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1 Introduction. Macrophages play a crucial role in the immune surveil- lance. Tissue-specific ..... 100 PFU of the slowly replicating LCMV strain Armstrong.
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Peter Seiler', Peter Aichele', Bernhard Odermatt', Hans Hengartner', Rolf M. Zinkernagel' and Reto A. Schwendene8

Crucial role of marginal zone macrophages and marginal zone metallophils in the clearance of lymphocytic choriomeningitis virus infection

' Institute of Experimental

Macrophages play a key role in the immune defense against pathogens. They control early invasion by antigen-unspecific phagocytosis of pathogens and act as professional antigen-presenting cells to induce antigen-specific T cell responses. To investigate the involvement of particular subsets of the splenic macrophages in an antiviral immune response, we selectively depleted mice of splenic marginal zone macrophages (MZM) and marginal zone metallophils (MM) using the clodronate liposome depletion technique. MZM- and MM-depleted mice were not able to control an infection with lymphocytic choriomeningitis virus (LCMV). In these mice, LCMV spread from the spleen to peripheral organs at an early phase of infection. The virus-specific cytotoxic T lymphocyte (CTL) response was induced initially, yet was exhausted in parallel with the overwhelming virus replication. These findings suggest that MZM and MM play a crucial role in the early control of a LCMV infection by preventing immediate virus spread to peripheral organs, but are not essential for the induction of the LCMV-specific CTL response.

Immunology, Department of Pathology, University Hospital Zurich, Zurich, Switzerland Department of Pathology, Division of Cancer Research, University Hospital Zurich, Zurich, Switzerland

1 Introduction Macrophages play a crucial role in the immune surveillance. Tissue-specific mononuclear cells act as a first line of defense by antigen-unspecific phagocytosis of invading pathogens (reviewed in [l-81). They also function as professional antigen-presenting cells (APC) by providing the optimal signals for efficient induction of antigen-specific T cell responses [9-181. Detailed knowledge of the spectrum of functions expressed by particular subsets of macrophages in vivo is difficult to obtain. Most information on the functions of macrophages in vivo was acquired using animals experimentally depleted of macrophages. Efficient depletion of macrophages is achieved by the clodronate liposome depletion technique [19-21]. Liposomes containing clodronate (dichloromethylene-bisphosphonate) are preferentially taken up by macrophages and induce apoptotic cell death after delivery to the cytoplasm [20]. Depending on the route and dose of application, different subsets of macrophages in various tissues are eliminated [21]. Upon macrophage depletion, clearance of a variety

[I 171901 Received June 18, 1997; in revised form July 21, 1997; accepted July 2.5, 1997. The first two authors contributed equally to this work.

Correspondence: Peter Seiler and Peter Aichele, Institute of Experimental Immunology, Department of Pathology, University Hospital Zurich, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland Fax: +41-1-25.5 4420 e-mail: [email protected] and paic@pathol .unizh.ch Abbreviations: MZM: Marginal zone macrophages MM: Marginal zone metallophils RPM: Red pulp macrophages DC: Dendritic cells FDC: Follicular dendritic cells Key words: Lymphocytic choriomeningitis virus / Macrophagc dcpletion technique / Clodronate liposome I Marginal zone macrophage I Marginal zone metallophil 0014-2980/Y7/1O10-2626$17.S0+ ,5010

of microbial pathogens was impaired [5-8, 10,221. In antiviral immune responses, the antigen presenting function seemed to be of primary importance. Macrophage depletion reduced the host capacity to control viral infections due to ineffective induction of T cell responses [13, 14, 22, 231. A recent study demonstrated that splenic red pulp macrophages are critically involved in induction of virusspecific T cell responses [14]. The function of marginal zone macrophages and marginal zone metallophils remained unclear. Lymphocytic choriomeningitis virus (LCMV) is a model infection to investigate antiviral immunity. The natural host of LCMV is the mouse. Acute infection is predominantly controlled by virus-specific CTL [24-281. Viral persistence is established after systemic or peripheral infection of mice with low CTL precursor frequencies or impaired interferon type I or type I1 responses and after high-dose infection with rapidly replicating virus isolates [29-321. The contribution of macrophages to virus control is not completely understood. Whereas experiments with pure lymphocyte-free populations of unprimed bone marrow-derived mononuclear phagocytes have shown that bacteria and some of their products were potent in modulating the expression of surface molecules and in stimulating the secretory and cell-mediated activities, LCMV was poor in this respect [33, 341. However, in mice experimentally depleted of whole splenic macrophages, elimination of LCMV was delayed [23, 351. These results were interpreted as ineffective induction of virus-specific CTL. Thus, macrophages may have an important role in induction of virus-specific immune responses. To analyze the contribution of macrophages to the control of a LCMV infection in detail, and in particular the role of different subsets of splenic macrophages, we selectively depleted mice of splenic marginal zone macrophages (MZM) [36,37] and marginal zone metallophils (MM) [38, 391 using the clodronate liposome depletion technique. Selective depletion of these two macrophage subsets was achieved by intravenous application of clodronate encap0WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1997

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Macrophagcs of thc marginal zone in LCMV clearance

sulated into small unilamellar liposomes. MZM- and MMdepleted mice were not able to clear low dose LCMV infection. Immediately after infection, LCMV spread to peripheral organs. Although antiviral CTL were induced initially, the virus-specific CTL were exhausted because of the overwhelming virus replication. Therefore we conclude that MZM and MM function as 'phagocytic filter' in the spleen controlling systemically spreading viruses including LCMV and preventing early generalized virus spread to peripheral organs until an effective CTL response is mounted.

membranes. Liposome size and homogeneity were routinely measured with a Nicomp 370 Submicron Particle Sizer (Particle Sizing Systems, Sta. Barbara, CA). Depletion of splenic MZM and MM was achieved by i.v. application of liposomes containing 2-2.5 mg clodronate at day-3 before infection with LCMV. If not otherwise mentioned, depletion was repeated 2 days after infection by i.p. application of liposomes containing 0.8-1 mg clodronate. The lower dose of clodronate used for the second treatment was sufficient to maintain depletion of MZM and MM.

2 Materials and methods 2.4 Immunohistology 2.1 Animals C57BL/6 (H-2b) mice were obtained from the breeding colony of the Institut fur Zuchthygiene (Labortierkunde, Tierspital Zurich, Switzerland). The mouse line 318 expressing a transgenic TCR specific for the LCMV peptide GP33-41 (KAVYNFATC) in association with H-2Dh has been described previously [40]. Animals were bred under special pathogen-free conditions and used in the experiments at 12-18 weeks of age under conventional conditions. 2.2 Virus The LCMV-WE strain was originally obtained from Dr. F. Lehmann-Grube (Hamburg, Germany) and was propagated on L929 fibroblast cells [41]. Virus stocks were diluted in MEM supplemented with 2 YO FCS.

Freshly removed organs were immersed in HBSS and snap frozen in liquid nitrogen. l h u e sections of 5 pm thickness were cut in a cryostat, placed on siliconized glass slides, air dried, fixed with acetone for 10 min and stored at -70°C. Secondary affinity-purified polyclonal anti-Ig antisera were diluted in Tris-buffered saline (TBS, pH 7.4) containing 5 % normal mouse serum. All other dilutions were made in TBS alone. Incubations were done at room temperature for 30 min; TBS was used for all washing steps. Alkaline phosphatase was visualized using naphthol AS-BI phosphate and New Fuchsin as substrate. Endogenous alkaline phosphatase was blocked by levamisole. All color reactions were performed at room temperature for 15 min with reagents from Sigma (St. Louis, MO). Sections were counterstained with hemalum. Coverslips were mounted with glycerol/gelatin.

Rehydrated tissue sections were incubated with the following rat primary mAb: anti-MZM (ERTR-9; [43]), antiMM (MOMA-1; Biomedicals, Augst, Switzerland), antired pulp macrophages (RPM) (F4/80; ATCC HB198), 2.3 Preparation of clodronate liposomes anti-follicular dendritic cells (FDC) (4C11; [44]). Primary Small unilamellar liposomes composed of soy phosphati- rat mAb were revealed by a twofold sequential incubation dylcholine (SPC), cholesterol and D,L-a-tocopherol con- with rabbit anti-rat Ig and rat alkaline phosphatase anti taining 40 mg SPC, 6 mg cholesterol and 0.2 mg D,L-CL- alkaline phosphatase complex (DAKO, Glostrup, Dentocopherol per ml (1 :0.3 :0.01 molar ratio) were prepared mark). C D llc on dendritic cells (DC) was stained with the by freeze-thawing and filter extrusion [42]. The dry lipid hamster mAb N418 (ATCC HB-224). Primary hamster Ig mixture was solubilized by addition of 1 ml clodronate were detected by alkaline phosphatase-labeled rabbit antisolution (37.5 mg sodium clodronate, Ostac, Boehringer hamster Ig followed by alkaline phosphatase-labeled goat Mannheim). The resulting multilamellar vesicles were anti-rabbit Ig. freeze-thawed in three cycles of liquid nitrogen and water at 40 "C, followed by repetitive fivefold filter extrusion through 400-nm membranes (Nuclepore, Sterico AG, Die- 2.5 LCMV infectious focus formation assay tikon, Switzerland) using a LipexTMextruder (Lipex Biomembranes Inc., Vancouver). For the determination of LCMV titers from organs of infected mice were deterclodronate encapsulation efficiency the preparations were mined as described [45]. Briefly, organs were homogetrace labeled with 45CaC12(Amersham Int., Amersham, nized and serial tenfold dilutions of the homogenate were GB). Unencapsulated clodronate was removed in two plated and incubated on a MC57G cell monolayer for 48 h steps by concentration to 4-5 ml with an Amicon (Amicon under an overlay of 1% methyl cellulose in DMEM/5 % Corp., Lexington, MA) ultrafiltration cell using a YMlOO FCS. Cells were fixed with 4 YOformalin in PBS and infec(cutoff 100 kDa) membrane followed by size exclu- tious foci were detected by intracellular LCMV staining of sion chromatography on a Sephadex G-25 column infected cells with the rat anti LCMV mAb VL-4. (30 X 2.5 cm) using phosphate buffer (67 mM, pH 7.4) as eluent. The diluted liposomes collected after column elution were re-concentrated by ultrafiltration to obtain lipo- 2.6 Restimulation of primed antiviral CTL in vitro somes containing 3-5 mg/ml encapsulated clodronate. All preparations were sterilized through 0.45-pm filters (Gel- Spleen cells (4 X 10h/well)from LCMV-primed mice were man Sciences, Ann Arbor, MI). Accordingly, empty con- restimulated in 24-well tissue culture plates with 2 x lo5 trol liposomes were prepared by extrusion through 400-nm irradiated (3000 rad), LCMV-infected macrophages in

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Iscove's modified Dulbecco's medium supplemented with 10 Yo FCS, penicillin/streptomycin, 0.001 M 2-ME and 1 YO ConA supernatant. After 5 days, restimulated spleen effector cells from two wells were pooled and resuspended in 1 ml of complete MEM. Serial threefold dilutions were made (indicated as dilution of culture) and tested in a "Crrelease assay.

untreated

clodronate treated

MZM

2.7 Cytotoxicity assay The cytolytic activity of virus-specific spleen cells isolated ex vivo or restimulated in vitro was determined in a 5'Crrelease assay as described [27]. EL-4 cells or MC57G cells were coated with LCMV-derived peptides GP 33-41 [46] or NP 396-404 [47]. Negative control targets were either coated with a H-2Dh-binding adenovirus peptide [48] or left uncoated. All peptides were used at concentrations of M. Target cells were labeled with 250 pCi "Cr for 2 h at 37°C on a rocking platform, washed three times and counted. Target cells (lo4) were incubated in 96-well round-bottom plates with a serial threefold dilution of spleen effector cells in a final volume of 200 pl. After a 5-h incubation at 37"C, 70 p1 of the supernatants were harvested and assayed. Spontaneous release was always