Virally Induced Inflammation Triggers Fratricide of Fas-LigandExpressing ß-Cells
Urs Christen1,
Rima Darwiche2,
Helen E. Thomas2,
Tom Wolfe1,
Evelyn Rodrigo1,
Alexander Chervonsky3,
Richard A. Flavell3, and
Matthias G. von Herrath1
1 Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California
2 St. Vincents Institute, Fitzroy, Victoria, Australia
3 Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut
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ABSTRACT
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Tissue-specific expression of Fas-ligand (Fas-L) can provide immune privilege by inducing apoptosis of "invading" lymphocytes expressing Fas. However, accelerated diabetes has been reported in transgenic mice expressing Fas-L in islets (RIP-Fas-L) as a result of Fas-dependent fratricide of ß-cells after transfer of diabetogenic clones. Here we studied whether Fas-L could protect islets from autoaggressive CD8 lymphocytes in a transgenic model of virally induced diabetes (RIP-LCMV-NP transgenic mice), in which the autoaggressive response is directed to a viral nucleoprotein (NP) expressed as a transgene in ß-cells. Indeed, disease incidence after viral (lymphocytic choriomeningitis virus [LCMV]) infection was reduced by
30%, which was associated with a decrease of autoaggressive CD8 NP-specific lymphocytes in islets and pancreatic draining lymph nodes. However, surprisingly, a high degree (50%) of diabetes was seen in mice that expressed only Fas-L but not the viral transgene (NP) in ß-cells after infection with LCMV. This was due to induction of Fas on ß-cells after LCMV infection of the pancreas, resulting in Fas/Fas-Lmediated fratricide. Thus, although Fas-L can lend some immune privilege to islet cells, local virus-induced inflammation will induce Fas on ß-cells, leading to their mutual destruction if Fas-L is present. Expression of Fas-L therefore might not be protective in situations in which viral inflammation can be expected, resulting in Fas induction on the targeted cell itself.
Expression of Fas-ligand (Fas-L) on the surface of cells has been shown to provide immune-privilege by inducing death of lymphocytes that express Fas (CD95). For example, expression of Fas-L is increased in the eye, and, consequently, local immunity is suppressed (13). It is not clear whether a similar protective mechanism is operative in other sites, for example, the brain. Therapeutically, this concept has elicited interest, because one could envision preventing immune-mediated destruction of transplanted cells or organs by expression of Fas-L (46). However, there has been only limited success using this strategy (4,5,79). Some insight into the potential problems with this approach was provided by a recent study in which Fas-L was expressed under the rat insulin promoter in ß-cells (4,10). Unexpectedly, such mice were not protected from spontaneous diabetes and several NOD congenic lines even exhibited accelerated disease. In addition, diabetogenic T-cell clones were not eliminated by Fas-mediated killing but induced more rapid diabetes in RIP-Fas-L transgenics. The reason was that arrival of T-cells in the islets would induce upregulation of Fas on ß-cells, which resulted in their ability to kill each other ("fratricide"). Thus, overexpression of Fas-L bears the inherent danger of locally increasing the risk of Fas-mediated death of any cell that expresses Fas. This can lead to undesirable demise of useful cells, among them possibly also regulatory lymphocytes.
For the pathogenesis of type 1 diabetes, the role of Fas/Fas-L interactions in ß-cell destruction is controversial (1115). It has been observed that Fas and Fas-L are upregulated on human islets (14), in particular after in vitro manipulation using cytokines (16) or nitric oxide (17). Furthermore, on the lymphocyte side, there is some evidence for altered apoptosis in patients with type 1 diabetes (18), which supports the therapeutic concept of inducing immune privilege by killing invading T-cells through apoptosis using Fas/Fas-L interactions (6).
We sought to evaluate two outstanding issues with this present investigation. First, we wanted to study the in vivo effect of RIP-expressed Fas-L on a defined population of autoaggressive "driver" CD8 lymphocytes. Second, we wished to test whether virally induced local inflammation of the pancreas would be capable of upregulating Fas on ß-cells and in this way lead to their mutual demise in RIP-Fas-L transgenics. This allowed us to judge the effect of inflammation on cells or organs engineered to express Fas-L and capable of possibly expressing Fas as well, which is of clinical relevance. We chose to use the well-established RIP-LCMV model for virally triggered type 1 diabetes, because it was ideally suited to address these questions. These mice express the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) in ß-cells as an islet-specific autoantigen to which the immune response can be tracked easily (1921). Diabetes usually develops only after infection with LCMV that activates NP-specific autoaggressive CD4 and CD8 lymphocytes, which both are required for disease (19). Indeed, cytotoxic killing of ß-cells is a major requirement for diabetes development along with local inflammation of the pancreas and islets. As early as 2 days after LCMV infection, "preconditioning" of the pancreas is occurring, reflected by upregulation of major histocompatibility complex class II molecules on activated antigen-presenting cells (22). This LCMV-induced inflammation is transient (day 2 through 6 postinfection) and is alone insufficient to cause diabetes in nontransgenic mice (23,24). However, in RIP-LCMV mice this activation of APCs and secretion of interferon and other inflammatory molecules is important to "entice" autoaggressive (LCMV-NP specific) lymphocytes to the pancreas and islets, where they will continue to attack ß-cells that express the NP antigen until all of them have been destroyed.
For the present study, RIP-LCMV mice (LdKdIAd) were crossed to RIP-Fas-L transgenic mice (DbKdIAg7). We present two interesting observations that clearly illustrate the dual and opposing mechanisms that local Fas-L expression can elicit. First, virally induced disease in RIP-NP mice is significantly delayed by Fas-L expression in ß-cells mediated by reduction of autoaggressive CD8 cells locally in islets and pancreatic draining lymph nodes. However, LCMV infection can induce diabetes in Fas-L expressor mice in complete absence of the LCMV-NP transgene. This occurs in conjunction with Fas upregulation on ß-cells that enables Fas-L transgenic ß-cells to kill their neighbors/brothers (fratricide).
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RESEARCH DESIGN AND METHODS
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Mice breeding scheme and origin.
Generation of H-2b RIP-LCMV-NP transgenic mice used for this study has been described previously (19). RIP-Fas-L mice expressing Fas-L under the control of the rat insulin promoter had been generated previously (4). NOD (DbKdIAg7) RIP-Fas-L mice were intercrossed with the H-2d (DbKdIAd) RIP-LCMV-NP line, resulting in double-transgenic mice. These mice were used for all studies here as littermates.
Mice genotyping.
The presence of transgenic RIP-LCMV-NP or Fas-L sequences was determined by performing two independent standard PCR reactions with genomic DNA obtained from mouse tails as described (4,19).
Virus.
Virus used was LCMV strain Armstrong (arm), clone 53b. LCMV was plaque purified three times on Vero cells, and stocks were prepared by a single passage on BHK-21 cells. Mice were infected with a single intraperitoneal dose of 105 pfu LCMV-arm unless indicated otherwise (20).
Viral titers.
LCMV viral titers of organ homogenates were determined by infection of Vero cells as described elsewhere (18). Briefly, organ homogenates were diluted serially and cultured with Vero cells for 5 days. Viral titers were determined from the number of counted plaques (20).
Blood glucose measurements.
Blood samples were obtained from the retro-orbital plexus, and plasma glucose concentration was determined using Accucheck III (Roche, Indianapolis, IN). Mice with blood glucose values >300 mg/dl were considered diabetic (24).
Islet cell enrichment.
Islets were isolated as previously described (25). Briefly, the pancreas was removed, cut into little pieces, and digested with collagenase P (Roche). Islets were purified on histopaque-1077 density gradients (Sigma, St. Louis, MO). The obtained islet-enriched fraction and the remaining portion of the pancreas containing mainly acinar cells were either immediately homogenized in tri-reagent (Molecular Research Center) for subsequent isolation of total RNA or islets were further digested with trypsin (Sigma) to obtain single ß-cell suspensions (26).
RNase protection assay.
Total RNA was isolated either from whole pancreas homogenates or from islet or acinar cellenriched fractions using tri-reagent (Molecular Research Center). RNA was extracted with chloroform followed by isopropanol precipitation and washing with ethanol. Twenty micrograms of total pancreatic RNA was used for hybridization with a 32P-UTPlabeled multitemplate set containing specific probes for Fas, Fas-L, tumor necrosis factor-
,
-interferon (IFN-
), and lymphotoxin-ß provided by a commercial kit (Riboquant, mCK-3b and mAPO-3; Pharmingen, La Jolla, CA). The RNase protection assay was carried out according to the manufacturers guidelines. The resulting analytical acrylamide gel was scanned using a Storm-860 phospho-imaging system (Molecular Dynamics), and the intensity of bands corresponding to protected mRNAs was quantified using the Imagequant image analysis software (Molecular Dynamics) using L32 as a reference gene (23).
Immunohistochemistry.
Organs were harvested at week 6 after LCMV infection unless indicated otherwise, immersed in Tissue-Tek OCT (Bayer, Elkhart, IN), and quick frozen on dry ice. Six- to 10-µm tissue sections were cut using a cryomicrotome and sialin-coated "superfrost plus" slides (Fisher Scientific, Pittsburgh, PA). Sections were fixed with 90% ethanol at -20°C, and after washing in PBS an avidin-biotin blocking step was included (Vector Laboratories, Burlingame, CA). Primary and biotinylated secondary antibodies (Vector Laboratories) were reacted with the sections for 30 min each, and color reaction was obtained by sequential incubation with avidin-peroxidase conjugate (Vector Laboratories) and diaminobenzidine-hydrogen peroxide. Primary antibodies were rat anti-mouse CD8a (ly2), rat anti-mouse CD8b (ly3), and antiLCMV-NP Alexa 1.1.3 antibody (19,27).
Cytotoxicity assays.
LCMV-specific cytotoxic T-cell (CTL) activity in spleen was analyzed in a 5-h in vitro 51Cr-release assay (19). All samples were run in triplicate. Primary CTL activity was tested by harvesting spleens at day 7 after intraperitoneal infection with 105 pfu LCMV-arm. Splenocytes were co-incubated with major histocompatibility complexmatched (Balb/C17 H-2d) and mismatched (MC57 H-2b) target cells that had been loaded with 51Cr. Target cells were either LCMV-infected, uninfected but coated with the immune dominant LCMV major histocompatibility complex class I peptide NP118 (RPQASGVYM), or uninfected and uncoated. For determination of secondary CTL activity, spleens were harvested 6 weeks after LCMV infection and splenocytes were cultured for 8 days on LCMV-infected, irradiated peritoneal exudate cells before testing CTL activity in a 51Cr release assay. For precursor frequency analysis, spleen cells were serially diluted and cultured in 96-well flat bottom plates in the presence of T-cell growth factor (primarily containing interleukin-2), irradiated LCMV-infected peritoneal exudate cells and spleen feeder cells. After 810 days, cultures were assayed for CTL activity on LCMV-infected and uninfected target cells. Precursor frequencies were calculated as described (28).
Flow cytometry.
Spleen and pancreatic draining lymph nodes were harvested at week 6 after LCMV infection. Single-cell suspensions were stimulated with 100 µ/ml interleukin-2 and 2 µg/ml Brefeldin A (Sigma) for 516 h at 37°C. Cells were stained for cell surface markers using monoclonal antibodies (mAbs) against CD8 and CD4, permeabilized and fixed with paraformaldehyde/saponin, and stained for intracellular cytokines using fluorescein isothiocyanateconjugated anti-mouse tumor necrosis factor-
mAb and PE-conjugated anti-mouse IFN-
mAb (Pharmingen). Cells were acquired and analyzed on a FACSort or FACS-Calibur flow cytometer (Becton Dickinson) using cell quest software (Becton Dickinson) (29).
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RESULTS
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Expression of Fas-L by ß-cells leads to elimination of autoaggressive CD8 lymphocytes and lower diabetes incidence in RIP-LCMV mice.
Littermates from F1 mating of RIP-LCMV-NP (H-2d) x RIP-Fas-L (NOD) transgenic were infected with LCMV. As shown in Fig. 1, LCMV infection resulted in 80% diabetes in RIP-NP+ Fas-L- controls within 28 weeks. In contrast, type 1 diabetes incidence was significantly reduced in littermates that expressed Fas-L in addition to NP antigen in ß-cells. Thus, expression of Fas-L has a clear protective effect in the RIP-LCMV diabetes model. Importantly, systemic numbers of cytotoxic CD8 cells were equivalent when comparing Fas-L+ versus Fas-L- NP expressors (Table 1). Comparable numbers of CTL precursors and primary killing activity was detected in spleens. In contrast, locally in the pancreatic draining lymph node, we detected a significant reduction of NP pCTL (Table 1) and, accordingly, a lesser degree of CD8 infiltration in Fas-L+NP+ protected compared with Fas-L-NP+ diabetic mice (Fig. 2A versus B). This reduction of NP CTLs was not only site specific but also antigen specific, because CD8 lymphocytes producing IFN-
in response to LCMV NP but not glycoprotein peptides were reduced (Table 1). Thus, local expression of Fas-L by ß-cells can exert some protective capacity by eliminating autoaggressive CD8 lymphocytes in an islet antigen-specific manner. The antigen specificity of the process argues for the fact that interaction of Fas-expressing LCMV-NPspecific CD8 lymphocytes directly with Fas-Lexpressing NP+ ß-cells leads to their demise.

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FIG. 1. Incidence of diabetes in RIP-Fas-L x RIP-LCMV-NP lines after LCMV infection. Ten to 20 mice per group were infected with 1 x 105 pfu LCMV intraperitoneally. All experimental mice are littermates from F1 mating of RIP-LCMV-NP (H-2d) x RIP-Fas-L (NOD) transgenics. Spontaneous diabetes (without LCMV infection) did consequently not occur in any of the offspring. Blood glucose was assessed once every 2 weeks, and mice with values >300 mg/dl were considered diabetic.
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TABLE 1 Enumeration of autoaggressive (LCMV-NPspecific) CD8 lymphocytes in RIP-Fas-L x RIP-LCMV-NP transgenic mice after LCMV infection
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FIG. 2. Histologic findings in RIP-Fas-L x RIP-LCMV-NP transgenic mice after LCMV infection. Representative sections are shown in each panel; at least 10 islets and three mice for each group or time point were evaluated. Immunohistochemistry was performed as described by us previously. The positions of the individual pancreatic islets of Langerhans are indicated by dotted lines. A: CD8 lymphocytes in an islet of a RIP-NP+ (H-2d) x Fas-L- (NOD) F1 mouse 2 weeks after LCMV infection, diabetic. B: CD8 lymphocytes in an islet of a RIP-NP+ (H-2d) x Fas-L+ (NOD) F1 mouse 2 weeks after LCMV infection, no diabetes. C: Lack of lymphocytic infiltration into islets of a RIP-NP- (H-2d) x Fas-L+ (NOD) F1 littermate 5 days after LCMV infection, diabetic. D: Strong CD8 lymphocyte infiltration in an islet of a "regular" RIP-NP+ (H-2d) transgenic mouse at week 2 after infection with LCMV, diabetic. E: LCMV infection of pancreas and islets at day 2 after inoculation of 105 pfu LCMV intraperitoneally. LCMV-NP expression was detected by the anti-NP antibody (clone 1.1.3).
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Viral (LCMV) infection of the pancreas induces Fas on ß-cells, leading to fratricide by Fas-Lexpressing transgenic islet cells.
To our surprise, we observed almost 50% diabetes development in Fas-L+, NP-nonexpressing littermates 610 months after LCMV infection (Fig. 1). This was not a spontaneous disease comparable to that found in the original Fas-L+ NOD lines, because the mice under observation were intercrosses between the original NOD and nondiabetes-prone Balb/c mice, and spontaneous disease without LCMV infection was never seen in these mice (data not shown). It is interesting that the diabetes in the Fas-L+ NP- mice was not associated with marked CD8 (Fig. 2C) and other lymphocytic infiltration (not shown) as compared with more extensive CD8 infiltrates in NP+ littermates (Fig. 2A and B) or in LCMV-infected RIP-NP Balb/c mice (Fig. 2D). This observation indicated that ß-cell fratricide had occurred as a consequence of virally induced inflammation of the pancreas, because LCMV infects the pancreas and the islets directly as shown by immunohistochemical stains of pancreas sections with anti-NP antibody (clone 1.1.3.; Fig. 2E) (19,27). Because earlier studies had demonstrated expression of Fas by ß-cells after transfer of diabetogenic clones, we hypothesized that LCMV-induced inflammation of the pancreas would lead to Fas induction by ß-cells.
Indeed, we could detect Fas expression on both RNA and protein level by RNase protection assay (RPA) and flow cytometry of isolated islets, respectively. First, Fig. 3A shows that Fas gene expression, as detected by RPA, is seen on islet cells early after LCMV infection (day 4) immediately after viral antigen is first expressed (day 2 after infection) and interferons, chemokines, and other inflammatory mediators are induced. Because Fas upregulation is induced by IFN-
and can be blocked by neutralizing antibodies (30), we next treated LCMV-infected mice with neutralizing antibodies against IFN-
and analyzed Fas gene expression by RPA. As expected, Fas gene expression at day 4 after LCMV infection was significantly lower in the pancreatic draining lymph node of mice that received four doses of antiIFN-
mAb at days 0, 1, 2, and 3 after infection than in mice that received an isotype-matched control antibody (Fig. 3B). However, no change in Fas gene expression was observed in the pancreas (Fig. 3B). Second, for detection of Fas expression on the ß-cell surface, whole islets obtained by collagenase P digestion of pancreata were further treated with trypsin to obtain a single-cell suspension. Islet cells were stained for Fas surface expression by flow cytometry, and viable ß-cells were identified through their high autofluorescence detectable in the FL1 channel (Fig. 4A). Increased Fas expression was first noted at day 5 after LCMV infection, and it was >10-fold higher than on ß-cells isolated from uninfected mice at day 7 after infection (Fig. 4B).

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FIG. 3. Expression of Fas in pancreatic acinar and ß-cells after systemic LCMV infection. A: Groups of three mice were infected with 105 pfu LCMV intraperitoneally, and RNA was collected from enriched fractions of isolated islets (>90% pure after handpicking of islets) and acinar cells and from whole spleen at the times indicated and was pooled for RPA as described in RESEARCH DESIGN AND METHODS. B: Groups of four mice were infected with 105 pfu LCMV intraperitoneally, and neutralizing antiIFN- mAb (75 µg/dose intraperitoneally; Pharmingen #554409) was administered 6 h before and 1 day after LCMV infection. Groups of two LCMV-infected mice were administered with an isotype-matched control antibody at the same conditions. RPA was performed with total RNA isolated from pancreas and pancreatic draining lymph nodes (PDLN) at day 3 after infection. Elevation of Fas levels is statistically significant (P < 0.05).
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FIG. 4. Expression of Fas on the cell surface of isolated ß-cells. A: Pancreatic islet cell preparations were stained with anti-Fas antibody and incubated with propidium iodine (PI). Viable ß-cells were identified by flow cytometry on the basis of their side scatter (SSC) versus forward scatter (FSC) pattern (R1; left), low propidium iodine incorporation (R2; middle), and high autofluorescence in channel FL1 (R3; right). B: Fas expression was analyzed in ß-cells (gated in R3) at several days after infection with 105 pfu LCMV. Data shown are average (±SE) of the mean fluorescence intensity (MFI) (n = 35 mice/group).
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DISCUSSION
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We demonstrate in this study a dual effect of Fas-L expression on ß-cells. On one hand, Fas-L provides immune privilege by eliminating autoaggressive CD8 lymphocytes. This finding would encourage the use of Fas-L to protect ß-cells and other cell types/organs from autoimmune attacks and fits well with the known role of Fas-mediated apoptosis in CD4 populations. On the other hand, viral infection of the pancreas but not the islets can lead to induction of Fas on ß-cells, which renders them susceptible to Fas/Fas-Lmediated apoptosis. This results in our system in a significant degree of clinically manifest diabetes after LCMV infection. The mechanism of virally mediated Fas upregulation in ß-cells is not completely clear at this point but is likely not caused by direct viral infection of islets (Fig. 2E), because in vitro infected islets do not exhibit increased Fas levels (25). Rather, we propose an action of virally induced inflammatory mediators such as nitric oxide (NO) and/or cytokines or viral dsRNA (31). Experimentally, using neutralizing antibodies, we excluded a role for IFN-
(Fig. 3B) or lymphotoxin-ß (not shown), because no change in pancreatic/islet FAS levels was observed after their blockade. It is unlikely that virally induced NO plays a role in islet FAS upregulation as suggested by studies with islets from inducible NO synthase knockout mice (32). Our observation thus underlines an important additional caveat when using topical expression of Fas-L to provide immune protection. Viral infections of the target area might abolish any advantage by inducing Fas on selective cell types. This possibility should be taken into account before using Fas-L expression therapeutically.
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ACKNOWLEDGMENTS
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This is Publication No. 550 from the Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology (San Diego, CA).
This work was supported by National Institutes of Health Grants U19 AI51973, 2 PO1 AG04342-19, and R29 DK51091 to M.G.V. and Juvenile Diabetes Research Foundation Grant 3-2000-510 to U.C.
We thank Diana Frye for excellent assistance with manuscript preparation.
Address correspondence and reprint requests to Matthias G. von Herrath, MD, Division of Developmental Immunology, DI-3, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121. E-mail: matthias{at}liai.org
Received for publication July 30, 2003
and accepted in revised form November 26, 2003
CTL, cytotoxic T-cell; Fas-L, Fas-ligand; IFN-
,
-interferon; LCMV, lymphocytic choriomeningitis virus; mAb, monoclonal antibody; NP, nucleoprotein; RPA, RNase protection assay
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