Brief Definitive Report |
Address correspondence to Emil R. Unanue, Dept. of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. Phone: (314) 362-7442; Fax: (314) 362-4096; email: unanue{at}pathology.wustl.edu
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Abstract |
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Key Words: apoptosis cytokines Listeria inflammation T lymphocytes
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Introduction |
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Much is known about the role of type I IFNs (IFN- and IFN-ß) in the antiviral response and type II IFN (IFN-
) in the antiviral and antibacterial response (36). However, there exists a paucity of data on the role of type I IFNs during bacterial infection. Here we report that the absence of the shared IFN-
/ß receptor (IFN-
ßR/) provides mice with an advantage during Listeria infection. This resistance was accompanied by an amelioration of the lymphocyte apoptotic process that took place during the early exponential growth of Listeria in tissues.
We and others have described an early transitory phase of lymphocyte apoptosis in infective foci that peaks at 48 h after infection (7, 8). Experiments ex vivo indicated that listeriolysin O (LLO), a pore-forming molecule and major virulence factor, caused lymphocyte apoptosis particularly in cells that were replicating (9). Moreover, injection of pure LLO subcutaneously led to lymphocyte apoptosis in the T cellrich zones of draining lymph nodes. Of note is that during the in vivo infection the apoptotic lesions were either not affected or were increased in mice in which IL-1, IL-12, or IFN- were either neutralized or not produced (7). The lesions were unrelated to activation-induced cell death, to FasFas ligand interactions, or to the production of reactive oxygen or nitrogen intermediates. In toto the evidence points to the release of soluble LLO during the early robust growth of Listeria as the mechanism leading to the apoptotic death of lymphocytes in infective foci. In fact, neutralization of LLO by injection of monoclonal antibody to LLO controlled the infection as well as the intensity of the apoptotic lesions (10). The data presented herein suggests that type I IFN sensitizes lymphocytes to undergo apoptosis during Listeria infection, and that this has a negative effect on bacterial handling in the mouse.
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Materials and Methods |
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Cell Cultures.
A CD4 T cell line reactive to ovalbumin was generated from normal 129Sv/J mice (The Jackson Laboratory) immunized with an ovalbumin emulsion in complete Freund's adjuvant using previously described techniques (9). The line was passaged every 1012 d by the addition of irradiated 129Sv/J splenocytes (3,000 rads), 10 µM ovalbumin, and 50 U/ml IL-2. The usual behavior was that of proliferation that slowly stopped by about day 8. On day 10 after stimulation, the T cells were harvested and were not in cell cycle. Assays with the T cell line were performed as described previously (9). Whole splenocytes were isolated from 129Sv/Ev or IFN-ßR/ mice. Single cell suspensions were made and cells were plated at a density of 5 x 106 cells/ml in DMEM and 10% FCS, and treated with recombinant mouse IFN-
A at 1, 10, or 100 U/ml (specific activity: 4.8 x 107 U/mg; PBL Biomedical Laboratories). Cells were incubated for 24 h, and then the nonadherent cells were removed and purified. Both the T cell line and the splenocytes were purified over a Histopaque 1119 gradient (Sigma-Aldrich). After Histopaque, cells were resuspended in DMEM containing 1% FCS, and then treated with 250 ng/ml of purified recombinant LLO (4.4 nM) for 6 h. Cells were stained with annexin V-PE and 7-AAD (BD Biosciences), and analyzed by flow cytometry. For assays involving splenocytes, cells were also stained with antiCD3-APC (BD Biosciences) to identify T cells.
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Results and Discussion |
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During early Listeria infection, one sees a nonspecific stage of T lymphocyte activation, made evident by the up-regulation of CD69 cell surface expression (8). It has also been shown that type I IFN signaling can induce CD69 expression on T cells (12). Moreover, in vivo Listeria infection induces type I IFN production (13, 14) and, in vitro, Listeria-infected macrophages produce type I IFN after detecting the presence of an intracellular pathogen (15). Therefore, we assayed Listeria-infected wild-type and IFN-ßR/ mice for the percentage of CD4 and CD8 T cells expressing CD69. Infected wild-type mice showed a three- to sixfold increase in the number of CD69+ T cells in their spleens, whereas the increase in infected IFN-
ßR/ mice was only two- to threefold. In uninfected 129Sv/Ev mice, 4% of the CD8+ T cells were CD69+. This number increased to 13 and 25% at days 1 and 2 after infection, respectively. In uninfected IFN-
ßR/ mice, 4% of the CD8+ T cells were CD69+, which increased to 8 and 12% at days 1 and 2, respectively. The picture was similar for CD4+ T cells, where in 129Sv/Ev mice, 10% of the CD4+ cells were CD69+ before infection, 18% were CD69+ at day 1, and 30% were CD69+ at day 2 after infection. In IFN-
ßR/ mice, 8% of the CD4+ cells were CD69+ in the uninfected mouse, with 10% CD69+ at day 1 and 15% CD69+ at day 2 after infection. This difference in CD69 may reflect a higher level of activation of T lymphocytes in wild-type mice, a step which could enhance their susceptibility to Listeria-induced apoptosis.
Response of T Cells to LLO in Culture.
We demonstrated previously that treatment with sublytic doses of LLO induced apoptosis in T cells (9). In that study, we observed a difference in the extent of apoptosis between resting lymphocytes and those recently activated by exposure to antigen. Because type I IFN is produced early during Listeria infection (1315), we hypothesized that the early burst of IFN could enhance the susceptibility of lymphocytes to apoptosis.
We chose to monitor apoptosis in lymphocytes treated with type I IFN by flow cytometry using annexin V that binds to phosphatidylserine exposed on the outer leaflet of the plasma membrane, and 7-AAD, which measures nuclear permeability. This assay distinguishes apoptotic cells, which stain with annexin V but not 7-AAD, from late apoptotic or necrotic cells, which stain positive for both markers.
We first tested CD4+ T lymphocytes from ex vivo culture 10 d after antigen stimulation by incubating them in the presence or absence of IFN-A for 24 h, and then exposing them to 250 ng/ml LLO for 6 h. We selected this time point on the basis of previous kinetic studies on cultured T cells treated with LLO (9). In the absence of LLO, 810% of cells underwent apoptosis (i.e., became annexin V+/7-AAD). 6 h of treatment with LLO increased this number to
22%, in accordance with our previous study (Fig. 3, A, D, and J; reference 9). The set of cells staining with both annexin V and 7-AAD also increased upon LLO treatment. Our previous study demonstrated that LLO-treated cells shifted over time from the single positive (annexin V+/7-AAD) to the double positive (annexin V+/7-AAD+), the latter of which contains late apoptotic as well as necrotic cells (9).
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To extend our results to primary cells, we isolated splenocytes, cultured them for 24 h in increasing doses of IFN-A, treated them with LLO, and assayed them for apoptosis by annexin V/7-AAD staining. As an average, 13% of untreated CD3+ cells were annexin V+/7-AAD (Fig. 3, B and K). This number increased to 24% upon LLO treatment (Fig. 3, E and K). The addition of type I IFN increased the response of T cells to LLO-induced apoptosis in a dose-dependent manner. At the highest IFN-
A dose tested (100 U/ml), 37% of cells CD3+ became annexin V+/7-AAD (Fig. 3, H and K). This increase, albeit modest, was highly reproducible in three experiments. In contrast, type I IFN did not increase LLO-induced apoptosis in splenocytes isolated from IFN-
ßR/ mice. The percentage increase in annexin V+/7-AAD cells taken from IFN-
ßR/ spleen after treatment with LLO was the same in the absence of IFN-
A (Fig. 3, F and L) and at all doses of IFN-
A tested (Fig. 3, I and L).
(Spleen cells from wild-type mice cultured for 2, 4, and 6 h with LLO resulted in 26, 29, and 24% of annexin V+/7-AAD cells, respectively. The addition of IFN-A increased the number of cells at the same time points to 35, 42, and 37%. Spleen cells from the IFN-
R/ mice cultured for 2, 4, and 6 h with LLO resulted in 30, 32, and 30%, respectively. However, this number did not increase after culture in IFN-
A: 29, 31, and 30%. Both untreated spleen cell populations were 16% annexin V+/7-AAD cells after 6 h of culture. Thus, there were no major changes in kinetics during the 26-h time period. Fig. 3 shows the 6 h results.)
Comments.
Our experiments with IFN-ßR/ mice indicate a deleterious effect on early resistance to Listeria infection by type I IFNs. Fehr et al. (16) reported that IFN-
ßR/ mice were not impaired in their resistance to Listeria infection. Moreover, their results suggested an increased resistance to Listeria at day 5 after infection when compared with wild-type controls. They hypothesized that this was a function of type I and II IFN receptor competition for components of signaling pathways. However, that study, which focused on the role of transcription factors and nitric oxide in resistance to Listeria, did not fully explain the increased resistance of IFN-
ßR/ mice.
Our report expands on previous findings in the following ways. In the absence of a response to type I IFNs (i.e., in the receptor null mice), the infective foci contained fewer TUNEL+ lymphocytes as well as fewer activated T cells. Type I IFN unresponsive mice were also more resistant to infection. We believe that early type I IFN production during Listeria infection results in nonspecific activation of T cells. The provocative finding is that this early activation also sensitizes these cells to the apoptogenic actions of LLO. Furthermore, macrophages treated with type I IFN were shown to be susceptible to Listeria killing in vitro (17). These results suggest that type I IFN could enhance the susceptibility to death of multiple leukocyte subsets during Listeria infection.
The immunological consequences of cellular death during Listeria infection of the mouse must be further examined, but could involve the development of responses that are inhibitory for bacterial clearance. Clearance of apoptotic bodies by macrophages is generally thought to down-regulate inflammation through macrophage release of TGF-ß, prostaglandin E2, and platelet-activating factor (1820). Apoptotic lymphocytes themselves may also down-regulate inflammation through the release of preformed TGF-ß upon apoptosis induction (21). We suggest an inverse relationship between death by apoptosis, in part enhanced by type I IFNs and the growth of Listeria. In the absence of type I IFN signaling, apoptosis is limited, and less inhibition of phagocyte antimicrobial processes takes place. In support of these hypotheses, we have observed that SCID mice, lacking T and B lymphocytes, do not show apoptotic lesions after Listeria infection, and are more resistant than wild-type mice during the first 24 d after infection (22 and unpublished data).
The cellular source of type I IFN during Listeria infection and the Listeria component(s) inducing its production remain unknown at this time. Candidate ligands would include Toll-like receptor (TLR)4 or TLR9 agonists such as lipoteichoic acid or CpG DNA motifs, which have been shown to induce type I IFN expression by dendritic cells (23). Analysis of TLR4/, TLR9/, and MyD88/ mice or cells may yield insight into the generation of type I IFN in response to Listeria infection. Macrophages recognizing cytosolic Listeria also generate IFN-ß (15), suggesting this cell type as a source for the cytokine. Alternatively, Listeria could generate proteins that promote expression of type I IFN to enhance its virulence and dissemination.
A classical role ascribed to type I IFN signaling is the enhancement of cancer cell and virus-infected cell apoptosis. One possible mechanism for type I IFN's sensitization of T cells to undergo apoptosis would be the accumulation of the p53 tumor suppressor gene (24). After p53 expression, cellular stress caused by LLO disruption of the plasma membrane might then lead to apoptosis induction. Further studies will examine the role of type I IFNs in LLO-induced apoptosis.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health.
The authors have no conflicting financial interests.
Submitted: 16 April 2004
Accepted: 7 July 2004
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References |
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