Marginal Zone Macrophages and Immune Responses Against Viruses Stephan Oehen, Bernhard Odermatt, Urs Karrer, Hans Hengartner, Rolf Zinkernagel and Constantino López-Macías This information is current as of September 23, 2021. J Immunol 2002; 169:1453-1458; ; doi: 10.4049/jimmunol.169.3.1453 http://www.jimmunol.org/content/169/3/1453 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Marginal Zone Macrophages and Immune Responses Against Viruses1

Stephan Oehen,2* Bernhard Odermatt,* Urs Karrer,3* Hans Hengartner,* Rolf Zinkernagel,* and Constantino Lo´pez-Macı´as†

The effective establishment of antiviral protection requires a coordinated interplay between the innate and adaptive immune system. Using osteopetrotic (op؊/؊) mice, this study investigated the influence of marginal zone macrophages in controlling and initiating a protective immune response against a cytopathic vs a non- or low-cytopathic virus. Despite the generation of potent adaptive immune responses, antiviral protection against cytopathic vesicular stomatitis virus critically depended on the presence of marginal zone macrophages. Infection with low doses (100 PFU) of non- or low-cytopathic lymphocytic choriomeningitis virus was rarely cleared and usually resulted in a carrier state in the majority of mice. This shows that the early innate immune system provides an important preparatory phase to the adaptive immune system and is particularly important for antiviral Downloaded from protection. The Journal of Immunology, 2002, 169: 1453–1458.

or efficient immunity, the different components of the im- be experimentally induced by the administration of clodronate en- mune system have to cooperate. The interplay among lym- capsulated in liposomes (15Ð17). Using this experimental ap- phokines, chemokines, and cellular and humoral immunity proach, it has been shown that such mice fail to control a systemic

F http://www.jimmunol.org/ has been studied in numerous models of natural and targeted mu- lymphocytic choriomeningitis virus (LCMV) infection (18). How- tations (1Ð3). However, collaboration between the innate and the ever, long-term studies are difficult to carry out in MZM-depleted adaptive immune system is not well explored because defects af- mice because the MZM are replenished and because repeated treat- fecting the innate immune system, particularly macrophages or ment with clodronate liposomes results in a high mortality rate neutrophils, are often embryonically lethal or severely immuno- with severe side effects resulting in noninterpretable results. Nev- compromise the host, leading to death soon after birth. Osteope- ertheless, studies in MZM-depleted mice and in opϪ/Ϫ mice re- trotic (op)4 mice carry a natural point mutation in the M-CSF gene vealed that MZM play a role in controlling Listeria monocytogenes (4Ð7) and fail to develop marginal zone macrophages (MZM) in infections (19, 20). Additionally, earlier studies had shown that the spleen and the corresponding sinusoidal macrophages in the LCMV-infected MZM were destroyed by virus-specific CTLs and lymph nodes (8, 9); these mice are severely deficient in functional caused systemic immunosuppression (21). Non- or low-cytopathic by guest on September 23, 2021 osteoclasts and therefore form inadequate bone marrow cavities. viruses such as LCMV differ from cytopathic viruses such as ve- Despite these defects, opϪ/Ϫ mice develop normal in vivo phago- sicular stomatitis virus (VSV) in that the former are usually elim- cytic functions but have impaired release of TNF and G-CSF (10). inated by CTLs, whereas neutralizing Abs induced during the Although B cell lineage precursors are present in the bone marrow course of the primary immune response usually control the latter. of these mice, B cell lymphopoiesis in adult opϪ/Ϫ mice is ob- Common to both viruses is that they are initially recruited to lym- served only in the liver (11). Despite this, opϪ/Ϫ mice demonstrate phoid organs by natural Abs, where they are thought to be removed normal B cell responses in vivo (10, 12). Normal MLRs in vitro by MZM to reduce dissemination of virus to vital organs and at the indicated that T cells from opϪ/Ϫ mice are functional (12). same time enhance induction of an immune response (22Ð24). The It has been observed that macrophage depletion reduces the host present study used opϪ/Ϫ mice to evaluate the contribution of MZM capacity to control viral infections (13, 14). Depletion of MZM can to the elimination of a non- or low-cytopathic virus vs a cytopathic virus and to investigate the effect of impaired removal of infectious virus on antiviral immunity and disease manifestation. *Institute for Experimental Immunology, University Hospital, Zurich, Switzerland; and †Unidad de Investigacio«n Me«dica en Inmunoquõ«mica, Hospital de Especialidades, Me«xico City, Me«xico Materials and Methods Received for publication January 14, 2002. Accepted for publication May 30, 2002. Mice and viruses The costs of publication of this article were defrayed in part by the payment of page C57BL/6 mice were obtained from the breeding colony of the Institut fu¬r charges. This article must therefore be hereby marked advertisement in accordance Labortierkunde (Faculty of Veterinary Medicine, University Zurich-Irchel, with 18 U.S.C. Section 1734 solely to indicate this fact. Zurich, Switzerland). Experiments were conducted with age- and sex- 1 C.L.-M. was a recipient of a Bundesstipendium from the Eidgeno¬ssiche Stipendien matched animals kept under specific pathogen-free conditions. Heterozy- Kommission (Bern, Switzerland) and acknowledges the support received from the gous op/ϩ mice were obtained from The Jackson Laboratory (Bar Harbor, Heuberg Stifftung and the Coordinacion de Investigacion en Salud del Instituto Mexi- ME), embryo-derived, back-crossed onto C57BL/6 for two generations, cano del Seguro Social, the Sistema Nacional de Investigadores, and the Consejo and kept under specific pathogen-free conditions according to institutional Nacional de Ciencia y Tecnologõ«a of Me«xico. guidelines. Heterozygous breeding pairs were identified by PCR as previ- 2 Address correspondence and reprint requests to Dr. Stephan Oehen at the current ously described (5). Inspecting them for the lack of incisors identified address: Cytos Biotechnology, Wagistrasse 25, 8952 Zurich, Switzerland. E-mail ad- homozygous opϪ/Ϫ offspring. They were fed powdered mouse food and dress: [email protected] water supplemented with 5% glucose. Heterozygous and wild-type litter- 3 Current address: Infektiologie, University Hospital, Zu¬rich, Switzerland. mates were separated and sacrificed as early as possible from opϪ/Ϫ lit- Ϫ/Ϫ 4 Abbreviations used in this paper: op, osteopetrotic; VSV, vesicular stomatitis virus; termates to increase the survival rate of op mice and to obtain a suffi- LCMV, lymphocytic choriomeningitis virus; MZM, marginal zone macrophage; i.f., cient number of homozygotes. For this reason, i.e., lack of littermates, all via the footpad; FDC, follicular dendritic cell. experiments were conducted with age-matched (date of birth within the

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 1454 M-CSF-DEFICIENT MICE ARE HIGHLY SUSCEPTIBLE TO VIRAL INFECTION range of 2 wk) C57BL/6 control mice. Blood samples from experimental supernatant was harvested and radioactivity determined in a gamma opϪ/Ϫ mice were checked for the presence of H-2b by FACS analysis. counter, and the percentage of specific release was calculated. Despite the possibility that inbred mouse strains may show altered kinetics of virus clearance, our longstanding experience with transgenic and knock- Secondary in vitro restimulation out mouse lines generated on mixed genetic backgrounds showed that the ϫ 6 ϫ 5 immune responses against LCMV and VSV were identical to C57BL/6 A total of 5 10 spleen cells and 2 10 LCMV-infected peritoneal after the first, and even more so the second, backcross to C57BL/6; thus, exudate macrophages were cocultured in 24-well tissue culture plates in a C57BL/6 were considered to serve as appropriate controls in our total volume of 2 ml IMDM supplemented with 10% FCS, penicillin/strep- ϫ Ϫ5 experiments. tomycin, glutamine, and 5 10 M 2-ME. Macrophages were obtained by injecting mice i.p. with 1 ml thioglycolate on day Ϫ6 and with 200 PFU VSV-IND (Mudd-Summers isolate) was originally obtained from D. Ϫ ␮ Kolakofsky (University of Geneva, Geneva, Switzerland) and was grown LCMV on day 4. After 5 days, 1 ml medium was removed and 100 l on BHK cells in MEM with 5% FCS to virus stocks containing 109 PFU/ of these standard cultures were diluted in duplicate (four steps, 3-fold di- ml. LCMV-WE was originally provided by F. Lehmann-Grube (University lutions) in MEM supplemented with 2% FCS in 96-well round-bottom Hamburg, Hamburg, Germany) and was grown on L-929 cells for 48 h in plates and specific cytotoxicity was determined as described above. MEM with 5% FCS after infection with an initial multiplicity of infection of 0.01. Immunohistochemistry Freshly removed organs were immersed in HBSS and snap-frozen in liquid VSV neutralization assay nitrogen. Tissue sections of 5-␮m thickness were cut in a cryostat, placed Mice were bled from the retroorbital cavity at the indicated time points. on siliconized glass slides, air dried, fixed with acetone for 10 min, and Ϫ Serial 2-fold dilutions of serum samples (previously diluted 1/40) were stored at 70¡C. Second-stage affinity-purified polyclonal anti-Ig antisera mixed with equal volumes of VSV containing 500 PFU/ml and the mix- were diluted in Tween-buffered saline (TwBS; pH 7.4) containing 5% nor- mal mouse serum. All other dilutions were made in TwBS alone. Incuba- tures were incubated for 90 min at 37¡C in an atmosphere containing 5% Downloaded from ␮ tions were performed at room temperature for 30 min; TwBS was used for CO2. A total of 100 l of the mixture was transferred onto Vero cell monolayers in 96-well plates and incubated for1hat37¡C. Monolayers all washing steps. Alkaline phosphatase was visualized using naphthol were overlaid with 100 ␮l of DMEM containing 1% methylcellulose and AS-BI phosphate and New Fuchsin as substrate, which yields a red pre- incubated for 24 h at 37¡C, after which the overlay was removed and the cipitate. Endogenous alkaline phosphatase was blocked by levamisole. All monolayer was fixed and stained with 0.5% crystal violet dissolved in 5% color reactions were performed at room temperature for 15 min with re- formaldehyde, 50% ethanol, and 4.25% NaCl. The dilution reducing the agents from Sigma-Aldrich (St. Louis, MO). Sections were counterstained number of plaques by 50% was taken as titer (25). To determine IgG titers, with hemalum. Coverslips were mounted with glycerol and gelatin. Stain- undiluted serum was treated with an equal volume of 0.1 M 2-ME in MEM ings for the cell differentiation markers MOMA-1 (mouse metallophilic http://www.jimmunol.org/ tissue culture medium for1hatroom temperature. macrophages) and 4C11 (follicular dendritic cells (FDC)) were performed exactly as previously described (31). The detection of VSV-Ag has been Determination of VSV and LCMV titers in tissues described previously (32). Mice were sacrificed by cervical dislocation. Brain tissue was homoge- nized in balanced salt solution, and serial 3-fold dilutions were plated on Vero cell monolayers in 96-well plates. Viral titers were calculated by multiplying counted plaques by the dilution factor. Mice without detectable virus in the brain (Ͻ103 PFU per brain) were scored negative (26, 27). LCMV titers of organs of infected mice were determined in an infectious focus formation assay as described previously (28). Briefly, spleen homog- by guest on September 23, 2021 enates were serially diluted 10-fold and grown on an MC57G cell mono- layer for 48 h under an overlay of 1% methylcellulose in DMEM. Cells were fixed with 4% formalin in PBS, and infectious foci were detected by intracellular LCMV staining of infected cells with the rat anti-LCMV mAb VL-4. Cytotoxicity assay The cytolytic activity of spleen cells was tested in a 51Cr release assay as described previously (29). Briefly, EL-4 target cells were coated with LCMV-derived peptide gp33Ð41 (30) at concentrations of 10Ϫ6 M. A total of 104 target cells were incubated in 96-well round-bottom plates with serial 3-fold dilutions of spleen effector cells starting at an E:T ratio of 70:1 in a final volume of 200 ␮l. After5hofincubation at 37¡C, 70 ␮lof

FIGURE 2. A, opϪ/Ϫ mice develop strong but slightly reduced CTL responses. opϪ/Ϫ mice were injected i.f. with 100 PFU LCMV. CTL re- sponses were measured in the spleen 9 days later on EL-4 target cells FIGURE 1. opϪ/Ϫ mice fail to develop a footpad swelling reaction. coated with LCMV peptide p33 (op if p33) or were left untreated (op if). opϪ/Ϫ (op) and C57BL/6 (B6) mice were injected into the hind footpad C57BL/6 served as controls (B6 if p33 and B6 if, respectively). Data with 100 PFU LCMV and the footpad swelling reaction was monitored shown represent one of two independent experiments. B, opϪ/Ϫ mice fail to daily for the indicated time period. Each line represents one animal. Some efficiently control LCMV during the acute phase. opϪ/Ϫ (op) and C57BL/6 mice were sacrificed for other experiments (discontinued lines). Data from (B6) control mice were injected into the footpad with 100 PFU LCMV and two independent experiments are shown. virus titers were measured in the indicated organs 9 days later. The Journal of Immunology 1455

Footpad swelling reaction uate whether opϪ/Ϫ mice generated LCMV-specific CTL responses measurable in vitro, cytotoxicity assays were performed Mice were injected into the hind footpad with 100 PFU LCMV WE in 30 Ϫ/Ϫ ␮l of MEM supplemented with 2% FCS. The footpad thickness was mea- with spleen cells from op and C57BL/6 mice infected i.f. with Ϫ Ϫ sured with a spring-loaded caliper (Kroeplein; Schluchtern, Hessen, Ger- LCMV (Fig. 2A). The data show that op / mice generated many). Values were taken as the mean of both hind footpads (29, 33). slightly reduced but still strong CTL responses. Thus, the lack of a footpad swelling reaction in opϪ/Ϫ mice cannot be explained by Results the lack of CTL induction but may rather reflect the failure of CTL Ϫ/Ϫ op mice fail to generate a primary footpad swelling reaction homing to the footpad. Ϫ/Ϫ To assess the capacity of op mice to mount an immune re- Ϫ/Ϫ sponse against non- or low-cytopathic LCMV, mice were infected op mice are unable to control LCMV during the acute phase via the footpad (i.f.) with LCMV. After footpad infection, immu- of the infection nocompetent mice develop a footpad swelling reaction due to a A possible explanation for the homing deficiency of CTL into the biphasic CD8ϩ and CD4ϩ T cell response (29, 33). The strength footpad is that virus spreads throughout all organs and recruits and the kinetics of the swelling reaction correlate with the induc- CTL away from the site of infection. To assess this possibility, tion and recruitment of T cells to the site of infection. Naive mice virus titers in opϪ/Ϫ mice were determined in different organs of show a strong swelling reaction on day 8 after infection, whereas mice infected i.f. with 100 PFU LCMV (Fig. 2B). LCMV titers LCMV-immune mice immediately clear the virus and therefore were 10- to 50-fold increased in thymus, spleen, and liver of opϪ/Ϫ fail to generate a strong swelling reaction. To assess priming and mice compared with C57BL/6 controls, suggesting that opϪ/Ϫ homing of T cells in opϪ/Ϫ mice, 100 PFU LCMV WE were in- mice could not efficiently control virus spread during the acute Downloaded from jected into the footpad of opϪ/Ϫ and C57BL/6 control mice (Fig. phase. This observation was even more pronounced when 100 PFU 1). While C57BL/6 mice generated a strong swelling reaction on LCMV were injected i.v., in which case virus titers in opϪ/Ϫ mice day 8, opϪ/Ϫ mice failed to produce a swelling reaction, suggest- were 100Ð10,000 times higher than in C57BL/6 controls: thymus ing that in opϪ/Ϫ mice LCMV either did not induce a T cell re- op, 2.68 ϫ 106 Ϯ 9.1 ϫ 105 PFU; thymus B6, 1.1 ϫ 104 Ϯ 8.9 ϫ sponse or induced T cells did not migrate to the footpad. To eval- 103 PFU; liver op, 4.9 ϫ 107 Ϯ 2.6 ϫ 106 PFU; liver B6, 3.1 ϫ

103 Ϯ 2.9 ϫ 103 PFU; spleen op, 2.5 ϫ 107 Ϯ 2.2 ϫ 107 PFU; http://www.jimmunol.org/ spleen B6, 1.2 ϫ 104 Ϯ 1 ϫ 104 PFU. Taken together, uncon- trolled virus dissemination may lead to increased Ag loads in pe- ripheral organs and deviation of CTL into these organs. by guest on September 23, 2021

FIGURE 3. A, A proportion of opϪ/Ϫ mice recovering from generalized immunopathology become virus carriers. Three groups of a total of 11 opϪ/Ϫ mice were injected i.f. with 100 PFU LCMV. Each group was tested FIGURE 4. A, opϪ/Ϫ mice raise normal VSV-specific IgM and IgG Ab in a separate experiment. The LCMV titers in the indicated organs were responses. Groups of three op and C57BL/6 mice were injected i.v. with determined from eight surviving opϪ/Ϫ mice on days 21Ð28. Shown are the 2 ϫ 106 PFU VSV. On the indicated days neutralizing IgM plus IgG and titers from six carrier op mice; two op mice cleared the virus as C57BL/6 IgG titers were determined. One of three independent experiments is control mice did. B and C, LCMV-specific CTL are recovered from virus- shown. B, expt. 1, Virus spreads to the brain in opϪ/Ϫ mice. Three op and free opϪ/Ϫ mice (B) but not from carrier opϪ/Ϫ mice (C). Spleen cells from the C57BL/6 mice were injected i.v. with 2 ϫ 106 PFU VSV. Moribund the surviving eight opϪ/Ϫ (op) from the groups described in A and the mice were sacrificed and virus titers were measured in the brain. Expt. 2, C57BL/6 control (B6) mice were restimulated in vitro with LCMV-in- op mice control low doses of VSV. Groups of three opϪ/Ϫ and C57BL/6 fected macrophages. After 5 days the cultures were tested in the indicated mice were injected i.v. with 2 ϫ 104 PFU VSV or 2 ϫ 106 PFU VSV, serial dilutions for the presence of LCMV-specific CTL in 51Cr release respectively. Virus titers were measured in the brain of moribund mice. assays using EL-4 target cells coated with LCMV peptide p33 (op p33 and Animals that remained free of clinical symptoms were sacrificed on day 20 B6 p33) or untreated cells (op and B6). for virus titer determination. 1456 M-CSF-DEFICIENT MICE ARE HIGHLY SUSCEPTIBLE TO VIRAL INFECTION

After LCMV infection opϪ/Ϫ mice developed a generalized opϪ/Ϫ mice mount normal B cell-neutralizing Ab responses to immunopathology VSV but succumb to infection Mice that cannot efficiently control virus spread show signs of To evaluate the potential of opϪ/Ϫ mice to control infection by a generalized immunopathological disease. To evaluate whether cytopathic virus, 2 ϫ 106 PFU VSV IND were injected i.v. and opϪ/Ϫ mice eventually controlled LCMV infection, they were in- neutralizing Ab titers were monitored. Fig. 4A shows that opϪ/Ϫ fected i.f. with LCMV and clinical symptoms of generalized im- mice mount normal neutralizing IgM and IgG Ab responses. How- munopathology were monitored. In contrast to C57BL/6 mice, all ever, surprisingly, mice succumb to lethal VSV infection between LCMV-infected opϪ/Ϫ mice developed generalized immunopa- days 8 and 11 and VSV can readily be isolated from the brain of thology characterized by a hunched back, ruffled fur, and lethargy: opϪ/Ϫ mice but not from control C57BL/6 mice (Fig. 4B, expt. 1). 9of25opϪ/Ϫ mice succumbed to the infection between days 12 Early control of virus dissemination to avoid infection of vital and 18, and the remaining mice recovered completely from clinical organs by VSV is crucial for resistance to VSV infection (22, 37, symptoms by days 21Ð28. Thus, opϪ/Ϫ mice are immunocompro- 38). To establish whether opϪ/Ϫ mice were able to control dis- mised and have difficulties controlling virus spread despite being semination of VSV to the brain after infection with 100-fold lower able to generate significant primary CTL responses. doses of VSV, opϪ/Ϫ mice were infected i.v. with 2 ϫ 104 PFU of this virus (Fig. 4B, expt. 2). The data show that opϪ/Ϫ mice in- Ϫ/Ϫ Impaired virus clearance in op mice correlates with the lack fected with a low dose of VSV controlled the infection and did not of LCMV-specific CTL succumb to VSV-mediated encephalitis. Ϫ/Ϫ

To determine whether op mice, which recover from transient Downloaded from immunopathology, eliminated the virus or became virus carriers due to CTL exhaustion (34Ð36), a total of 11 opϪ/Ϫ mice were VSV Ag is efficiently recruited to the spleen injected with LCMV into the footpad, virus titers were determined Recruitment of VSV by natural Abs to secondary lymphoid organs in the organs between days 21 and 28, and the presence of CTL plays a key role in controlling disease by reducing virus spread and was assessed in secondary in vitro assays. Three of the opϪ/Ϫ mice enhancing induction of an immune response (22, 38). opϪ/Ϫ mice died with severe clinical symptoms of generalized immunopathol- lack metallophilic MZM in the spleen and subcapsular macro- http://www.jimmunol.org/ ogy. Of the remaining eight opϪ/Ϫ mice, six mice failed to elim- phages in the lymph nodes, and have normal FDC populations inate LCMV from all organs (Fig. 3A) and two mice completely (Fig. 5, upper panels). To investigate whether VSV was efficiently cleared the virus below detectable levels (data not shown). After in recruited to the spleen, opϪ/Ϫ mice were infected i.v. with 5 ϫ 108 vitro restimulation, LCMV-specific CTL could be recovered from PFU VSV. No difference in VSV titers was found in the spleens the mice that had eliminated the viruses (Fig. 3B), whereas no after6hofinfection. Titers were Ͼ2.1 ϫ 108 PFU per gram of LCMV-specific CTL could be recovered from the carrier mice organ in both opϪ/Ϫ and control mice. Consistent with this, cryo- (Fig. 3C). Thus, elimination of virus correlated with CTL activity, sections revealed that VSV Ag was recruited to the spleen. How- whereas impaired virus clearance correlated with the absence of ever, while VSV Ag was predominantly found in the marginal

LCMV-specific CTL. zone of C57BL/6 mice, VSV Ag could be found in the red pulp by guest on September 23, 2021

FIGURE 5. AÐF, Macrophage and FDC populations in spleen and lymph nodes of opϪ/Ϫ mice. Spleen (A, C, D, and F) and lymph node (B and E) sections from C57BL/6 (AÐC) and opϪ/Ϫ (DÐF) mice were stained with an Ab recognizing mouse metallophilic macro- phages (MOMA) or FDCs (4C11). GÐJ, Distribution of viral Ag in the spleen of opϪ/Ϫ mice. C57BL/6 (G and H) and opϪ/Ϫ (I and J) mice were injected i.v. with 5 ϫ 108 PFU VSV. After 6 h spleens were removed and sections were stained for VSV Ag (G and I) and FDC (4C11; H and J). Data shown are representative of one mouse from three independent mice. The Journal of Immunology 1457 and the white pulp of opϪ/Ϫ mice. Moreover, the Ag largely co- opϪ/Ϫ mice have been shown to generate normal MLR T cell localized with FDC in opϪ/Ϫ mice (Fig. 5). responses and normal B cell responses in vivo against OVA (12). The present study is consistent with these findings. However, de- spite generating normal T and B cell responses, opϪ/Ϫ mice were Discussion susceptible to viral infection. This points to the importance of the For non- or low-cytopathic viruses the relative balance between architecture of lymphoid organs for controlling viral infections in virus spread and CTL response critically defines whether the virus vivo (42Ð45). Crucial functions of structural components of lym- will be cleared, immunopathology will ensue, or the immune re- phoid organs may not be uncovered if nonreplicating Ags are used. sponse will be exhausted and the virus will persist (39). Because Although such experimental systems provide us with important cytopathic viruses directly destroy infected cells, they cannot es- information, they usually disclose rather a narrow window of the tablish a carrier state without killing the host; therefore, virus complex processes that are triggered by infections. spread has to be controlled rapidly. Taken together this study shows that the early innate immune Early CTL and B cell responses against LCMV and VSV were system provides an important preparatory phase for the adaptive largely unimpaired in op mice. The small reduction in the early day immune system: cooperation between the innate and the adaptive 8 cytotoxic activity of spleen cells against LCMV in opϪ/Ϫ mice immune system shifts the balance between induction of an immune is probably a consequence of virus dissemination and CTL extrav- response and virus spread in favor of the immune response. This is asation into peripheral infected tissues rather than inefficient CTL particularly important for cytopathic viruses where virus spread priming. This view is consistent with the observation that opϪ/Ϫ usually leads to death, while non- or low-cytopathic viruses, de- mice failed to generate a footpad swelling reaction, probably due pending on the relative balance between immune response and Downloaded from to CTL recruitment to many sites in addition to the infected foot- virus spread, can establish a virus carrier state without harming the pad (40). host if extensive immunopathology is avoided. All opϪ/Ϫ mice went through a critical phase of generalized immunopathology, which one-third of the mice did not survive. In Acknowledgments Ϫ/Ϫ a small proportion of the op mice an efficient CTL response We thank Karin Riem-Brudscha and Lenka Vlk for excellent technical dominated over virus spread, virus was cleared, and LCMV-spe- assistance. http://www.jimmunol.org/ cific CTL could be recovered. However, most of the mice failed to completely clear LCMV from all organs (virus persisted particu- References larly in the kidney) and no LCMV-specific CTL could be recov- 1. Carroll, M. C. 1998. The role of complement and complement receptors in in- ered, indicating that CTL had been exhausted. duction and regulation of immunity. Annu. Rev. Immunol. 16:545. 2. Fischer, A., M. Cavazzana-Calvo, B. G. De Saint, J. P. DeVillartay, It has previously been observed that T cell-dependent LCMV J. P. Di Santo, C. Hivroz, F. Rieux-Laucat, and F. Le Deist. 1997. Naturally clearance involves cooperation between T cells and probably mac- occurring primary deficiencies of the immune system. Annu. Rev. Immunol. 15: rophages (14). In addition, an earlier study by Seiler et al. (18) 93. 3. Rossi, D., and A. Zlotnik. 2000. The biology of chemokines and their receptors. investigated the role of MZM in immunity against LCMV. The Annu. Rev. Immunol. 18:217. by guest on September 23, 2021 authors depleted MZM by administration of liposome-encapsu- 4. Marks, S. C., Jr., and P. W. Lane. 1976. Osteopetrosis, a new recessive skeletal lated clodronate (15Ð17) and reported that CTL responses were mutation on chromosome 12 of the mouse. J. Hered. 67:11. 5. Yoshida, H., S. Hayashi, T. Kunisada, M. Ogawa, S. Nishikawa, H. Okamura, exhausted in all mice. There was no mention of generalized im- T. Sudo, L. D. Shultz, and S. Nishikawa. 1990. The murine mutation osteope- munopathology, most likely because the treatment of mice with trosis is in the coding region of the macrophage colony stimulating factor gene. clodronate liposomes is stressful to the animal and clinical symp- Nature 345:442. 6. Wiktor-Jedrzejczak, W. W., A. Ahmed, C. Szczylik, and R. R. Skelly. 1982. toms may have been masked due to this harsh treatment. In this Hematological characterization of congenital osteopetrosis in op/op mouse: pos- work we show that three clinical outcomes are possible: 1) virus sible mechanism for abnormal macrophage differentiation. J. Exp. Med. 156: 1516. elimination accompanied by intact LCMV-specific CTL re- 7. Wiktor-Jedrzejczak, W., A. Bartocci, A. W. Ferrante, Jr., A. Ahmed-Ansari, sponses, 2) virus persistence and CTL exhaustion, or 3) death as a K. W. Sell, J. W. Pollard, and E. R. Stanley. 1990. Total absence of colony- consequence of virus spread and immunopathological CTL stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse. Proc. Natl. Acad. Sci. USA 87:4828. response. 8. Witmer-Pack, M. D., D. A. Hughes, G. Schuler, L. Lawson, A. McWilliam, Ϫ Ϫ After infection with VSV, op / mice generated normal neu- K. Inaba, R. M. Steinman, and S. Gordon. 1993. 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