Choriomeningitis Virus Infection Wasting Disease During Lymphocytic ...

The Journal of Immunology

The Role of Proinflammatory Cytokines in Wasting Disease During Lymphocytic Choriomeningitis Virus Infection1 Cris Kamperschroer and Daniel G. Quinn2 Infection with pathogens often leads to loss of body weight, but the cause of weight loss during infection is poorly understood. We used the infection of mice with lymphocytic choriomeningitis virus (LCMV) as a model to study how pathogens induce weight loss. If LCMV is introduced into the CNS of CTL-deficient mice, the immune response against the virus leads to a severe weight loss called wasting disease. We planned to determine what components of this antiviral immune response mediate wasting disease. By adoptive transfer, we show that CD4 T cells activated by LCMV infection are sufficient to cause wasting disease. We examined the role of cytokines in LCMV-induced wasting disease using mice lacking specific cytokines or cytokine receptors. Results of adoptive transfer experiments suggest that TNF-␣ is not involved in LCMV-induced wasting disease and show that IFN-␥ contributes to the disease. Consistent with a role for IFN-␥ in wasting, we find that IFN-␥ is necessary for LCMV-specific CD4 T cell responses in the CNS, most likely because it is required to induce MHC class II expression. Our data also indicate that IL-1 is required for LCMV-induced wasting and that IL-6 contributes to the wasting disease. Additionally, our results identify ␣-melanocyte-stimulating hormone as a potential mediator of the disease. Overall, this work defines the critical role of virus-primed CD4 T cells and of proinflammatory cytokines in the pathogenesis of wasting disease induced by LCMV infection. The Journal of Immunology, 2002, 169: 340 –349.

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hronic weight loss is a debilitating symptom of a wide range of diseases (1–3), including those resulting from infections with bacteria (4, 5), parasites (6, 7), and viruses (8 –12). We have used the infection of mice with lymphocytic choriomeningitis virus (LCMV)3 as a model to study how immune responses against a persisting pathogen can lead to severe weight loss. Mice deficient in CD8 CTL activity cannot clear infection with LCMV and harbor consistently high viral titers in all tissues tested, including blood, spleen, liver, kidney, and brain (13–15). If LCMV is inoculated directly into the CNS by intracranial (i.c.) injection, CTL-deficient mice also develop severe weight loss (13, 14, 16, 17) termed wasting disease (16). Thus, i.c. infection of strains of mice deficient in ␤2-microglobulin (␤2m) (14, 16, 17), CD8 (17), or perforin (15), or of wild-type mice depleted of CD8 T cells (16) leads to wasting disease. This wasting disease does not occur after i.p. or i.v. infection with LCMV (18) (C. Kamperschroer, unpublished observations), suggesting that wasting is a pathology of the CNS. The wasting disease is not an effect of LCMV replication, but rather results from the immune response against the virus (17), and requires CD4 T cells (13, 14, 16, 17). However, the mechanism whereby the antiviral immune response leads to wasting disease is unknown. Department of Microbiology and Immunology, Loyola University Chicago Medical Center, Maywood, IL 60153 Received for publication October 23, 2001. Accepted for publication April 29, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

This work was supported by a First Award from the Crohn’s and Colitis Foundation of America and National Institutes of Health Grant AI44861 (to D.G.Q.), a Schmitt dissertation fellowship, and National Institutes of Health Training Grant T32 AI07508-01 (to C.K.). 2 Address correspondence and reprint requests to Dr. Daniel G. Quinn, Department of Microbiology and Immunology, Loyola University Chicago Medical Center, Maywood, IL 60153. E-mail address: [email protected] 3

Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; ␣-MSH, ␣-melanocyte-stimulating hormone; ␤2m, ␤2-microglobulin; i.c., intracranial; i.c.v., intracerebroventricular; NP, nucleoprotein; NPY, neuropeptide Y. Copyright © 2002 by The American Association of Immunologists, Inc.

In this study, we investigated which components of the immune response against LCMV contribute to the wasting disease. CD4 T cells are required for wasting (13, 14, 16, 17), but it is not clear whether CD4 T cells of any specificity allow for wasting or whether wasting disease is caused by CD4 T cells specific for LCMV. We therefore tested whether CD4 T cells activated by LCMV infection are sufficient to induce wasting disease. We also assessed the role of cytokines in the disease. A number of proinflammatory cytokines, such as IFN-␥, TNF-␣, IL-1, and IL-6, are involved in wasting syndromes (1–3). Infection of mice with LCMV generates T cells that produce IFN-␥ (19), and both IL-1 (18) and IL-6 (20) are expressed in the CNS during persistent LCMV infection. In these studies, we addressed the hypothesis that LCMV-induced wasting disease results from the action of proinflammatory cytokines produced in response to the virus. Our studies suggest that virus-specific CD4 T cells are critical for LCMV-induced wasting disease. We also show that the proinflammatory cytokines IFN-␥, IL-1, and IL-6 contribute to the wasting disease, whereas TNF-␣ does not. Our data further identify an appetite-suppressing factor, ␣-melanocyte-stimulating hormone (␣-MSH), as a potential mediator of wasting. This work provides insight into how immune responses against pathogens can cause severe weight loss, a debilitating or even fatal symptom of a number of disease states.

Materials and Methods Virus The Armstrong-3 strain of LCMV was propagated in BHK-21 cells (American Type Culture Collection, Manassas, VA), and viral titers of infectious supernatants were determined by plaque assay on Vero cell monolayers. Virus was diluted in serum-free DMEM to obtain appropriate titers for use in experiments.

Treatment of mice B6.129P2-B2mtm1Unc (␤2m⫺/⫺) (21), B6.129S2-Cd4tm1Mak (CD4⫺/⫺) (22), B6.129S7-Ifngtm1Ts (IFN-␥⫺/⫺) (23), B6;129S-Tnftm1Gkl (TNF-␣⫺/⫺) (24), B6.129-Tnfrsf1atm1Mak (TNFR-1⫺/⫺) (25), B6.129-Tnfrsf1btm1Mwm (TNFR2⫺/⫺) (26), B6.129S6-Il6tm1Kopf (IL-6⫺/⫺) (27), or B6;129S-Il1r1tm1Roml (IL0022-1767/02/$02.00

The Journal of Immunology 1R⫺/⫺) (28) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 (B6) mice were obtained from Taconic (Germantown, NY). B6.129-B2mtm1 or B6.SJL-Ptprca/BoAiTac-B2mtm1 (␤2m⫺/⫺) (29) mice obtained from Taconic were used for some experiments. Strains of ␤2m⫺/⫺ mice from either vendor gave similar results. Mice were maintained in specific pathogen-free, American Association for the Accreditation of Laboratory Animal Care-accredited facilities at Loyola University Medical Center on a 12-h light-dark cycle with continuous access to food and water. Mice were infected via the i.p. route with 4 ⫻ 105 PFU LCMV in a volume of 0.4 ml. For i.c. infection, mice were anesthetized by inhalation of Halothane (Sigma-Aldrich, St. Louis, MO) or by i.p. injection of Avertin (2, 2, 2-tribromoethanol), and were then injected i.c. with 2 ⫻ 104 PFU LCMV in a volume of 20 ␮l. Intracranial infection of wild-type mice with LCMV causes lethal meningitis due to CD8 CTL-mediated killing of brain cells (30, 31). To prevent this lethal meningitis in experiments with IL-1R⫺/⫺ or IL-6⫺/⫺ and B6 control mice, CD8 T cells were removed in vivo by injecting animals i.p. with the CD8-depleting Ab 2.43 (32) on days ⫺2, ⫹2, and ⫹7 relative to infection, and at weekly intervals thereafter. The effective dose of 2.43 was determined before experiments, and depletion of CD8 T cells was periodically confirmed on individual mice during experiments by flow cytometry.

Peptide synthesis Peptides corresponding to aa 61– 80 of the LCMV glycoprotein (gp61) or 309 –328 of the LCMV nucleoprotein (np309) were synthesized by the Loyola University Macromolecular Synthesis Facility using a Synergy 432A automated synthesizer. The gp61 and np309 are restricted by I-Ab (33).

Cell preparations Spleen cell suspensions were prepared as previously described (19). Mononuclear cells were isolated from brain tissue over a Percoll gradient using a modification of described methods (34). Mice were bled by cardiac puncture and perfused with PBS. Brains were disrupted in complete medium (HEPES-buffered RPMI 1640 (Mediatech, Herndon, VA) containing 10% (v/v) FCS (Atlanta Biologicals, Norcross, GA), 2 mM L-glutamine, and 50 ␮M 2-ME) by passage through a sterile nylon mesh (100 ␮m pore size; BD Biosciences, San Jose, CA), followed by sequential passages through 19and 23-gauge needles (BD Biosciences). Cell suspensions were centrifuged at 700 ⫻ g for 7 min at 4°C, and pellets were resuspended in a Percoll solution comprised of a 10:1 mixture of Percoll (Sigma-Aldrich) and 10⫻ RPMI 1640 (Sigma-Aldrich). Cell suspensions were then overlaid with 60%, 40%, and 0% dilutions of this Percoll solution. Following centrifugation at 1000 ⫻ g for 15 min at 4°C, mononuclear cells were harvested from the 40 – 60% interface. Cells were washed in complete medium before use in experiments.

Flow cytometry Aliquots containing 1 ⫻ 106 spleen cells in ice-cold PBS supplemented with 1% (v/v) FCS and 15 mM sodium azide (FACS buffer) were incubated with flourochrome-conjugated anti-CD4 (clone RM4-5; BD PharMingen, San Diego, CA) or anti-CD8 (clone 53-6.7; BD PharMingen) Abs at a concentration of 1 ␮g/106 cells, for 30 min at 4°C. Samples were washed with FACS buffer and were analyzed using a FACSCalibur flow cytometer (BD Biosciences). Dead cells were excluded on the basis of forward and side scatter, and data analyses were performed on ⱖ10,000 acquired events using the CellQuest software (BD Biosciences).

ELISPOT and intracellular cytokine staining to enumerate LCMV-reactive cells ELISPOT assays to detect cells producing IFN-␥ in response to antigenic peptides from LCMV were performed, as described previously (19), using cells isolated from spleen or brain, as described above. Intracellular cytokines were detected, as previously described (19), using the Cytofix/Cytoperm intracellular staining kit (BD PharMingen), according to the manufacturer’s instructions. Briefly, test cells were stimulated for 5 h with antigenic peptides in the presence of brefeldin A (Sigma-Aldrich) and IL-2 (Sigma-Aldrich) in wells of 96-well tissue culture plates. Where indicated, we also added 1 ⫻ 106 spleen cells from naive CD4⫺/⫺ mice as APC to each well during the stimulation period. Cell surface CD4 was stained using the Ab RM4-5. Cells were fixed and permeabilized, then Abs XMG1.2, MP6-XT22, and MP5-20F3 were used to detect IFN-␥, TNF-␣, and IL-6, respectively. Isotype-matched Abs were used as controls. All Abs for intracellular cytokine staining were purchased from BD PharMingen.

341 Adoptive transfer Donor spleen cells were obtained from mice 10 days after i.p. infection with LCMV, except where indicated in figure legends. In these cases, donor cells were obtained 9 days after i.p. infection and were cultured overnight at 5 ⫻ 106 cells/ml along with 10 U/ml human rIL-2 (Sigma-Aldrich). In our experience, this treatment enhances cell survival during culture. To prevent transfer of lethal meningitis, CD8 T cells were removed from donor populations before cell transfer by in vitro treatment with the anti-CD8 Ab 31M-6 (32) and baby rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada), as previously described (19). Similarly treated control donor cells were prepared from noninfected mice. Where indicated, donor spleen cells were additionally separated into CD4⫹ and CD4⫺ fractions using anti-CD4-coated magnetic beads before transfer, according to instructions supplied by the manufacturer (Miltenyi Biotec, Auburn, CA). Alternatively, CD4 cells were sorted using a FACStar cell sorter (BD Biosciences) after labeling with PE-conjugated anti-CD4 Ab (clone RM4-5; BD PharMingen). Isolated CD4⫹ fractions were ⱖ98% pure, and CD4⫺ fractions were ⬎99% CD4 deficient, as assessed by flow cytometry (data not shown). Recipient mice were infected i.c. with LCMV 2 days before cell transfer. Recipients were also given 650 rad gamma radiation using a GammaCell-40 irradiator 3 days before cell transfer. This dose of irradiation prevented immune responses against LCMV, as assessed by ELISPOT, and did not cause weight loss (data not shown). Gamma-irradiated mice were used as recipients instead of T cell-deficient RAG⫺/⫺ or SCID mice so that certain strains of gene-targeted mice could be used as recipients. To remove any residual transferred CD8 T cells capable of causing lethal meningitis (30, 31), recipient mice were injected i.p. with the anti-CD8 Ab 2.43 (32) on the day of cell transfer. Depletion of CD8 T cells was confirmed by flow cytometry. Donor cells were injected i.v. into recipient mice (⫽ day 0) in 400 ␮l PBS. Following adoptive transfer, recipient mice were weighed daily. Experiments were typically terminated at or before 8 –10 days after transfer to exclude the possibility that naive LCMV-specific CD4 T cells in various donor populations would become activated, expand, and contribute to weight loss.

Western blotting Lysates were prepared from brain homogenates and from the cell lines CH.B2 (H-2b) and CH.12 (H-2a) using previously described methods (35). Equal amounts of protein from each sample were separated on a 12% SDS-PAGE gel, and I-Ab␤ was detected by Western blot using culture supernatant from the anti-I-Ab␤-secreting hybridoma KL295, as described previously (35).

Measuring food and water intake ␤2m⫺/⫺ mice were housed in metabolism cages (Nalgene Company, Rochester, NY), and given continuous access to food and water. At 24-h intervals, food and water containers were weighed, and their masses were subtracted from those of the respective containers at the previous time point. This difference was taken to be the amount of food or water consumed during each 24-h interval. The mice also were weighed at each time point. Six days after the start of the experiment, mice were infected i.c. with LCMV. All measurements were taken at hour 11:00 of the light cycle.

Serum leptin quantification ␤2m⫺/⫺ mice were infected i.c. with LCMV, injected i.c. with PBS, or left untreated. At hour 11:00 of the light cycle on various days after i.c. infection, mice were bled from the retroorbital sinus. Serum was collected and stored at ⫺80°C until assayed. Leptin concentrations were measured by ELISA, according to the instructions provided with the Quantikine immunoassay for mouse leptin (R&D Systems, Minneapolis, MN).

Quantification of neuropeptides ␤2m⫺/⫺ mice were infected i.c. with LCMV, injected i.c. with PBS, or left untreated. At hour 11:00 of the light cycle on days 6, 12, or 16 after infection, mice were bled by cardiac puncture and perfused with PBS. Brains were removed, and tissue blocks containing the hypothalamus were prepared. Neuropeptides were isolated from tissues according to published methods (36). Samples were lyophilized and stored at ⫺80°C until subjected to RIA specific for each neuropeptide. Concentrations of the neuropeptides ␣-MSH, neuropeptide Y (NPY), and neuromedin U were measured using RIA kits, according to instructions provided by the manufacturer (Pheonix Pharmaceuticals, Belmont, CA).

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ROLE OF CYTOKINES IN LCMV-INDUCED WASTING DISEASE

Statistical analyses The statistical significance of differences observed between groups or of changes within individual groups over time was assessed by an ANOVA using the program Prism, version 3.0 (Graphpad Software, San Diego, CA).

Results Correlation between the LCMV-specific CD4 T cell response and wasting disease If wasting disease induced by LCMV infection results from the CD4 T cell response against the virus, then we would expect weight loss to correlate with the generation of the LCMV-specific CD4 T cell response. To determine whether this is the case, we enumerated LCMV-reactive CD4 T cells using IFN-␥ ELISPOT at various time points following infection of ␤2m⫺/⫺ mice. By 10 –12 days after infection, the number of splenic cells that reacted against the dominant class II-restricted LCMV epitope, gp61, peaked at ⬃2 ⫻ 106 (Fig. 1A). This peak in the CD4 T cell response correlated with the onset of wasting (Fig. 1B). After the antiviral response peaked, it rapidly declined so that by 20 days after infection the numbers of gp61-reactive CD4 T cells decreased nearly 45-fold from their peak numbers (Fig. 1A), which corresponded to the time when mice began to recover from the wasting disease (Fig. 1B). Mice generally regain the lost body weight by 40 –50 days after infection (16) (data not shown), and by this time gp61-reactive cells were nearly undetectable (Fig. 1A). CD4 T cell responses against the subdominant MHC class II-restricted LCMV epitope np309 followed the same trend, but at all time points ex-

amined, the numbers of np309-reactive cells were approximately one-half of the numbers of gp61-reactive cells (data not shown). These data indicate that the LCMV-specific CD4 T cell response correlates with the timing of wasting disease. Adoptive transfer of wasting disease using CD4 T cells activated by LCMV infection We found that if we adoptively transferred unfractionated spleen cells from LCMV-infected mice into infected and irradiated recipient mice, the recipients lost ⬃20% of their initial body weight within 6 days, and removal of donor CD4 T cells before injection prevented transfer of weight loss (Fig. 2). Transfer of equal numbers of cells from noninfected mice did not cause weight loss. These results indicate that the wasting disease can be transferred using cells primed during LCMV infection and confirm that CD4 T cells are required for the wasting disease. We reasoned that if antiviral CD4 T cells are responsible for inducing wasting disease, then purified CD4 T cells from LCMV-infected mice should cause wasting if adoptively transferred to another LCMV-infected mouse. When we transferred purified CD4 T cells from LCMVinfected mice, weight loss of recipients was comparable with that induced by transfer of whole spleen cells from infected mice (Fig. 2). The transfer of wasting required CD4 T cells primed during LCMV infection because transfer of purified CD4 T cells from noninfected mice did not cause weight loss. These data indicate that CD4 T cells primed during LCMV infection are sufficient to cause wasting disease. The roles of IFN-␥ and TNF-␣ in LCMV-induced wasting disease A major function of CD4 T cells is to secrete cytokines, so we examined whether cytokines contribute to LCMV-induced wasting disease. We first needed to identify cytokines produced by LCMVspecific CD4 T cells under conditions that lead to wasting. To do so, we isolated cells from brains or spleens of ␤2m⫺/⫺ mice after i.c. infection with LCMV. Cytokines produced by CD4 T cells following epitope-specific stimulation were detected using intracellular staining. CD4 T cells from spleens (Fig. 3A) and from

FIGURE 1. Correlation of virus-specific CD4 T cell responses with weight loss. A, LCMV-specific CD4 T cell responses in ␤2m⫺/⫺ mice. ␤2m⫺/⫺ mice were infected i.p. with LCMV. At indicated time points after infection, spleen cells were isolated, and the numbers of cells capable of reacting against gp61 were measured using ELISPOT. Symbols represent the numbers of gp61-reactive cells detected in spleens of individual mice, and the solid line indicates the mean number of reactive cells at each time point (n ⱖ 5, except on days 14, 17, and 60, in which n ⫽ 3). The dotted line represents the limit of detection of the assay. B, Change in body weight following infection of ␤2m⫺/⫺ mice. ␤2m⫺/⫺ mice were injected i.c. with LCMV or PBS, as indicated, and weighed every other day. Data are presented as the mean percent change from the initial (day 0) body weight ⫾ SEM. A statistically significant difference was observed between LCMVinfected (n ⫽ 8) and PBS-injected (n ⫽ 6) groups, p ⬍ 0.0001.

FIGURE 2. Adoptive transfer of wasting disease. Spleen cells isolated from B6 mice 9 days after i.p. infection with LCMV (INF), or from noninfected (NI) B6 mice as control, were depleted of CD8 cells and cultured overnight in medium containing IL-2. The cells were then separated into CD4⫹ and CD4⫺ fractions, or not fractionated (total). A total of 2 ⫻ 107 unfractionated cells, 2 ⫻ 107 CD4-depleted cells, or 5 ⫻ 106 purified CD4 cells were injected i.v. into recipient B6 mice. Transferred donor cell populations are indicated on the right. Recipients were infected i.c. with LCMV, irradiated, and injected with anti-CD8 Ab before cell transfer. Recipients were weighed daily following transfer. Data points represent the means ⫾ SEM for each group. Statistically significant differences were observed between total INF (n ⫽ 4) vs CD4⫺ INF (n ⫽ 5) and between CD4⫹ INF (n ⫽ 4) vs CD4⫹ NI (n ⫽ 4), p ⬍ 0.0001. Data shown are representative of two independent experiments.


FIGURE 3. Cytokines produced by LCMV-specific CD4 T cells during wasting disease. ␤2m⫺/⫺ mice were infected i.c. with LCMV. Twelve days after infection, cells were isolated from spleen (A) and brain (B). Cells were stimulated with gp61 or left unstimulated (no stim.). Surface CD4 was stained using anti-CD4 Ab, and cytokines produced by cells were detected by intracellular cytokine staining for IFN-␥ or TNF-␣, followed by flow cytometry. Numbers above each plot indicate the percentages of CD4⫹ cells staining positive for the indicated cytokine. Data shown are representative of two independent experiments.

brains (Fig. 3B) produced IFN-␥ and TNF-␣ following stimulation with gp61 (Fig. 3) or with np309 (data not shown), whereas unstimulated CD4 T cells did not produce either cytokine. We did not detect CD4 T cells producing IL-4 or IL-6 by intracellular cytokine staining following LCMV infection of ␤2m⫺/⫺ mice (data not shown). To determine whether TNF-␣ or IFN-␥ contributes to the LCMV-induced wasting disease, we adoptively transferred spleen cells from LCMV-infected TNF-␣⫺/⫺ or IFN-␥⫺/⫺ donor mice and assessed whether the transferred cells caused weight loss. Recipients of cells from LCMV-infected TNF-␣⫺/⫺ mice lost 25– 30% of their starting body weight within 6 days (Fig. 4A), which was indistinguishable from the weight loss induced by cells from infected wild-type B6 mice. Weight loss comparable with wildtype controls was observed regardless of whether the TNF-␣⫺/⫺ donor cells were transferred into wild-type recipients or into TNF receptor-deficient TNFR-1⫺/⫺ or TNFR-2⫺/⫺ recipients (Fig. 4A). These results strongly suggest that TNF-␣ is not required for the LCMV-induced wasting disease. Fig. 4B shows the results of experiments to assess the role of IFN-␥. As expected, transfer of cells from noninfected mice did not cause weight loss. Mice that received cells from infected wild-type mice lost ⬃25% of their body weight. In contrast, mice that received cells from infected IFN␥⫺/⫺ mice lost 10% of their starting body weight (Fig. 4B). This indicates that IFN-␥ contributes to the LCMV-induced wasting disease. We additionally transferred IFN-␥⫺/⫺ donor cells into TNFR-1⫺/⫺ recipient mice, but the weight loss of recipients observed under these conditions was indistinguishable from that observed when IFN-␥⫺/⫺ donor cells were transferred into wild-type recipients (data not shown). The results further suggest that TNF-␣ does not contribute to the wasting disease and confirm that IFN-␥ contributes to the wasting disease following LCMV infection. LCMV-specific CD4 T cell responses of adoptively transferred IFN-␥⫺/⫺ cells We have shown that transfer of LCMV-primed IFN-␥⫺/⫺ cells leads to less severe weight loss than does transfer of primed wildtype cells (Fig. 4B). One possible explanation for this is that without IFN-␥, insufficient LCMV-specific CD4 T cells are generated in the donor mice. However, when we compared spleen cells from wild-type B6 donor mice with IFN-␥⫺/⫺ donor mice, we did not

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FIGURE 4. Contribution of IFN-␥ and TNF-␣ to wasting disease. Spleen cells isolated from TNF-␣⫺/⫺ (A) or IFN-␥⫺/⫺ (B) donor mice 10 days after i.p. infection with LCMV or from control noninfected (NI) mice were depleted of CD8 cells. Recipient mice were infected i.c. with LCMV, irradiated, and injected with anti-CD8 Ab before cell transfer. Recipients were weighed daily following cell transfer. Donor and recipient mice are indicated in legends on the right, and each data point represents the mean ⫾ SEM (in which n ⱖ 3). A, 1 ⫻ 107 donor cells, or no cells (none), were injected i.v. into recipient mice. No statistically significant differences were observed between groups receiving B6 cells vs TNF-␣⫺/⫺ cells, regardless of the recipient strain (B63 B6, n ⫽ 4; B63 TNFR-1⫺/⫺, n ⫽ 5; B63 TNFR-2⫺/⫺, n ⫽ 2; TNF-␣⫺/⫺3 B6, n ⫽ 3; TNF-␣⫺/⫺3 TNFR1⫺/⫺, n ⫽ 5; TNF-␣⫺/⫺3 TNFR-2⫺/⫺, n ⫽ 3; none3 B6, n ⫽ 9; none3 TNFR-2⫺/⫺, n ⫽ 1). B, 2 ⫻ 107 donor cells were injected i.v. into recipient mice. Statistically significant differences were observed between IFN-␥⫺/⫺3 B6 (n ⫽ 5) vs B63 B6 (n ⫽ 4) and B6 NI3 B6 (n ⫽ 3), p ⬍ 0.0001. Data shown are representative of two (A) or three (B) separate experiments.

observe any consistent differences in the percentages or the numbers of donor CD4 T cells reacting against gp61 following infection (data not shown). This indicates that IFN-␥ deficiency does not impair the generation of LCMV-reactive CD4 T cells. It is also possible that IFN-␥⫺/⫺ cells are not maintained following i.v. transfer or that IFN-␥⫺/⫺ donor cells do not migrate to appropriate sites after transfer. Finally, transferred IFN-␥⫺/⫺ CD4 T cells may be unable to respond to Ag. To test these possibilities, we enumerated virus-reactive CD4 T cells in spleen and brains of each recipient mouse following transfer. LCMV-specific CD4 T cells produce TNF-␣ (Fig. 3), so we detected IFN-␥⫺/⫺ antiviral CD4 T cells by intracellular cytokine staining for TNF-␣. In the spleens of recipient mice 5 days after cell transfer, no significant differences were observed in the total number of cells or in the number of CD4 T cells between mice that received wild-type cells and mice that received IFN-␥⫺/⫺ cells. By intracellular staining, we found that ⬃5% of CD4 T cells in the spleen were gp61 specific when IFN-␥⫺/⫺ cells were transferred compared with ⬃4% if wild-type cells were transferred (Fig. 5A). This shows that there is no defect in the ability of LCMV-specific IFN-␥⫺/⫺ CD4 T cells to survive i.v. transfer or to populate the spleen following transfer. The number of mononuclear cells as well as the number and proportion of CD4 cells isolated from brains of recipients receiving either wild-type or IFN-␥⫺/⫺ cells were similar (Fig. 5B and data not shown). In contrast, we did not observe CD4 T cells infiltrating

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FIGURE 5. LCMV-specific CD4 T cell responses in recipient mice after transfer of LCMV-primed IFN-␥⫺/⫺ cells. Spleen cells from LCMV-infected IFN-␥⫺/⫺ or B6 mice or from noninfected B6 mice were adoptively transferred to recipients, as described in Fig. 4. Cells were isolated from spleens (A) or brains (B) of recipients 6 days after transfer of cells from the indicated donor, and were stimulated with gp61 or left unstimulated (no stim.). Surface CD4 was labeled with anti-CD4 Ab, and gp61-reactive cells were detected using intracellular staining for TNF-␣, followed by flow cytometry. Numbers above each plot indicate the percentages of CD4⫹ cells staining positive for TNF-␣. Data are representative of two independent experiments with at least three mice per group per experiment.

the brains of recipient mice if cells from noninfected mice were transferred. This indicates that LCMV-specific CD4 T cells migrate to the brains of infected recipient mice following transfer, and that this migration is not grossly affected by IFN-␥ deficiency. When we measured LCMV-specific responses, greater than 12% of CD4 T cells from brains of recipients receiving wild-type cells were gp61 specific. In contrast, few (2.4%) of the CD4 T cells in brains of mice receiving IFN-␥⫺/⫺ cells were able to respond against gp61 (Fig. 5B). Taken together, these results show that IFN-␥⫺/⫺ donor cells are capable of responding against LCMV in the spleen and are able to migrate to the brain, yet very few of these cells respond against LCMV when they reach the brain. IFN-␥ dependence of LCMV-specific CD4 T cell responses in the CNS during wasting disease IFN-␥ is a potent inducer of class II MHC expression in the CNS (37), so one possible explanation for the failure of transferred IFN␥⫺/⫺ T cells to respond in the CNS is that there is insufficient expression of I-Ab to present LCMV Ag to these T cells. To investigate this, we examined levels of I-Ab in the brains of recipient mice by Western blot after adoptive transfer of cells from infected IFN-␥⫺/⫺ mice, infected wild-type B6 mice, or noninfected wildtype mice, as described above. When no cells were transferred, I-Ab was almost undetectable, whereas I-Ab was dramatically upregulated in brains after transfer of wild-type cells (Fig. 6A). In recipients of IFN-␥⫺/⫺ cells, levels of I-Ab in brains were comparable with those in controls receiving noninfected spleen cells or no cells at all (Fig. 6A). This shows that production of IFN-␥ from the transferred cells is required for up-regulation of I-Ab in the brain following i.c. infection with LCMV. If antiviral IFN-␥⫺/⫺ CD4 T cells are not responding in the brain because of insufficient I-Ab expression, then their ability to respond should be restored by adding I-Ab-expressing cells during the intracellular staining assay. This was done by adding spleen cells from CD4⫺/⫺ mice so that we could distinguish brain-derived CD4 T cells from the added spleen cells. When we added I-Abexpressing spleen cells as APC (from CD4⫺/⫺ mice) to brainderived IFN-␥⫺/⫺ donor cells, the percentage of gp61-reactive CD4 T cells increased from 1.3% to 18.1% (Fig. 6B). Addition of APC had a modest effect on the proportion of transferred wild-type

CD4 T cells that reacted against gp61. These data indicate that LCMV-specific CD4 T cells from brains of mice receiving IFN␥⫺/⫺ cells are capable of responding against viral Ag, and suggest that they do not respond due to insufficient Ag presentation within the brain. Together, the data shown in Fig. 6 suggest that IFN-␥ promotes LCMV-specific CD4 T cell responses in the CNS during wasting disease by up-regulating MHC class II expression. Food and water intake during LCMV-induced wasting disease To this point, we had determined that CD4 T cells activated by LCMV infection are sufficient to cause wasting disease and that IFN-␥, which is produced by LCMV-specific CD4 T cells, contributes to the disease. However, it was still unclear how the CD4 T cells promote wasting. In an attempt to identify factors that could mediate LCMV-induced wasting, we investigated the physiological basis for the weight loss. One possible explanation is that mice decrease their consumption of food or water. To address whether wasting correlates with decreased intake of food or water, we measured changes in body weight and in the amount of food and water consumed daily before and after i.c. infection of ␤2m⫺/⫺ mice with LCMV. Before infection, mice consumed ⬃3– 4 g of food per day (Fig. 7B). Daily food intake was fairly constant until approximately day 6 after infection. By 8 days after infection, the amount of food consumed per day decreased 3- to 4-fold (Fig. 7B), and mice had lost ⬎15% of their starting body weight (Fig. 7A). Daily food intake increased after this time, but it took 10 additional days to return to preinfection levels of food intake (Fig. 7B). During these 10 days, mice reached their peak weight loss, which was maintained until 19 days after infection (Fig. 7A). We also observed a similar trend in water consumption (Fig. 7C), but the decrease of water intake was less pronounced. Together, these data show that daily intake of food, and to a lesser degree water, decreases as mice succumb to wasting disease. The contribution of IL-1 and IL-6 to LCMV-induced wasting disease The decrease of food and water intake observed as LCMV-infected mice lose weight (Fig. 7) suggests that the wasting disease results at least partly from a loss of appetite, or anorexia. IL-1 and IL-6 can cause anorexia (1–3), and both cytokines are expressed in the


FIGURE 6. MHC class II expression and responsiveness of cells in brains of recipient mice after adoptive transfer. Spleen cells isolated from LCMV-infected (INF) IFN-␥⫺/⫺ or B6 mice or from noninfected (NI) B6 mice were adoptively transferred to recipients, as described in Fig. 4. A, Six days following adoptive transfer of the indicated donor cells or of no cells (none), brains were removed from recipient mice and protein lysates were prepared. Lysates were also prepared from control cell lines that do (CH. B2) or do not (CH.12) express I-Ab. I-Ab protein from each sample was detected using Western blot. Data shown are representative of two independent experiments. B, Six days after transfer of the indicated donor cells, cells were isolated from brains of recipient mice. Cells were left unstimulated (no stim.) or were stimulated with gp61 without any added APC (top panels) or in the presence of naive spleen cells from CD4⫺/⫺ mice as APC (bottom panels). Surface CD4 was labeled with anti-CD4 Ab, and gp61reactive cells were detected using intracellular staining for TNF-␣, followed by flow cytometry. Numbers above each plot indicate the percentages of CD4⫹ cells staining positive for TNF-␣. Only CD4⫹ events were analyzed to distinguish CD4 cells isolated from brain from the added spleen cells.

CNS during persistent LCMV infection (18, 20). Therefore, we determined whether IL-1 or IL-6 contributes to LCMV-induced wasting disease. If IL-1 is required for the wasting disease, then IL-1R-deficient (IL-1R⫺/⫺) mice that cannot respond to IL-1 should not lose weight after infection. We infected CD8 T cell-depleted IL-1R⫺/⫺ mice i.c. with LCMV and measured subsequent changes in body weight. Infected IL-1R⫺/⫺ mice lost little or no weight compared with noninfected wild-type B6 control mice (Fig. 8A). In contrast, infected B6 control mice lost 20 –25% of their initial body weight. These results show that the IL-1R is required for wasting disease induced by LCMV. We tested the contribution of IL-6 to LCMV-induced wasting disease using a similar approach. If IL-6 mediates the wasting disease, then IL-6⫺/⫺ mice should be protected from weight loss upon infection. In the first 14 days after i.c. LCMV infection, weight loss among IL-6⫺/⫺ mice was similar to that of wild-type B6 mice (Fig. 8B). After day 14, however, IL-6⫺/⫺ mice quickly regained the weight lost, whereas wild-type mice did not begin to regain the weight lost until at least 24 days after infection. These

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FIGURE 7. Food and water consumption during LCMV-induced wasting disease. ␤2m⫺/⫺ mice (n ⫽ 4) were infected i.c. with LCMV. At 24-h intervals, mice were weighed (A). Amounts of food (B) and water (C) consumed by the same mice over each 24-h interval were also measured. Data points represent the mean ⫾ SEM. Statistically significant decreases in body weight (A) and food intake (B) were observed, p ⬍ 0.0001. Data are representative of two independent experiments.

results indicate that IL-6 contributes to the wasting disease, particularly in the later stages of the disease. Levels of factors that control appetite during LCMV-induced wasting disease If anorexia contributes to the LCMV-induced wasting disease, as our data suggest (Fig. 7), then there should be a change in the amounts of one or more of the factors that control appetite as mice lose weight. The hypothalamus is the main appetite control center of the brain, so we measured levels of several factors that act within the hypothalamus to control appetite. We chose to measure levels of leptin, ␣-MSH, NPY, and neuromedin U because these factors strongly influence appetite (38, 39). Leptin is produced systemically and is a potent suppressor of appetite (38). Thus, if leptin were mediating the weight loss, leptin levels should increase as mice lose weight. Serum concentrations of leptin in untreated ␤2m⫺/⫺ mice were 7– 8 ng/ml before infection (Fig. 9A). Conversely, by 8 –10 days after i.c. infection with LCMV, serum leptin concentrations were ⬃20-fold lower than preinfection concentrations and ⬃10-fold lower compared with those of mock-infected ␤2m⫺/⫺ mice (Fig. 9A). The observation that serum leptin levels drastically decrease following infection is inconsistent with leptin being a mediator of LCMV-induced wasting disease. NPY, neuromedin U, and ␣-MSH are neuropeptides that act within the hypothalamus to affect appetite (38, 39). To assess the levels of these neuropeptides during wasting, we measured the amounts of each neuropeptide present in brain tissues containing the hypothalamus isolated at various time points after i.c. infection of ␤2m⫺/⫺ mice. For each time point, these amounts were compared with the amounts of weight lost during the preceding 2 days. In this way, we assessed whether mice were in the process of losing weight when the neuropeptide was measured. We detected

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FIGURE 8. Contribution of IL-1 and IL-6 to LCMV-induced wasting disease. The indicated strains of mice were infected i.c. with LCMV or injected i.c. with PBS as control (NI). All mice were also depleted of CD8 cells. Mice were weighed every other day following infection. Data points represent the mean ⫾ SEM. A, Statistically significant differences were observed between IL-1R⫺/⫺ (n ⫽ 4) vs B6 (n ⫽ 6), p ⬍ 0.0001. B, Statistically significant differences were observed between all groups, p ⬍ 0.0001 (IL-6⫺/⫺, n ⫽ 5; B6, n ⫽ 5; B6 NI, n ⫽ 4). Data shown are representative of two independent experiments.

no change in the levels of NPY or neuromedin U following infection, suggesting that neither of these factors mediates LCMV-induced wasting disease. ␣-MSH suppresses appetite (38). Thus, if ␣-MSH mediates wasting, then we would expect hypothalamic ␣-MSH levels to increase at times when mice are losing weight. We detected 2 ng ␣-MSH/mg protein isolated from untreated (day 0) brain tissues before infection (Fig. 9B). Amounts of ␣-MSH remained low (from 4 to 7 ng/mg protein) at days 6 and 12 after infection and at day 16 after control injection with PBS. In this particular experiment, we also did not observe weight loss during the 2 days preceding these time points. In contrast, a 14-fold increase in ␣-MSH from preinfection levels to 27 ng/mg protein was observed at 16 days after LCMV infection, and this correlated with significant loss of body weight (2.3 g) from day 14 to day 16 (Fig. 9B). These data are consistent with ␣-MSH being a mediator of wasting disease induced by LCMV infection.

Discussion To determine the mechanism of LCMV-induced wasting disease, we assessed what cell types are involved in the disease and tested whether several proinflammatory cytokines contribute to the disease. We have determined that CD4 T cells primed during LCMV infection are sufficient to cause wasting disease. Our data show that IFN-␥, IL-1, and IL-6 contribute to wasting and suggest that the disease is at least partly due to anorexia, possibly via the action of ␣-MSH. Although CTL-deficient mice cannot control LCMV infection (13–15), viral replication itself does not cause wasting disease (17). Rather, the observed weight loss results from the immune

FIGURE 9. Levels of serum leptin and brain ␣-MSH during wasting. A, Serum leptin concentrations. ␤2m⫺/⫺ mice were infected i.c. with LCMV (filled symbols), injected i.c. with PBS (E), or left untreated (䡺). Leptin concentrations in serum samples taken at various time points after infection were measured by ELISA. Each set of filled symbols represents an individual mouse. Data points indicated by open symbols represent the mean ⫾ SEM. Statistically significant differences were observed between LCMV (n ⫽ 6) vs PBS (n ⫽ 3) and untreated (n ⫽ 5) groups, p ⱕ 0.001. B, Levels of ␣-MSH in the brain during wasting. ␤2m⫺/⫺ mice (n ⫽ 4) were infected i.c. with LCMV, injected i.c. with PBS, or left untreated (day 0). At indicated time points after infection, blocks of brain tissue containing the hypothalamus were prepared, and the amount of ␣-MSH in each tissue was measured by RIA. Data are expressed in ng ␣-MSH detected/mg total protein isolated from each tissue (left y-axis and open bars). Mice were also weighed following infection. Shown is the change in body weight during the 2 days before the indicated time points (right y-axis and f). Values shown represent the mean ⫾ SEM.

response against the virus (17). We therefore focused on what components of the immune response against LCMV contribute to the disease. The requirement for CD4 T cells for wasting suggests that the CD4 T cell responses against LCMV may induce disease. Following infection, we found that the expansion of large numbers of LCMV-reactive CD4 T cells correlates with induction of wasting disease, and that the loss of these LCMV-reactive CD4 T cells correlates with recovery from wasting. Further evidence that the LCMV-specific CD4 T cell response leads to wasting comes from our adoptive transfer studies. We showed that the wasting disease could be adoptively transferred with purified CD4 T cells taken from LCMV-primed donor mice, but not with those taken from noninfected donors. These results indicate that CD4 T cells activated by LCMV infection are sufficient to cause wasting disease. At 9 –10 days after infection, when we isolated donor cells for adoptive transfer, LCMV is undetectable by plaque assay (data not shown). However, it is possible that in our adoptive transfer experiments, residual virus is transferred along with the donor cells. Regardless, the transfer of virus to recipient mice would not affect these experiments because the recipients are already infected with LCMV at the time of transfer and because LCMV replication by itself does not cause wasting disease (17). It is possible that CD4 T cells that are not specific for LCMV become activated in a bystander fashion during LCMV infection, and that they are the cells

The Journal of Immunology that are responsible for inducing wasting disease. However, a recent report has estimated that greater than 80% of the CD4 T cells that proliferate in response to LCMV infection react against either gp61 or np309 (40). This estimate argues against substantial bystander activation of CD4 T cells during LCMV infection. Therefore, we think it is unlikely that bystander-activated CD4 T cells cause wasting disease following LCMV infection. To elucidate functions of LCMV-specific CD4 T cells that induce wasting disease, we first determined what cytokines are produced by LCMV-specific CD4 T cells during the disease. We found that LCMV-specific CD4 T cells from brains and spleens of ␤2m⫺/⫺ mice produce IFN-␥ and TNF-␣ during wasting. Hildeman et al. (41) have shown using ELISA that cells from the cerebrospinal fluid of ␤2m⫺/⫺ mice 7 days after i.c. infection produce IFN-␥, but these authors were unable to detect TNF-␣ production by cells from spleen or from cerebrospinal fluid of these animals (18, 41). A recent report showed that the percentage of CD4 T cells producing TNF-␣ in response to gp61 increases from 0.9 to 3.3% from day 7 to day 9 following infection (40). We find that by day 10 –12 after i.p. infection of both B6 and ␤2m⫺/⫺ mice, 6 – 8% of splenic CD4 T cells produce TNF-␣ in response to gp61 (data not shown), so it is likely that the time points examined by Hildeman et al. (3 and 7 days after i.c. infection) were too early in the antiviral CD4 T cell response to detect TNF-␣ production by CD4 T cells. From our data, we conclude that under conditions that lead to wasting disease, LCMV-specific CD4 T cells produce IFN-␥ and TNF-␣. We next determined whether IFN-␥ or TNF-␣ contributes to wasting. In our adoptive transfer studies, the weight loss induced by TNF-␣⫺/⫺ cells was equivalent to that induced by wild-type cells. This was the case regardless of whether the recipient mice lacked the ability to respond to TNF-␣ through TNFR-1 or TNFR-2. These results strongly suggest that TNF-␣ is not required for wasting. It is formally possible, however, that TNF-␣ produced by radioresistant recipient cells induces the transferred cells to produce another factor that causes wasting. IFN-␥⫺/⫺ cells adoptively transferred minimal weight loss compared with that transferred by wild-type cells, so we conclude that IFN-␥ contributes to wasting. Removing IFN-␥ did not provide complete protection from wasting disease because recipients of IFN-␥⫺/⫺ cells lost more weight than did recipients of noninfected cells. It is unlikely that recipient cells produced sufficient IFN-␥ to induce the minimal weight loss observed because we detected no IFN-␥-producing cells in recipients after transfer of noninfected cells. The most likely explanation, then, is that another factor acts independently of IFN-␥ to promote weight loss. Regardless, little weight loss was observed in the absence of IFN-␥, so IFN-␥ is an important contributing factor to the wasting disease. To determine how IFN-␥ contributes to the wasting disease, we examined whether removing IFN-␥ prevents CD4 T cells from responding against LCMV in recipient mice after adoptive transfer. To detect LCMV-reactive CD4 T cells in these experiments, we relied on their ability to produce TNF-␣ after antigenic stimulation. We have observed that 10 days or more after infection, the ratio of the percentage of TNF-␣-positive CD4 T cells to the percentage of IFN-␥-positive CD4 T cells is constant (Fig. 3 and data not shown). This indicates that for our experiments, TNF-␣ production is a reliable indicator of LCMV-reactive CD4 T cells. Our data indicate that while the transferred IFN-␥⫺/⫺ CD4 T cells are capable of responding against LCMV in the spleen, few do so once they reach the brain. Fewer LCMV-responsive CD4 T cells in the CNS correlate with relatively little weight loss in recipients of IFN-␥⫺/⫺ cells, and is consistent with the idea that LCMV-induced wasting disease is a pathology of the CNS. Upon further

347 investigation, we found that levels of I-Ab were substantially lower in brains of mice receiving IFN-␥⫺/⫺ cells when compared with those receiving wild-type cells. Additionally, we could restore antiviral responses of brain-derived IFN-␥⫺/⫺ CD4 T cells if we added spleen cells as APC. Together, these results suggest that IFN-␥ contributes to the LCMV-induced wasting disease by inducing sufficient class II MHC expression to drive LCMV-specific CD4 T cell responses within the CNS. Although we identified IFN-␥ as a factor produced by CD4 T cells that contributes to the LCMV-induced wasting disease, the mechanism by which the weight loss occurs was still unclear. One possible explanation is that mice decrease food or water intake, so we measured food and water intake over the course of wasting. We observed that ␤2m⫺/⫺ mice decreased food intake at the onset of wasting, in agreement with a recent report (18). By restricting uninfected mice to the same amount of food intake as LCMVinfected mice, the authors of this report found it possible to account for weight loss by the observed decrease in feeding. Although this result does not demonstrate that the wasting disease is due to decreased food intake, it suggests, as our data do, that decreased food intake contributes to the weight loss. We additionally found that water intake also decreased during wasting, although this decrease was less pronounced than the decrease in food intake. Decreased water intake is consistent with the severe dehydration we observe during LCMV-induced wasting (C. Kamperschroer, unpublished observations). Taken together, our data show that intake of food and water decreases during wasting disease, suggesting that anorexia (appetite suppression) is at least partly responsible for the wasting disease. IL-1 can induce anorexia (1–3), and we found that IL-1R⫺/⫺ mice did not lose weight after i.c. infection, demonstrating that the IL-1R is required for wasting. A recent study by Hildeman and Muller (18) reported that treatment of mice via intracerebroventricular (i.c.v.) cannulae with neutralizing Ab against IL-1␤ lessened the severity of LCMV-induced wasting. However, there was no statistically significant difference in weight loss between mice treated with anti-IL-1␤ and untreated controls at any time points analyzed, except at day 8 after infection, and both groups ultimately succumbed to wasting. IL-1␣ also binds IL-1R, so one explanation consistent with both our data and that of Hildeman and Muller is that IL-1␣ is the major mediator of LCMV-induced wasting disease, whereas IL-1␤ plays a minor role in the disease. Another possibility is that these authors did not achieve complete neutralization of IL-1␤. Regardless, because IL-1R⫺/⫺ mice were completely protected from weight loss after i.c. infection with LCMV, we infer that IL-1 is essential for the LCMV-induced wasting disease. IL-1␤ directly acts upon neurons in the hypothalamus during the weight loss that results from i.c.v. injection of this cytokine (42). It is possible that IL-1 mediates LCMV-induced wasting in this manner. Our results demonstrate that IL-6 contributes to the wasting disease, and that its effect is most prominent beyond the first 2 wk after infection. IL-6 is induced in the brain later than IFN-␥ or IL-1 following i.c. infection of B6 mice (43), consistent with IL-6 having its major effect later in the disease course. During LCMV infection, the IL-6 produced in the CNS is largely derived from microglia and astrocytes (44), so these cell types may produce the IL-6 that contributes to wasting disease. The involvement of IL-1 and IL-6 in LCMV-induced wasting is consistent with the CD4 T cell dependence of this disease. It has been shown that production of IL-1 and IL-6 in the LCMV-infected CNS requires T cells (43, 44). We know that LCMV-specific CD4 T cells produce IFN-␥ and that IFN-␥ can induce cells to produce IL-1 (45, 46), so it is possible that the IFN-␥ secreted

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by LCMV-specific CD4 T cells induces IL-1 production. This could explain why removing IFN-␥ prevents severe weight loss. However, another CD4 T cell-derived factor must also induce IL-1 production in this scenario because the protection afforded by removing IFN-␥ is not complete. The observation that LCMV-infected mice decrease food and water consumption at the onset of wasting (18) prompted us to examine factors involved in the regulation of appetite. We observed increased amounts of the appetite-suppressing neuropeptide ␣-MSH in brain tissue containing the hypothalamus only at a time when mice were losing weight. This is consistent with the idea that LCMV-induced weight loss is mediated by ␣-MSH. The increase in ␣-MSH is unlikely to directly result from viral replication because at all time points examined in this experiment, brain viral titers are equivalent (data not shown). There is evidence that i.c.v. injection of IL-1 causes weight loss through the ␣-MSH pathway (47), so during LCMV-induced wasting disease, IL-1 may mediate weight loss by directly stimulating hypothalamic neurons (41) to produce ␣-MSH. Additional experiments are needed to determine whether this is the case. With the data presented in this study, we propose a model for the pathogenesis of LCMV-induced wasting disease (Fig. 10). Following i.c. infection with LCMV, virus replicates within the CNS. Activated CD4 T cells migrate to the CNS, where they encounter LCMV Ag. These antiviral CD4 T cells react against LCMV by producing IFN-␥ and TNF-␣. IFN-␥ up-regulates MHC class II on cells within the CNS, which in turn drives the CD4 T cell response. The antiviral CD4 T cell response cannot control the virus, and instead initiates the wasting disease. IFN-␥ or other CD4 T cellderived factors induce other cells within the CNS, such as microglia or infiltrating macrophages, to produce IL-1 and IL-6. IL-1 directly mediates weight loss by inducing neurons in the hypothalamus to produce ␣-MSH or other anorectic neuropeptides. IL-6 also contributes to the wasting disease, particularly later in the disease. Through the actions of IL-1 and IL-6, mice succumb to severe weight loss due at least partly to anorexia. Despite continued viral replication, antiviral CD4 T cell responses wane by 2–3 wk after infection, possibly due to overwhelming Ag load (48). Following the loss of the CD4 T cell response against LCMV, the inflammatory response in the brain subsides, and production of IL-1 and IL-6 within the brain decreases. In the absence of these cytokines, the mouse begins to recover from disease and regains the body weight lost. This study demonstrates how the immune response against a persisting pathogen can lead to wasting, a symptom of many disease states. The findings made using our system of infection may be applicable to other infections, and identify potential targets for

FIGURE 10. Model for LCMV-induced wasting disease.

therapies aimed at minimizing the debilitating or even fatal consequences of severe weight loss during disease.

Acknowledgments We thank John Dye, Emma Nielsen, and Paul Jasper for critical reading of the manuscript, and John Dye for help with some of the experiments. We also thank P. Simms for assistance with flow cytometry.

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