavity. In the spleen, the number of bacteria increased to a maximum level and remained at the relatively higher level on day 6, followed by a rapid decrease from day 6 to day 10 after infection. Appearance of CD3* TCRa/3' T cells in the peritoneal cavity and the liver in rats infected with L. monocytogenes We examined the kinetics of T cells subsets in the peritoneal cavity, liver, and spleen after an i.p. infection with L. monocytogenes using FCM analysis. A typical result from three different experiments is shown in Fig. 2. Only a few CD3+TCRa/3" T cells were detected in the resident PECs from normal F344 rats. The proportion of CD3+TCRa/S~ T cells in the PECs increased from 9.5% on day 3 to 17.1% on day 6 after
infection, and thereafter decreased on day 10 after infection. The absolute number of non-adherent cells only slightly increased from 8.0 x 1 C^/rat in normal rats to 1.2 x IC^/rat on day 6, and 1.7 x lO^rat on day 10 after infection. The number of liver lymphocytes remained at a stable level ( - 2 x 1 C^/rat) from day 3 to day 10 after infection. Similar to that seen in the PECs, the proportion of CD3+TCRa/3" T cells in the liver increased to 12.0% on day 6 and then decreased to normal levels on day 10 after infection. In addition, a significant increase in the proportion of CD3+TCRa/3+ T cells was evident in the liver on day 3 after infection. Thus, the absolute number of CD3+TCRa/3" T cells in the peritoneal cavity and liver temporarily increased on day 6 after infection. However, an increase in the proportion of the CD3+TCRaj3- T cells was not evident in the spleen (Fig. 2) and mesenteric lymph node (data not shown) at any stage after listerial infection. Next, we examined expression of CD3 and CD8 on the T cells appearing in the peritoneal cavity, liver, and spleen during listerial infection. As shown in Fig. 3, which represents in three different experiments, the proportion of CD3 + CD8 + T cells in the peritoneal cavity was significantly increased from 20.5% in normal rats to 36.1 % in rats on day 6 after infection. Similar to that seen in the peritoneal cavity, the liver showed a significant increase in the proportion of CD3 + CD8 + T cells on day 6 after infection, whereas such an increase was not evident in the spleen at this stage. Notably, an appreciable level of CD3~CD8+ cells was detected in all tissues that we examined (Fig. 3). This population
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38.3
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yS T cells during listeriosis in rats
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24.5
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g U
19.9
201
21.3
24.7
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ii m
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24.3
272
16.4
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3.7
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3d
6d
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Days after infection Fig. 3. Two-color FCM analysis of the expression of CD8 and CD3 on non-adherent PECs, liver lymphocytes, and spleen lymphocytes of F344 rats inoculated i.p. with 1 x 108 L monocytogenes. Non-adherent PECs, liver lymphocytes, and spleen ceOs on days 3, 6, and 10 after i.p. injection viable L. monocytogenes were stained with FITC-conjugated anti-CD8 and txotin-conjugated CD3 (1F4) mAb, then with streptoavidm-PE.
is thought to belong to natural killer cells in rats (31). The percentage of CD3~CD8 + cells in the peritoneal cavity decreased on day 6 after infection. The profile of expression of CD4 and CD8 on CD3 + T cells in the peritoneal cavity, liver, and spleen on day 6 after infection is shown in Fig. 4. Most CD3 + T cells in the peritoneal cavity expressed either CD4 or CD8, and only a few CD4" CD8~ cells were detected in the CD3 + T cells. Similar to those in the peritoneal cavity, liver and spleen contained very few CD4~CD8~ T cells at any stage after infection. In contrast to mouse listeriosis, CD3 + CD4"CD8- T cells were not induced during listeriosis in rats. We further determined the expression of CD4 and CD8 on CD3+TCRa/3~ T cells in the peritoneal cavity and liver on day 6 afer infection. The results of these experiments are summarized in Table 1. More than 70% of CD3+ TCRa/3" cells were found to express CD8 on their surface.
a relatively higher proportion of CD3 + TCRa/3" T cells (Fig. 5). These results suggest that the early appearing CD3 + TCRa/3 T cells in the peritoneal cavity and liver of rats during listeriosis may express TCR76 on the their surface. Effects of pretreatment with anti-TCRa/3 mAb in vivo on the growth of bacteria in rats infected with L. monocytogenes
Our results indicated that the CD3 + TCRa/9- T cells bearing CD8 and presumably TCR76 appeared temporarily at the inflamed sites such as the peritoneal cavity and liver at day 6 after i.p. infection with L. monocytogenes. To further investigate the protective role of the 76 T cells appearing in the peritoneal cavity during listeriois, TCRa0+ T cell-supressed rats were prepared by an i.p. injection of anti-rat TCRa/3 mAb. As shown in Fig. 6, CD3 + TCRa/3+ T cells were hardly detected in the peritoneal cavity, liver, and spleen until day 6 after treatment wrth 1.5 mg R73. CD3 + T cells in the peritoneal cavity were almost completely deleted on day 6 after treatment, while the liver and Expression of TCR y and S genes in peritoneal non-adherent cells spleen on day 3 and on day 6 after the treatment contained a and liver lymphocytes significant number of CD3 + TCRa/3" T cells, which might + comprise both TCR76 T cells and T cells modulated in expression To confirm that the CD3 TCRa/?~ T cells appearing in the of TCRa/3 by the treatment with the mAb. An appreciable level inflamed sites during listeriosis expressed TCR76, we examined of CD3 + TCRa/3+ T cells was detected in these organs on day the expression of TCR 7 and 6 genes of the lymphocytes in the 10 after treatment. The number of bacteria was counted on days peritoneal cavity and liver of rats inoculated i.p. with 3, 6, and 10 after i.p. infection with a sublethal dose of viable L. monocytogenes 6 days previously by Northern Wot analysis L. monocytogenes (1 x 107). As shown in Fig. 7, the number using murine TCR 7, 5, and /3 chain cDNA probes. The abundant of bacteria in the peritoneal cavity of the control rats was expression of TCR y and 5 genes was detected in the non4.3 x 10" c.f.u. on day 3 and 1.6 x 104 c.f.u. on day 6 after adherent PECs and liver lymphocytes on day 6, which contained
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yS T cells during listeriosis in rats infection, and then decreased to 3.0 x 102 c.f.u. by day 10. The numbers of L. monocytogenes in the peritoneal cavity of TCRa0 T cell-suppressed rats were much the same as those of the control rats on day 3 and day 6 after infection, while the numbers of bacteria were significantly increased in the peritoneal
3.6
4.4
: 36J
t
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Discussion 48.8
Spleen
CD8 Fig. 4. Three-color FCM analysis for expression of CD3, CD4, and CD8 on non-adherent PECs, liver lymphocytes, and spleen cells on day 6 after infection with 1 x 108 L. monocytogenes. The non-adherent PECs, liver lymphocytes, and spleen cells were stained with FITC-conjugated a/3. PE-conjugated CD4, and biotin-conjugated anti-CD3 (1F4), then with streptoavidin-RED613. The profile of CD4 and CD8 expression was displayed after gating on the CD3 + cells.
We obtained the first evidence that yd T cells appeared in the peritoneal cavity and liver at an early stage after listerial infection in rats, as in the case of mice (25,26). The a/3 T cell-suppressed rats showed much the same resistance at the early stage after infection as that seen in control rats. At this stage, a significantly increased number of y8 T cells were detected in the a/3 T cellsuppressed rats. During listeriois, the early appearing yd T cells may participate in the host defense at an early stage of primary infection with L. monocytogenes among various species. L. monocytogenes is a facultative intracellular Gram-positive bacterium that evokes a strong T cell-mediated immune responses in mice (1,32-36). The kinetics of bacterial growth in rats infected with L. monocytogenes showed much the same pattern as that seen in mice, although the LD 50 was considerably higher in rats than in mice. The number of bacteria in the liver and spleen increased to a maximum by day 3 after i.p. infection with L. monocytogenes and then gradually decreased to an undetectable level by day 10 after infection. At
Table 1. The proportion of CD4 + T cells, CD8 + T cells, or CD4-CD8" T cells in CD3 + a/3" or CD3 + a/3 + cells in non-adherent PECs and liver lymphocytes from rats inoculated i.p. with 1 x 108 L. monocytogenes 6 days previously* CD3+a0Normal rat CD4 + CD8 + CD4-CD8" Non-adherent PECs Liver lymphocytes
11.1 kb R g . 5. Expression of TCR 7 and 5 genes in non-adherent PECs and liver lymphocytes of F344 rats inoculated i.p. with 1 x 108 L. monocytogenes 6 days previously. Total RNA (10 ^g) was electrophoresed and transferred to GeneScreen Plus, then hybridized with murine V 2 - J 2 - C 2 cDNA probes (NG6) or murine V ^ - D j i - D S 2 - J 5 1 - C 6 (RD11V) and munne C^I cDNA (NB2)
>, PEC
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40.8
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14.4
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255
24.7
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.7
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6d
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Days after R73 treatment Fig. 6. Two-color FCM analysis of the expression of TCRa/3 and CD3 on non-adherent PECs, Dver lymphocytes, and spleen cells of F344 rats treated i.p. with anti-ral TCRa£ mAb. F344 rats were injected i.p. with 1.5 mg of anti-rat TCRa/3 mAb. Two-color FCM analysis of the expression of TCRa/3 and CD3 was performed on days 3, 6, and 10 after infection.
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o
this stage, a strong delayed footpad reaction could be observed (data not shown), indicating that cell-mediated immunity against L. monocytogenes developed on day 10 after infection. Our present data clearly show that, similar to mouse listeriosis, the y8 T cells appeared in the inflamed sites at the early stages after infection with L. monocytogends in rats. The early appearing yd T cells may cover the gap between the very early phase protection mediated by non-specific inflammatory phagocytes and the late phase protection mediated by /./sfena-specific immunity. Anti-TCR a/3 mAbs can be used in vivo depletion/modulation of a/3 T cells in the peripheral lymphoid organs. Our present results showed that i.p. injection with antiTCR a/3 mAbs depletes/modulates the a/3 T cells in the peritoneal cavity, liver, and spleen for at least 6 days after administration. Strikingly, the anti-listeria resistance on day 10 after infection was severely impaired by pretreatment with anti-TCRa/3 mAb, confirming that the a/3 T cells play a crucial role in protection against listenal infection at the late stage of infection. However, the numbers of bacteria in the TCRa/3+ T cell-suppressed rats on days 3 and 6 afer infection were much the same as seen in control rats; however, athymic nude rats showed significantly increased number of bacteria in the PECs and liver on day 6 after infection. At these stages after infection, components other than the a/3 T cells may contribute to the resistance against listerial infection. L. monocytogenes usually escape from the host phagosome and exploit the cytoskeletal machinery, and then grow in the resident macrophages (37). However, the bacteria are easily killed by macrophages activated by interferon-y (38).
yd T cells during listeriosis in rats 1135
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91.0
22.6
w
*/&mil ^jWfcHl
0
0.1 • Moas
• TCR a P •
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R73 Ircaunciil
Fig. 7. Bactena growth in the per'itoneaJ cavity of anti-TCRa/3 mAb (R73)-treated F344 rats inoculated i p. with 1 x 107 L. monocytogenes 6 days previously. F344 rats were injected i p with 1.5 mg of R73 on day - 3 Rats were inoculated i.p with 1 x 107 L monocytogenes on day 0. (a) The number of Listeria from peritoneal cavities of infected rats on days 3, 6, and 10 was determined by colony formation assays. Each point indicates the mean of three rats, (b) Two-color FCM analysis of the expression of TCRa/3 and CD3 on non-adherent PECs which passed through nylon wood column. Rats were inoculated i.p. with 1 x 10' L. monocytogenes on day 0 and thei non-ai non-adherent PECs passed through nylon wool columns on day 6 after infection were stained with FITC-conjugated TCRa/3 and CD3.
NK cells may play an important role in protection through production of a significant level of interferon-7 at the very early stage of listerial infection in mice (39). However, in the case of rats in our experiments, any increase in the number of CD3~CD8+ cells, presumably NK cells, was not evident at day 6 after infection. On the contrary, a significantly increased number of CD3+TCRa/3~ T cells was detected in the peritoneal cavity in the TCRa/3+ T cell-suppressed rats at the early stage after infection, suggesting that the early appearing yd T cells may play a protective role in listerial infection at this stage, just before the appearance of Listeria TCRa/3+ T cells. It is another notable finding that the number of TCRa/3+ T cells in the liver was significantly increased on day 3 after listerial infection. Recently, Abo et a!. (40) have reported the predominant appearance of unique T cells with an intermediate intensity of TCRa/3 in the liver of mice after bacterial stimulation, which are thought to differentiate extrathymically in the sinusoid of the liver. Although no difference was observed in the intensity of the TCR/CD3 between liver T cells and spleen T cells in the case of rats, it is possible that the early increase in TCRa/3+ T cells in the liver after listerial infection may be due to an increase in T cells differentiating extrathymically in the liver. So far the specificity of the early appearing 76 T cells in rats during listeriois remains to be elucidated. In the case of mice, the early appearing 76 T cells respond to PPD, mycobacterial 65 kDa HSP, and oligopeptides spanning amino acids 180 - 1 9 6
of the 65 kDa HSP, and produce a significant level of interferon-7 (26). We speculate that the 76 T cells appearing at the early stage afer listerial infection in rats may also show specificity for exogenous and/or endogenous 65 kDa HSP and produce interferon-7 in response to the HSP, and consequently contribute to the host defense against listerial infection in rats. Ongoing analysis of cloning the yd T cells at the early stage of listerial infection in rats and the consecutive functional analysis may provide more information not only on elucidating the protective mechanism against the bacteria but also on clarifying the role of TCR yd recognition in the first lines of defense in rats. Acknowledgements We thank Dr T. Hunig for proving anti-TCRa0 mAb (R73). This work was supported in part (to Y.Y.) from the Ministry of Education, Science and Culture, the Ministry of Heafth and Welfare, The Nitto Foundation, The Ishida Foundation, The Arima Memorial Foundation, Japanese Foundation for Multidisciplinary Treatment of Cancer, and Special Coordination Funds of the Science and Technology Agency of the Japanese Government
Abbreviations FCM HBSS HSP MCF
flow cytometry Hank's balanced salt solution heat shock protein macrophage chemotacttc factor
Downloaded from http://intimm.oxfordjournals.org/ at Indiana University Library on July 18, 2015
b)
t
1136
yS T cells during listeriosis in rats
NK PE PEC TL
natural killer phycoerythrin peritoneal exudate cell thymus leukemic
References
Downloaded from http://intimm.oxfordjournals.org/ at Indiana University Library on July 18, 2015
1 Lane, F. C. and Unanue, E.R. 1972 Requirement of thymus (T) lymphocytes for resistance to listeriosis. J. Exp. Med. 135-1104. 2 Hahn, H and Kaufman, S. H. E. 1981. The role of cell-mediated immunity on bacterial infections. Rev. Infect. Dis 3'1221. 3 Modlin, R. L, Pirmez. C , Hofman, F. M., Torigian, V., Uyemura, K., Rea, T. H., Bloom, B R., and Brenner, M. B. 1989. Lymphocytes bearing antigen-specific yS T cell receptors accumulate in human infectious disease lesions. Nature 339.544. 4 O'Brien, R. L., Happ, M. P , Dallas, A., Palmer, E., Kubo, R , and Born, W. K 1989. Stimulation of a major subset of lymphocytes expressing T cell receptor 76 by an antigen derived from Mycobacterium tuberculosis. Cell 57:667. 5 Janis, E. M., Kaufmann, S. H. E., Schwartz, R H., and Pardoll.D. M. 1989. Activation of 76 T cells in the primary immune response to Mycobacterium tuberculosis. Science 244:713. 6 Haregewoin, A , Soman, G., Horn, R. C , and Finberg, R. W. 1989. Human y&* T cells respond to mycobacterial heat-shock protein. Nature 340.309. 7 Augustin, A., Kubo, R. T., and Sim, G. K. 1989. Resident pulmonary lymphocytes expressing the gamma/delta T-cell receptor Nature 340:239. 8 Holoshitz, J., Konnmg, F., Colligan, J. E., Bruyn, J D , and Strober, S. 1989. Isolation of CD4~CD8~ mycobacteria-reactive T lymphocyte clones from rheumatoid arthritis synovia! fluid. Nature 339:226. 9 Fowlkes, B. J and Pardoll, D. M. 1989. Molecular and cellular events of T cell development. Adv. Immunol. 44:207 10 Brenner, M. B., Strominger, J. L., and Krangel, M. S. 1988 The 76 T cell receptor. Adv. Immunol 43 132 11 Konig, F., Stingl, S., Yokoyama, W. M., Yamada, H., Tschachler, E , Scjvach, E. M., Coligan, J. E., and Maloy, W L 1987. Identification of a T3-asscciated 76 T cell receptor on Thy-1 + epidermal cell line. Science 236834. 12 Stingl, G , Konig, F., Yamada, H , Yokoyama, W. M., Tschachler, E , Bluestone, J. A., Steiner, G., Samelson, L. E., Lew, A M., Coligan, J. E , and Schvach, E. M. 1987 Thy-1 + dendritic epidermal cells express T3 antigen and the T-cell receptor 76 chain Proc. NatlAcad. Sci. USA 84:4586 13 Goodman, T. and Lefrancois, L. 1988. Expression of the 76-T cell receptor on intestinal CD8 + intraepithelial lymphocytes. Nature 333:855. 14 Bonneville, M C , Janeway, J C A , Ito, K , Haser, W., Ishida, I., Nakanishi, N., and Tonegawa, S. 1988. Intestinal intraepithelial lymphocytes are a distinct set of 76 T cells. Nature 336:479. 15 Lafaille, J. J., DeCloux, A., Bonneville, M., Takagaki, Y, and Tonegawa, S. 1989. Junctional sequences of T ceS receptor 76 genes implications for 76 T cell lineages and for a novel intermediate of V - ( D ) - J joining. Cell 59859. 16 Asarnow, D. M., Kuziet, W. A., Bonyhadi, M., Tigelaar, R. E., Tucker, P. W., and Allison, J P. 1988. Limited cfiversrty of gamma delta antigen receptor genes of Thy-1 + dendritic epidermal cells. Cell 55:837. 17 Bucy, R P., Chen, C.-L H., Cihak, J., Losch, U., and Cooper, M. D 1988. Avian T cells expressing gamma delta receptors localize in the splenic sinnsoids and the intestinal epithelium. J. Immunol. 141:2200. 18 Groh, V., Porcelli, S., Fabbi, M., Lanier, L, Piker, L J., Anderson, T., Warnke, R. A., Bahn, A. K., Strominger, J. L., and Brenner, M. B. 1989. Human lymphocytes beanng T cell receptor gamma/delta are phenotypicaBy diverse and evenly distributed throughout the tymphoid system. J. Exp. Med. 169:1277. 19.ltohara, S., Farr, A. G., Lafaille, J J., Bonneville, M., Takagaki, Y., Haas, W., and Tonegawa, S. 1990, Homing of a gamma delta thymocyte subset with homogeneous T-cell receptors to mucosal eptthelia. Nature 343:754.
20 Le francois, L. 1991. Extrathymic differentiation of intraepithelial lymphocytes: generation of a separate and unequal T-cell repertoire? Immunol Today 12436. 21 Kaer, L V., Wu, M. Ichikawa, Y., Ito, K , Bonneville, M., OstrandRosenberg, S., Murphy, K., and Tonegawa, S. 1991. Recognition of MHC TL gene products by 75 T cells. Immunol. Rev 12089. 22 Born, W., Hall, L, Dallas, A., Boyme), J., Shinnick, T. Young, D., Brennan, P., and O'Brien, R. 1990. Recognition of a peptide antigen by heat-shock-reactive 76 T lymphocytes. Science 249.67. 23 Rajasekar, R , Sim, G , and Augustin, A 1990 Self heat shock and 76 T cell reactivity. Proc. Nat! Acad. Sci. USA 87.1767. 24 Ohga, S , Yoshikai, Y., Takeda, Y., Hiromatsu, K., and Nomoto, K. 1990. Sequential appearance of 7*- and a/3-beanng T cells in the peritoneal cavity during an intraperitoneal infection with Listeria monocytogenes. Eur. J. Immunol 20:533. 25 Hiromatsu, K., Yoshikai, Y , Matsuzaki, G., Ohga, S., Muramori, K., Matsumoto.K., Jeffrey, A. B , and Nomoto, K. 1992. A protective role of 7* T cells in primary infection with Listena monocytogenes. J. Exp Med 175:49-56. 26 Tanaka, T., Masuko, T., Yagita, H., Tamura, T., and Hasimoto, Y. 1989. Characterization of a CD3-like rat T cell surface antigen recognized by a monoclonal antibody. J. Immunol. 142-2791 27 Huntg, T., Wallny, H.-J , Hartley, J. K., Lawetzky, A., and Tiefenthaler, G. 1989 A monoclonal antibody to a constant determinant of the rat T cell antigen receptor that induces T cell activation. Differential reactivity with subsets if immature and mature T lymohocytes. J Exp. Med. 16973. 28 Chomczynski, P. and Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156. 29 Yoshikai, Y , Reis, M., and Mak, T. W. 1986. Athymic express a high level of functional 7-chain but greatly reduced levels of a- and 0-chain T-cell receptor messages. Nature 324:482. 30 Kishihara, K , Yoshikai, Y., Matsuzaki, G., Tomooka, S., and Nomoto, K. 1988. 'Radioresistant' intrathymic T cell percursors express T cell receptor C gamma 4- and C delta-specific gene messages. Eur J. Immunol. 18:841. 31 Bouwens, L and Wisse, E. 1987. Immuno-electron microscopic characterization of large granular lymphocytes (natural killer cells) from rat liver. Eur J Immunol 17:1423. 32 Mitsuyama, M , Takeya, K., Nomoto, K., and Shimoton, S 1978 Three phases of phagocyte contribution to resistance against Listeria monocytogenes. J. Gen. Microbiol., 106:165. 33 Kaufmann, S H E , Simon, M. M., and Hahn, H. 1979. Specific Lyt 123 T cells are involved in protection against Listeria monocytogenes and in delayed-type hypersensitivity to listerial antigens. J. Exp. Med. 150:1033 34 Kaufman, S. H. E., Hug, E., Vath, U , and Muller, I. 1985. Effective protection against Listeria monocytogenes and delayed-type hypersensitivity to listenal antigens depend on cooperation between specific L3T4 + and Lyt2 + T cells. Infect. Immun 48:263. 35 Stolpmann, R. M , Naher, H , Osawa, H., Herrmann, T., Hahn, H., and Diamantstein, T. 1985. Production of Listera-specific rat T-cell clones and role of interieukin-2 receptors in regulation of Listeriadependent T-cell done growth in vitro. Infect. Immun 47:822 36. Melissa, C. W. and Douglas, D. M. 1984 The mediators of acquired resistance to Listeria monocytogenes are contained within a population of cytotoxic T. cells. Cell. Immunol. 87:538. 37 Hahn, H and Kaufman, S. H. E. 1981. The role of cell-mediated immunity in bacterial infections. Rev. Infect. Dis. 3:122 1. 38 Buchmaier, N. A. and Schreiber, R. D. 1985. Requirement of endogenous interferon-7 production for resolution of Listena monocytogenes infection. Proc Natl Acad Sci. USA 82:7404. 39 Dunn, P. L. and North, R. J. 1991. Early gamma interferon production by natural kHIer ceHs is important in defense against murine fisteriosis. Infect. Immun. 59:2892. 40 Abo, T., Ohteki, T., Seki, S., Koyamada.N., Yoshikai, Y., Matsuda, T., Rikiishi, H., and Kumagai, K. 1991. The appearance of T cells bearing self-reactive T cell receptor in the livers of mice injected with bacteria J. Exp. Med. 174:417.
