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y/6-bearing cells during listeriosis

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Shouichi Ohga, Yasunobu Yoshikai, Yasuyuki Takeda, Kenji Hiromatsu and Kikuo Nomoto

Sequential appearance of y/6- and alp-bearing T cells in the peritoneal cavity during an i.p. infection with Listeria monocytogenefl

Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka

To search for a potential role of Tcell antigen receptor (TcR) y/6-bearing cells in host-defense against Listeriu monocytogenes,we analyzed the sequential appearance of y/6 and a / p Tcell in the peritoneal exudate cells (PEC) during an i.p. infection with sublethal dose (2 x 103) of viable Listeriu organisms in mice. The PEC on day 1 after the infection consisted of 48% macrophages and 50% lymphocytes, most of which were surface IgM+ (B) cells. The number of PEC increased to the maximal level by day 3. The PEC at this stage contained an appreciable number of CD3+ T cells in addition to a large number of macrophages. Of the CD3+ cells, the proportion of CD4-CD8- cells, most of which expressed no TcR a / p , iqcreased to the maximal level on day 3 after the infection. In correlation with an increased number of CD3+CD4-CD8-TcR a/pcells, high level of TcR $6 chain gene messages was detected in the nonadherent population of the PEC on this stage. On the other hand, the PEC on day 8 contained an increased number of CD4+CD8- and CD4-CD8+ cells which expressed TcR a/p chain on their surface.These results suggest that the y/6 Tcells precede the a / p Tcells in appearance during listerial infection. The y/6 Tcells may be involved at the first line of the host-defense against Listeriu.

1 Introduction Murine host response to Listeriu monocytogenes has been a useful model for studying protective mechanisms against facultative intracellular pathogens [1-71. The early response, which occurs during the first 48 h after onset of primary infection with Listeriu, has been attributed to resident M@ and early influx of BM-derived phagocytes in the liver and spleen of mice [3-51. The late response, beginning at about day 4, is characterized by the proliferation of listerial antigen-dependent T cells which further enhance bacterial killing in vivo. The Listeriu-immune T cells and their related cytokines represent the major mediators that confer protection against the disease [ M I . Of the Listeriu-immune T cells, class II-restricted CD4+ T cell subsets are considered to be main effector cells that secrete IFN-y capable of activating listericidal capacity in M@. However, there are several lines of evidence that besides CD4+ T cells, CD8+ T cells also participate in protective immunity to Listeriu [ l , 71. Although the functional properties of the Listeriu-immune T cells have been studied extensively, the basis for coordination of various T cell subsets in the Listeriu-immune T cells in host-defense against listerial infection remains uncertain. T cells recognize nominal antigens in the context of self-MHC molecules by TcR which is composed of a and p chains [8,9]. Another type of TcR, which is composed of

y and 6 chains, has also been identified [lo, 111. TcR y/6 appears to represent the first CD3-associated TcR in ontogeny and might represent the ancestral TcR in evolution. There have been several lines of evidence suggesting that MHC antigens are an important component of theTcR y/6 ligand [12, 131. It is possible that the y/6 Tcells are functionally similar to TcR a/p Tcells although these cells are only present in much smaller numbers and display more limited diversity as compared t o a / p Tcells. The subtype of y/6 Tcells has been recently demonstrated to be distributed in different anatomical sites such as epidermis [14, 151 and intestine [16,17]. More recently, there has been convincing evidence that at least a significant fraction of y/S Tcells are specialized to recognize epitopes on mycobacterial antigens [18-201. It is suggested that the appearance of broadreactive y/&bearing cells in microbial infections may serve as a first line of defense against invasion of the various pathogens [21-241.

In the present study, t o elucidate a potential role of the y/6 Tcells in host-defense against Listeriu, we examined the kinetics of y/6 and a/@ Tcells during an i.p. infection with Listeriu. Our results demonstrated that y/6 Tcells preceded the a/p Tcells in appearance after primary infection with Listeriu. The y/6 cells may play a role for covering the gap between phagocytic system and highly evolved type immune responses mediated by a / p T cells in host-defense against listerial infection.

[I 80081

*

This work was supported in part by grants toYYoshikai from the Ministry of Education, Science and Culture, Japan (62480167, 01015081), and from Special Coordination Funds of the Science and Technology Agency of the Japanese Government.

Correspondence: Yasunobu Yoshikai, Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812, Japan Abbreviation: hsp: Heat-shock protein 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

2 Materials and methods 2.1 Animals and microorganisms Male mice of C3H/He were obtained from the Breeding Unit of Kyushu University Fukuoca. Eight- to ten-week-old mice were used for the experiments. L. monocytogenes strain EGD was maintained by serial passage in BALB/c mice. Fresh isolates were obtained from spleen, grown once 0014-2980/9O/0303-0533$02.50/0

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S. Ohga, Y.Yoshikai,Y. Thkeda et al.

in tryptic soy medium (Difco, Detroit, MI) and stored at - 70 "C in PBS until use. Fifty percent lethal dose (LD50) was 5 x 104 in C3H/He mice. 2.2 Kinetics of bacterial growth after i.p. infection with L. monocytogenes Mice were i.p. injected with a sublethal dose (2 x 103) of viable Listeria on day 0 , and killed by cutting the cervical artery at intervals after the inoculation. To observe the serial course of bacterial growth, peritoneal contents were lavaged with PBS and spleen cells were recovered on day 1, 3 and 8 after the inoculation. PEC were lysed by freezing and thawing and the fluid was diluted 10-fold with PBS. One-tenth milliliter of each dilution was spread on nutrient agar plates containing 0.4% (w/v) glucose. Colonies were counted after incubation for 20 h at 37 "C. Bacterial growth in spleen homogenates was determined as described previously [4]. 2.3 Preparation of peritoneal cells PEC were recovered by lavage of the peritoneal cavity with RPMI 1640 medium (Gibco Laboratories, Grand Island, NY) supplemented with 10% FCS, 5 X lop5 M 2-ME, containing 2 U/ml of heparin. The cells were collected by centrifuging at 110 x g for 10 min, washed once with medium and counted in a hematocytometer. Smear specimens for differential counts were stained with Giemsa solution. PEC suspended in the medium were then spread on a plastic plate (2 x 106 - 3 x 106 cells/plate) to separate plastic-nonadherent from adherent cells after 2 h of incubation in the COz incubator at 37 "C. The adherent and nonadherent cells of PEC were used after being washed twice with the medium and collected to an optimal concentration. 2.4 FACS analysis A single-cell suspension from the nonadherent population of the PEC was stained with FITC-conjugated anti-Thy-1.2, anti-IgM mAb and anti-CD3 (145-2C11). Anti-CD3 directed against E chain of CD3 complex was the gift of Dr. J. A. Bluestone. On three-color analysis, the cell suspension was stained with FITC-conjugated anti-CD3, PE-anti-CD4 and biotin-antLCD8 mAb (Becton Dickinson, Sunnyvale, CA), thereafter with DuoCHROMEstreptavidin (Becton Dickinson). In some experiments, we used the PE-anti-CD3, PE-anti-CD4 and FITC-conjugated anti-TcR a / p mAb (H57-597),which was the gift of Dr. R.T. Kubo [25]. The FACScan (Becton Dickinson) was set to analyze only viable lymphocytes by forward light scatter gate. Thus, fluorescence signals from dead cells or other background can be excluded. The percentage of fluorescence-positive cells was determined by integration from profiles based on 2 x 104 viable cells. 2.5 Northern blot analysis Using the guanidinium thiocyanate and CsCl gradient centrifugation procedure [26], total RNA was extracted

Eur. J. Immunol. 1990.20: 533-538

from nonadherent cells of the PEC,which were collected on day 1, 3 and 8 after an i.p. inocultion with Listeria. The RNA (17 pg) was electrophoresed on 0.8% agarose in a 10 mM sodium phosphate buffer, pH 7, and transferred to Gene Screen Plus (NEN, Boston, MA).The filter containing RNA obtained from nonadherent PEC was hybridized with 32P-labeled6, y, p and a chain C probes derived from the 6 cDNA (RADllC, 0.9-kb Eco RI fragment [27], y cDNA (MNG8, 1.6-kb Eco RI fragment) [28], p cDNA (NYB2, 0.7-kb Eco RI fragment) [29], a cDNA (NYA2, 0.8-kb Eco RI fragment) [29], and then hybridized with mouse IFN-y probe (pmc 14,0.7-kb, Pst I fragment) [30], which was the gift of Dr. D.V. Goeddel. Ethidium bromide staining of the gel before and after transfer and hybridization of the filter with p-actin probe were performed to confirm that nearly equal amounts of RNA were blotted to the screens. Following hybridization for 16-24 h at 60 "C in 1%, 1 M NaCl, 10% dextran sulfate and 100 pg/ml heat-denatured salmon sperm DNA, the filters were washed for 10 min in 2 x SSC, 1% SDS at 60°C and exposed to X-ray films at -70°C in the presence of intensifying screens. The probes were removed by washing for 15 min in boiling water. The removal of the previously used probes was checked by autoradiography. These blotting analyses were repeated using different filters for confirmation.

3 Results 3.1 Kinetics of the bacterial growth and the PEC after an i.p. infection with Listeria To examine the serial changes of bacterial growth during listerial infection, the number of bacteria in the peritoneal cavity or spleen was determined after i.p. inoculation with 1 X lo3 Listeria. The number of Listeria in the peritoneal cavity was increased on day 1, followed by the rapid decrease from day 3 to day 8 after infection. In the spleen, the number of bacteria peaked on day 3 and gradually decreased by day 8 after infection (Fig. la). Next, to examine the inflammatory cell influx in the peritoneal cavity during listerial infection, we analyzed the kinetics of PEC from mice i.p. inoculated with 1 x 103 Listeria. The number of peritoneal leukocytes increased to 3.82 x 106/mouseon day 3 after infection. Forty-eight percent of the PEC on day 1 and 64% of the PEC on day 3 were monocytes/M@, as judged by morphological characteristics. On the other hand, M@ in the PEC decreased in proportion to 30% on day 8 and nonadherent cells, most of which were lymphocytes, increased remarkably in proportion at this stage (Fig. lb). 3.2 Surface marker analysis of nonadherent cells of the PEC To characterize the inflammatory leukocytes in the peritoneal cavity during listeriosis, we carried out the FACS analyses on the nonadherent population of the PEC. Sixty to seventy percent of IgM+ cells, representing B cells, were consistently detected throughout the first 7 days after infection with Listeria, similar to those in resident PEC (data not shown). On the other hand, the number of CD3+

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y/&bearing cells during listeriosis

535

lymphocytes progressively increased (10% +43%) during the course of the infection (Fig. 2a). Next, an analysis gate was set on the CD3+ cells to display the profile of PE-CD4 and biotin-CD8 staining in three-color FACS analysis. As shown in Fig. 2b, the number of CD4-CD8+ or CD4+CD8- cells gradually increased by day 8 after infection, while the CD3+CD4-CD8- cells increased in proportion to the maximal level (13.7%) on day 3 after infection and thereafter decreased by day 8 (8.6%).

0

1

3

To determine whether the CD3+CD4-CD8- cells on day 3 express TcR a@or TcR ylS on their surface, the nonadherent PEC on day 3 were analyzed by two-color staining with PE-CD3 and FITC-TcR dB mAb and three-color staining with PE-CD4, biotin-CD8 and FITC-TcR a@ mAb. When the CD3+ lymphocyteswere gated on to be counted in this two-color analysis, a significant number of the CD3+ lymphocytes were found to express no TcR a/p (Fig. 3a). On the contrary, the three-color analysis revealed that almost all lymphocytes bearing either CD4 or CD8 determinant expressed TcR a@ after gating on CD4+ or CD8+ lymphocytes (Fig. 3b). These results indicated that CD3+CD4-CD8- cells on day 3 expressed TcR y/6 on their surface.

a

Days after infection

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3.3 Expression of TcR and IFN-y mRNA in nonadherent population of the PEC from mice after listerial infection

my3

Days after Infection

In order to confirm the appearance of the y/S-bearingTcells in the peritoneal cavity at the early phase of primary Figure 1. (a) Kinetics of bacterial growth at the early phase after i.p. inoculation with 1 X 103viable L. rnonocytogenesin spleen ( 0 ) infection with Listeria, the expression of the TcR genes on the nonadherent cells was examined using Northern blot and peritoneal cavity (0)of mice. Each point and bar indicated the analysis (Fig. 4). NoTcR gene transcripts were detected in mean of five mice k SE. (b) Kinetics of the PEC obtained from mice i.p. inoculated with 1 X 103 viable L. monocytogenes. ( Q ) nonadherent population of the PEC on day 1,while y and 6 PMN, ( N I ) monocyte-Ma. (0)lymphocytes. Each value repregene transcripts were highly expressed in the nonadherent sents average of 50 mice. population of the PEC on day 3. On the other hand, the

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Figure 2. Three-color FACS analysis of CD3, CD4 and CD8 expression in the nonadherent population of PEC obtained from mice after i.p. inoculation with L. monocytogenes. Nonadherent population from the PEC on day 1, 3 and 8 after i,p. injection of 1 x 103 L. monocytogenes was stained with FITC-anti-CD3 and PE-anti-CD4 mAb and biotin-CD8 mAb, then with DuoCHROME-conjugated streptavidin. (a) Relative cell number of CD3+ was presented on day 1, 3 and 8 after the infection. (b) The profile of PE-CD4 and DuoCHROME-CD8 was displayed after gating on the CD3+ cells using forward light scatter to exuclude dead cells and red blood cells.

Eur. J. Immunol. 1990. 20: 533-538

S. Ohga,Y.Yoshikai,Y. Bkeda et al. (a)

(a) (b) (c)(d)

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TcR a/I3 Figure3. (a) Two-color FACS analysis of CD3 and TcR dp expression in the nonadherent PEC obtained from mice on day 3 after i.p. inoculation with L. monocytogenes. The nonadherent population from PEC on day 3 after i.p. injection with 1 X 103 LLteria was stained with PE-anti-CD3(145-2C11), FITC-anti-TcR alp (H57-597).After the gating on CD3+ lymphocytes, relative cell number of TcR alp+ is presented. (b) Three-colorFACS analysis of TcR a@, CD4 and CD8 expression in the nonadherent PEC obtained from mice on day 3 after i.p. inoculation with L. monocytogenes.The nonadherent PEC on day 3 after i.p. injection with 1 x 103 Listeriu were stained with FITC-anti-TcR a l p (H57-597), PE-anti-CD4 and biotinylated anti-CD8 mAb with DuoCHROME-avidin. Relative number of TcR alp' cells was presented after the gating only on CD4+ or CD8+ cells, respectively.

amount of a / P gene messages increased and that of $6 genes reciprocally decreased in those on day 8. Thus, the amount of TcR y/6 gene messages increased in correlation with the increased number of CD3+CD4-CD8- lymphocytes in the course of listerial infection.

To determine the pattern of related cytokine production during listerial infection, we further examined sequential expression of I F N y in the peritoneal cavity listerial infection. Northern blot analysis revealed that IFN-y transcripts in the nonadherent PEC were first detected on day 3 and increased to the highest level in those on day

8.

Figure 4. Northern blot analysis of nonadherent PEC obtained from mice on day 1 (b), 3 (c) and 8 (d) after i.p. inoculation with L. monocytogenes. Equal volume (17 pg) of total RNA extracted from C3H/He control thymus (a) or nonadherent population of the PEC was electrophoresedand transferred to Gene Screen Plus.The same filter was hybridized in sequence to the 32P-labeled6, y, p, a cDNA and IFN-y probes after washing the filters for 15 min in boiling water.

4 Discussion The notable finding in our study is that the inflammatory cells expressing TcR y and 6 chain appeared in the inflamed sites at the early phase of listerial infection. An ordered appearance of $6- then a/P-bearing T cells is seen in the fetal thymus [31-331, and in the thymus of irradiated recipient mice at the early stage after BM transplantation [34]. Similar t o the processes of T cell differentiation in ontogeny, the appearance of TcR y/6 cells may precede that of a / P T cells in the infected sites during listeriosis. TcR y/b-bearing T cells which express CD4-CD8- or CD4-CD8+ are demonstrated to be populated in epidermis [14, 151 and in intestinal epithelium [16, 171 as well as in other peripheral lymphoid organs [35, 361. Recently, $6 T cells have been demonstrated t o increase in number in the draining LN after infection with Mycobacteriurn tuberculosis [19]. The early appearing y/6 T cells during infection may serve as a first defense against infectious agents.

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Although several data [12,13] suggested that the ligand of TcR y/6 may be MHC class I or class I1 proteins, molecular natures recognized remain to be defined. Recently, there has been convincing evidence that at least significant fractions of y/6 Tcells are specialized to recognize epitopes on mycobacterial protein [ 18-20]. Among the many mycobacterial antigens, heat shock protein (hsp) has been found to be a major component as immunodominant antigens [37]. Human synovial Tcells cloned by Holoshitz et al. [20] have been shown to react to a purified preparation of the hsp60 stress protein from M. bovis as well as to M. tuberculosis antigens. Antigen-unselected y/6 T cells from newborn murine thymus have been reported to respond to the recombinant M. bovis hsp [18]. hsp has been phylogenically conserved by prokaryotes and eukaryotes [38]. It is possible that the early appearing y/6 T cells in mice infected with Listeriu may recognize hsp from Listeria or autologous stress proteins which are highly homologous to various hsp. We cannot rule out the possibility that the y/6 T cells may recognize newly expressed or modified MHC molecules on the infected cells. Several reports have shown that NK-like activities were detected in y/&T cell clones stimulated with anti-CD3 mAb andor IL 2 [39]. Kaufmann [l] also described that broad-reactive killer Tcells may be involved in infection with intracellular bacteria. The y/6 T cells may participate in immune surveillance at the early stage against the invasion of Listeriu by elimination of infected cells. Cytokines play an important role as mediators in cell to cell interaction in the host response to Listeriu infection. Among various cytokines for the effective protection, IFN-y plays a crucial role in host defense against Listeriu infection through activating listericidal capacity in M a [40, 411. Listeriu-specific MHC class 11-restricted CD4+ cells are known to represent a major source for IFN-y production. Besides CD4+ T cells, CD8+ T cells are also reported to be activated during listerial infection and produce IFN-y. Kratz et al. [6] reported that IFN and IL 2 production did not increase in supernatants of spleen cells cultured with heat-killed Listeria until around day 4 and 5 after primary infection. Consistent with their results, a high level of expression of IFN-y transcripts, detected in the nonadherent cells on day 8 after infection, contained an increased level of TcR a@ chain gene messages. Murine T h cells can be divided into two subsets on the basis of their capacity for secreting interleukins [42].T Hcells ~ synthesize IL 2, IFN-y and lymphotoxin, w h e r e a s T ~ 2cells synthesize detectable amounts of IL 4 and IL 5. Our results suggested that T Hcells ~ might preferentially appear at the late stage after primary infection. A variety of y/6 cell lines has been documented to produce T cell-derived cytokines such as IL 2, IFN-y, IL 4 and GM-CSF. Modlin et al. [43] have reported that y/S T cells enriched in active granulomatous lesions of patients with leprosy or leishmaniasis secrete cytokines that cause MQ adhesion, aggregation and proliferation in the presence of GM-CSE We have previously reported that acquired resistence raised early after primary infection with Listeria depended mainly on MQ accumulation augmented by macrophage chemotactic factor (MCF) [44]. It is possible that the y/6 Tcells may produce MCF in response to Listeria. At present we cannot define what kinds of cytokines the early appearing y/6 T cells produce during listerial infection. Intensive study of the cytokineproducing ability in the fractionated population of locally

ylb-bearing cells during listeriosis

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accumulated or proliferated cells may be helpful for understanding the relationship between cytokines and effector cells for protection against Listeriu in vivo. Ongoing analysis of cloning the ylbbearing T cells at the early stage of listerial infection, and the consecutive functional analysis may provide more information not only on elucidating the protective mechanism against intracellular bacteria, but also on clarifying the role of TcR y/S recognition in the first defense lines. We thank Drs. J. A . Bluestone, i? W Mak, D. K Goeddel and R. i? Kubo for providing the monoclonal antibodies and probes. S. Ohga is grateful to Prof. K . Ueda, Department of Pediatrics, Kyushu University, Faculty of Medicine, for encouragement. Received October 12, 1989; in revised form November 14, 1989.

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