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Oct 5, 2007 - model for the formation of organ inffammation states that ... Immunotherapy Center, Medical College of Georgia, Augusta, GA. 30912. Phone: ... CyJ (Thy1.1) mice were purchased from Jackson Laboratories (Bar Harbor,. ME).
JOURNAL OF VIROLOGY, Mar. 2008, p. 2952–2965 0022-538X/08/$08.00⫹0 doi:10.1128/JVI.02191-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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Regulation of Immune Response and Inflammatory Reactions against Viral Infection by VCAM-1䌤 Rong Ou,1† Menghua Zhang,1† Lei Huang,1 Richard A. Flavell,2 Pandelakis A. Koni,3* and Demetrius Moskophidis1* Center for Molecular Chaperones/Radiobiology and Cancer Virology, Medical College of Georgia, Augusta, Georgia 309121; Department of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 065102; and Immunotherapy Center, Medical College of Georgia, Augusta, Georgia 309123 Received 5 October 2007/Accepted 21 December 2007

The migration of activated antigen-specific immune cells to the target tissues of virus replication is controlled by the expression of adhesion molecules on the vascular endothelium that bind to ligands on circulating lymphocytes. Here, we demonstrate that the adhesion pathway mediated by vascular cell adhesion molecule 1 (VCAM-1) plays a role in regulating T-cell-mediated inflammation and pathology in nonlymphoid tissues, including the central nervous system (CNS) during viral infection. The ablation of VCAM-1 expression from endothelial and hematopoietic cells using a loxP-Cre recombination strategy had no major effect on the induction or overall tissue distribution of antigen-specific T cells during a systemic infection with lymphocytic choriomeningitis virus (LCMV), except in the case of lung tissue. However, enhanced resistance to lethal LCM and the significantly reduced magnitude and duration of footpad swelling observed in VCAM-1 mutant mice compared to B6 controls suggest a significant role for VCAM-1 in promoting successful local inflammatory reactions associated with efficient viral clearance and even life-threatening immunopathology under particular infection conditions. Interestingly, analysis of the infiltrating populations in the brains of intracerebrally infected mice revealed that VCAM-1 deletion significantly delayed migration into the CNS of antigen-presenting cells (macrophages and dendritic cells), which are critical for optimal stimulation of migrating virusspecific CD8ⴙ T cells initiating a pathological cascade. We propose that the impaired migration of these accessory cells in the brain may explain the improved clinical outcome of infection in VCAM-1 mutant mice. Thus, these results underscore the potential role of VCAM-1 in regulating the immune response and inflammatory reactions against viral infections. ical deficit) remain poorly defined, a consensus is that the development of tissue pathology coincides with a massive extravasation of mononuclear cells to the site of virus replication, a process regulated by the increased expression of adhesion molecules on the nearby vasculature (62, 68). Thus, a general model for the formation of organ inflammation states that proinflammatory cytokines, released either by virally infected cells or by a few activated antigen-specific T cells that gain access to infected tissue through the constitutive expression of receptors for tissue-specific trafficking, result in the upregulation of a distinct array of adhesion molecules on endothelial cells in postcapillary venules at the target sites. This enables the influx of additional activated antigen-specific T cells and other cell types, including monocytes/macrophages, which may release other cytokines (68). Leukocyte trafficking is a dynamic multistep process of leukocyte rolling on the vascular endothelium, followed by activation, adherence, and transmigration into tissue (15, 31, 34, 35, 39, 51, 56, 66, 67). In particular, leukocyte recruitment into specific tissues is determined by integrin-mediated firm adhesion (2, 7, 20, 31, 33, 39, 42, 43, 46, 73). Integrin expression by leukocytes facilitates adherence to the endothelium and emigration through the vasculature. Other signals, which may include chemokines, direct the adhered leukocytes to migrate across the endothelium into the extravascular tissue space (15). Inflammation is thus a dynamic process involving parallel and/or stepwise activation of multiple signaling pathways governing the homing of various leukocytes to different sites of

Host recovery from viral infections is critically dependent on a coordinated and tightly regulated innate and adaptive inflammatory response, usually confined to the target organs of virus replication, as well as on the successful repair of tissue injury to limit the extent of cell damage (52, 75). In theory, organ pathology occurs as a direct consequence of the intrinsic ability of many viruses to destroy their cellular targets and is usually intensified by inflammatory cells recruited to sites of infection by chemokines released by infected cells (18, 69, 75). However, this paradigm is not universal, and several studies have indicated a significant role for antigen-specific T cells in organ pathology. Thus, cellular injury as a consequence of T-cellmediated inflammation and/or the destruction of virus-infected target cells by cytotoxic T cells has been associated with pathology in several experimental models and human diseases, including lethal viral infections of the central nervous system (CNS) (4, 6, 8, 9, 19, 21, 24, 44, 45, 50, 58, 65). Although the mechanisms by which T cells participate in the control of viral infection without causing collateral damage (such as neurolog* Corresponding author. Mailing address for Demetrius Moskophidis: Medical College of Georgia, 1410 Laney Walker Blvd., Augusta, GA 30912. Phone: (706) 721-8738. Fax: (706) 721-0101. E-mail: [email protected]. Mailing address for Pandelakis A. Koni: Immunotherapy Center, Medical College of Georgia, Augusta, GA 30912. Phone: (706) 721-6897. Fax: (706) 721-7959. E-mail: pkoni @mcg.edu. † These authors contributed equally to this work. 䌤 Published ahead of print on 23 January 2008. 2952

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inflammation. The patterns of expression of adhesion molecules, chemokines, and chemokine receptors that correlate with cell homing properties change during the course of infection, since the interplay between inflammatory cell and T-cell trafficking is also a dynamic process. Collectively, these studies indicate that the migration of leukocytes, in particular of antigen-specific T cells to sites of viral replication, is a wellregulated process at the cellular and molecular levels. However, the role of each molecule in the virally induced inflammatory reaction remains to be evaluated in vivo. While these mechanisms have been dissected extensively in vitro, as well as with in vivo homing assays relying on fluorescently labeled cells (51), the picture is far from complete in the context of viral infection in a whole animal. In this context, evaluation of the parameters controlling the inflammatory response in animal models is of particular interest. In this study, we aimed to directly assess the contribution of the adhesion cascade involving the ␣4␤1/vascular cell adhesion molecule 1 (VCAM-1) pathway in regulating T-cell-mediated inflammation and pathology in nonlymphoid tissues using the murine lymphocytic choriomeningitis virus (LCMV) model. Many classic paradigms for immune-mediated pathology were originally derived from experiments with this system, which is uniquely suited to the study of the dynamics of virushost interactions and to specifically demonstrating and evaluating the opposing roles of virus-specific T cells in initiating and maintaining tissue-specific inflammatory reactions associated with life-threatening pathology and in facilitating rapid viral clearance with minimal pathological consequences for the host (8, 75). The data obtained suggest that efficient migration of hematopoietic cells, in particular professional antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), and activated antigen-specific immune cells to the target tissues of virus replication, including the CNS, is a dynamic process and that VCAM-1 is involved in regulating this inflammatory response associated with pathology in target organs of virus spread and replication. MATERIALS AND METHODS Animals and virus. C57BL/6 (B6) (H-2b; Thy1.2) and congenic B6-PL-Thy1a/ CyJ (Thy1.1) mice were purchased from Jackson Laboratories (Bar Harbor, ME). LCMV glycoprotein (LCMV-GP)-specific CD8⫹ T-cell receptor (TCR) transgenic (Tg) (P14) and CD4⫹ TCR transgenic (SMARTA) mice were provided by Rolf M. Zinkernagel and Hans Hengartner and have previously been described (54, 55). The conditional deletion of both VCAM-1flox alleles was described previously (32) and was achieved by expression of a Cre recombinase transgene under the control of the TIE2 promoter (TIE2-Cre-Tg), which drives uniform expression in all endothelial cells during both embryogenesis and adulthood (32, 70). The VCAM-1flox allele is deleted in the germ line of the TIE2Cre⫹ mother mice. This allowed us to achieve complete deletion of VCAM-1 in hematopoietic tissues of TIE2-Cre⫹ VCAM-1flox/⌬ versus VCAM-1flox mice by subsequent breeding. Therefore, the mice used in our study had the TIE2-Cre⫹ VCAM-1flox/⌬ genotype, and controls were Cre⫺ VCAM-1flox/flox genotype or B6 mice. Mice were infected intravenously (i.v.) with 102 PFU or intracerebrally (i.c.) with 102 or 104 PFU of LCMV strain WE (LCMV-WE). Virus titers were determined with an immunological focus assay (3). Isolation of lymphocyte populations, adoptive transfer, and tissue processing. Mice were perfused with phosphate-buffered saline (PBS) containing heparin (75 U/ml) prior to tissue removal. Lung tissues were minced and then treated at 37°C for 1 h with collagenase (150 U/ml; Gibco) in RPMI medium with 5% fetal calf serum (FCS). The resulting suspension was pelleted by centrifugation, resuspended in 44% Percoll (Pharmacia) in PBS with heparin (200 U/ml), layered on 67.5% Percoll, and centrifuged at 600 ⫻ g for 20 min at 20°C. Lymphocytes were harvested from the gradient interface and washed extensively before their use.

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Liver, kidney, or brain tissues were mashed through a 70-␮m strainer in RPMI medium with 5% FCS. The resulting cell suspensions were centrifuged, and the pellet was resuspended in a 38% Percoll solution supplemented with heparin (200 U/ml) and centrifuged at 600 ⫻ g for 20 min at 20°C. The cell pellet was treated with 0.83% ammonium chloride to remove erythrocytes before its use. For adoptive transfer experiments, 104 CD8⫹ TCR-P14 or CD4⫹-SMARTA cells were transferred i.v. into B6 mice 24 h prior to i.c. LCMV infection. Assessment of virus-specific T-cell-mediated immune response. Phenotypic analysis of cell surface markers, gamma interferon (IFN-␥) intracellular staining, and major histocompatibility complex (MHC) class I tetramer staining was performed as previously described (53). Experiments utilized Db/GP133–41 (KAVY NFATM), Db/GP2276–286 (SGVENPGGYCL), or Db/NP396–404 (FQPQNGQFI) phycoerythrin-conjugated tetramers. Single-cell suspensions prepared from spleen or lymphocytes isolated from lung were stained with tetramer and antibody for CD8 (clone 53-6.72) in fluorescence-activated cell sorting (FACS) buffer (PBS with 1% bovine serum albumin and 0.2% sodium azide). After being stained for 1 h at 4°C, cells were fixed in PBS containing 0.1% paraformaldehyde, acquired on a FACSCanto flow cytometer (Becton-Dickinson, San Jose, CA), and data were analyzed using Diva software. For IFN-␥ intracellular staining, lymphocytes were cultured in 96-well flat-bottom plates at 1 ⫻ 106 cells/well in 200 ␮l RPMI 1640 (Gibco) supplemented with 10% FCS in the presence or absence of the indicated peptide at a concentration of 1 ␮g/ml. To quantify total virus-specific CD8⫹ T-cell responses, virus-infected DC stimulators (DC2.4 cell line kindly provided by K. Rock, Boston, MA) (63) were used in combination with intracellular cytokine staining. Effector cells (1 ⫻ 106) were incubated with 4 ⫻ 105 DC2.4 cells, uninfected or infected 48 h previously with LCMV at a multiplicity of infection of 0.5. Stimulations were performed for 6 h at 37°C in the presence of 10-unit/well murine interleukin 2 (IL-2) and 1-␮g/well brefeldin A (BD Pharmingen). After 6 h, cells were harvested, washed, and surface stained for CD8␣, prior to intracellular cytokine staining (fluorescein isothiocyanateconjugated antibody to murine IFN-␥, clone XMG1.2; eBioscience) using the Cytofix/Cytoperm kit (BD-Pharmingen). Stained cells were washed, fixed, and acquired as described above. Adoptive immunization. Recipient mice were infected in the footpad (i.f.) with 104 PFU of LCMV-WE and 24 h later were transfused i.v. with approximately 2 ⫻ 107 splenocytes containing an equivalent number of virus-specific CD8⫹ T cells (determined by IFN-␥ intracellular and MHC class I tetramer staining) isolated from day 9-infected mice. FACS analysis. Fluorochrome-conjugated antibodies for FACS were purchased from BD Pharmingen or eBioscience. CD8 T cells (CD45high Thy1.2⫹ CD8⫹), CD4 T cells (CD45high Thy1.2⫹ CD4⫹), B cells (CD45high NK1.1⫺ Thy1.2⫺ CD19⫹), DCs (CD45high NK1.1⫺ Thy1.2⫺ CD11c⫹), macrophages (CD45high NK1.1⫺ Thy1.2⫺ CD11c⫺ CD11b⫹), and microglia cells (CD45low NK1.1⫺ Thy1.2⫺ CD11b⫹) in the mononuclear cell preparations from the brains of infected mice were determined using multiparameter flow cytometry as previously described (37). Since CD11b and CD11c can be upregulated on cells other than APCs, a “dump channel” was included in the flow cytometric analysis to ensure a population of APCs that did not include contaminating leukocyte populations (e.g., CD3-PE, CD19-PE, and NK1.1-PE to exclude T, B, and NK cells, respectively). For the detection of granulocytes/neutrophils in the purified mononuclear brain cell populations, cells were stained with antibody specific to Gr-1 (RB6-8C5). Histological analysis of brain inflammation. Tissues harvested from mice that were infected i.c. and transfused with TCR-P14 or TCR-SMARTA transgenic cells were embedded in optimum cutting temperature compound, snap-frozen in a dry-ice 2-methyl-butane bath, sectioned, air dried, and fixed in 2% acetone. For immunofluorescence microscopy, tissue sections were reacted with antibody to CD8 (clone 2.43.5), CD90.1 (Thy1.1) (clone HIS51), CD11b (clone M1/70), CD106 (VCAM-1) (clone 429), or LCMV (clone VL4), and antigen-antibody complexes were detected with appropriate fluorescein isothiocyanate- or Cy3conjugated secondary antibody (Sigma). Primary antibodies were purchased from eBioscience, except for the monoclonal antibody specific to LCMV nucleoprotein (LCMV-NP) (VL4) provided by R. Zinkernagel (Zurich, Switzerland). For histology, sections were stained with hematoxylin and eosin and subjected to gross and microscopic pathological analyses.

RESULTS Expression pattern of VCAM-1 in tissues of normal or LCMV-infected VCAM-1⌬/⌬ and control VCAM-1⌬/flox or B6 mice. VCAM-1 is constitutively expressed at a relatively low level in resting vascular endothelial cells in many tissues and a

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FIG. 1. VCAM-1 expression in hematopoietic cells during an acute LCMV infection. The histograms reflect VCAM-1 expression in spleen or BM cells from uninfected VCAM-1⌬/⌬ (thin lines) and VCAM-1flox//flox control mice either uninfected (filled histograms) or infected with 102 PFU of LCMV i.v. and examined on day 9 (thick line). Cells were double stained with anti-VCAM-1 and antibodies specific to CD45, c-Kit, or lineage markers (TER119, B220, CD3, CD11B [Mac1], or Gr-1) (for BM) or CD3, CD8, CD4, B220, CD19, CD11B, or Gr-1 (for spleen) as shown. In addition, VCAM-1 expression on DC populations (CD8⫹ CD11c⫹, lymphoid DC or CD8␣⫺ CD11c⫹, and myeloid DC) in the spleen was measured by triple staining with anti-VCAM-1, CD8␣, and CD11c-specific antibodies. Data are representative of three individual mice.

fraction of hematopoietic cells in bone marrow (BM), spleen, and lymphoid tissues of normal mice (70). Induced VCAM-1 expression was observed on vascular endothelial cells in many tissues following infection and injury, and this elevated expression is considered a key regulatory event of cell trafficking and homing to sites of inflammation, especially in the CNS, that promote the rapid elimination of pathogens and recovery from infection (5, 36, 72). To examine the potential role of VCAM-1 in regulating virally induced tissue-specific inflammation and antigen-specific T-cell recruitment to target sites, we explored in great detail the LCMV-induced immune response and associated pathology in mice in which VCAM-1 ablation was achieved via the conditional deletion of VCAM-1flox alleles by breeding with TIE2-Cre transgenic mice (VCAM-1⌬/⌬). As controls, VCAM-1flox/flox or B6 mice were used. First we studied VCAM-1 expression patterns at basal levels in tissues of uninfected and LCMV-WE-infected (analyzed at day 9 after virus infection) B6 controls and VCAM-1⌬/⌬ mice. The expression of VCAM-1 in the hematopoietic cells of the BM, spleen, peripheral lymph nodes (PLN), and peripheral blood lymphocytes (PBL) was assessed by flow cytometry using antibodies specific for cell type markers (Fig. 1 and data not shown). In

agreement with earlier findings (32, 70), VCAM-1 expression was observed in nearly half of all cell lineages in the BM of uninfected B6 mice, and this expression level did not significantly change following LCMV infection (102 PFU i.v.). In spleen and PLN tissues, VCAM-1 expression was found largely on myeloid (CD11c⫹ CD8␣⫺) and lymphoid (CD11⫹ CD8␣⫹) DCs at comparable levels for uninfected and virus-infected B6 mice. In contrast to the constitutive VCAM-1 expression observed in lymphoid tissues, circulating cells in the PBL of normal and infected mice were negative for VCAM-1 expression. Moreover, the cells of VCAM-1⌬/⌬ mice were completely negative for VCAM-1 expression, and we did not observe significant differences in the distribution of T, B, and myeloid cells in lymphoid tissues between deficient and normal control mice (data not shown). VCAM-1 is expressed mostly by endothelial cells and the epithelium of the choroid plexus, and there is evidence that its expression is upregulated on endothelial cells in the brains of i.c. LCMV-infected mice (41). Importantly, no detectable VCAM-1-expressing endothelial cells were found in the tissues of ablated mice. In particular, brain endothelial cells in both normal and LCMV-infected VCAM-1⌬/⌬ mice remained negative (data not shown).

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FIG. 2. Virus-specific CD8⫹ T cells in lymphoid and nonlymphoid tissues during an acute LCMV infection. Virus-specific CD8⫹ T-cell populations in control VCAM-1flox//flox (F, broken lines) and VCAM-1⌬/⌬ (Œ, solid lines) mice infected with 102 PFU of LCMV were examined at the indicated time points. Total numbers of GP133–41, GP2276–286, or NP396–404 peptide-specific CD8⫹ T cells were measured by staining with H-2Db tetramers. Note that there was a significant delay in the recruitment and accumulation of virus-specific CD8⫹ T cells in the lungs of VCAM-1⌬/⌬ mice compared to controls (P ⬍ 0.05; indicated by arrows in lung panels). Data shown are means ⫾ standard errors of the means (log10) of virus-specific T cells per spleen of three to five mice.

Dynamics of virus-specific CD8ⴙ T-cell response in lymphoid versus nonlymphoid tissues during an acute LCMV infection of VCAM-1-deficient mice. To examine the potential of VCAM-1 to play an active role in the induction of virusspecific CD8⫹ T cells in lymphoid tissues and their subsequent recruitment to sites of virus replication in nonlymphoid tissues during an acute infection, we measured the kinetics of viral replication and virus-specific CD8⫹ T-cell responses in different tissues of VCAM-1⌬/⌬ mice compared to control B6 mice. Infection of mice with 102 PFU of LCMV-WE i.v. causes an

acute infection with initial viral replication in multiple tissues. Viral titers peaked between days 3 and 6, followed by a rapid decline below detectable levels by day 9 after infection in all tissues examined, with no significant differences in the kinetics of viral replication and clearance between VCAM-1⌬/⌬ and control mice (see Fig. 10A and data not shown). As shown in Fig. 2, the kinetics of proliferative expansion and memory T-cell development were also similar for virus-specific CD8⫹ T-cell populations (specific for GP133–41, GP2276–284, or NP396–404 epitopes) isolated from different tissues, except for the lungs,

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FIG. 3. Kinetics of total virus-specific CD8⫹ T-cell responses in different tissues. VCAM-1⌬/⌬ (Œ, solid lines) or control VCAM-1flox//flox (F, broken lines) mice were infected with 102 PFU of LCMV-WE. The percentages (A) and total numbers per tissue (B) of virus-specific CD8⫹ T cells producing IFN-␥ following stimulation with virus-infected DC2.4 cells were determined by concurrent analyses. Notably, there was a significant difference on days 6 and 7 after infection in the total numbers of virus-specific CD8⫹ T cells in the lungs of VCAM-1⌬/⌬ mice compared to controls (P ⬍ 0.05; indicated by arrows in lung panels). Data shown are means ⫾ standard errors of the means (log10) of virus-specific T cells per tissue of three to five mice.

of controls or VCAM-1⌬/⌬ mice. With lung tissue, we consistently observed significant delays in the recruitment and accumulation of virus-specific CD8⫹ T cells in VCAM-1⌬/⌬ mice compared to controls. Antigen-specific CD8⫹ T cells detected by MHC tetramer staining exhibited a functional phenotype, as assessed by staining for IFN-␥ secretion (data not shown). To further examine whether virus-specific CD8⫹ T-cell populations preferentially accumulate and/or are retained in tissues of infected mice, we evaluated the recruitment kinetics of specific CD8⫹ T cells by calculating the percentages of virusspecific T cells within the lymphocyte population in each tissue as a function of time. As a percentage of CD8⫹ T cells, total numbers of antigen-specific CD8⫹ T cells, identified by restimulation with virus-infected DCs (DC2.4) in combination

with intracellular IFN-␥ staining, varied significantly between different tissue compartments, but no significant differences were observed in VCAM-1⌬/⌬ mice compared to controls (Fig. 3A). When the total numbers of virus-specific CD8⫹ T cells per tissue were compared in this analysis, these values were similar for all tissues except lung, where differences in the kinetics of the overall virus-specific CD8⫹ T-cell populations were observed (Fig. 3B). This suggests that VCAM-1 ablation affects the overall influx of inflammatory cells, including antigen-specific CD8⫹ T cells, into the lung. Conditional VCAM-1 ablation in mice does not affect circulating antibody levels. A specific role for VCAM-1 in regulating the trafficking and retention of antigen-specific lymphocytes in vivo has been described using short-term migration

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FIG. 4. VCAM-1 ablation does not affect the levels of virus-specific IgM or IgG antibodies in serum. Virus-specific IgM or IgG antibody levels in the sera of VCAM-1⌬/⌬ (Œ, solid lines) or control VCAM1flox//flox (F, broken lines) mice following infection with 102 PFU of LCMV. Data points indicate enzyme-linked immunosorbent assay (ELISA) titers of virus-specific antibody (means ⫾ standard errors of the means [log10] of three to five mice) of the IgM or IgG isotype.

assays performed with labeled B or T cells (32, 38, 43). To assess whether the ablation of VCAM-1 in our study impacts the kinetics of antigen-specific antibody production, we measured antibody levels in the sera of VCAM-1⌬/⌬ mice compared to controls during LCMV infection. As shown in Fig. 4, VCAM-1 ablation did not significantly alter the levels of virusspecific immunoglobulin M (IgM) or IgG antibodies in the blood. Role of VCAM-1 in regulating virus-induced inflammation and associated pathology. A classic model of LCMV-induced CD8⫹ and CD4⫹ T-cell-mediated inflammation is the delayed type hypersensitivity (DTH) reaction detected as footpad swelling following local virus inoculation (27, 49). As shown in Fig. 5A, significant reductions in both the magnitude and duration of footpad swelling were observed in VCAM-1⌬/⌬ mice compared to controls, suggesting that this subdermal inflammatory reaction requires VCAM-1. It should be noted that the overall profile of the DTH reaction in VCAM-1⌬/⌬ mice appears to match the afferent plateau phase of the DTH reaction in control mice. Previous studies have clearly established that this response in normal mice is sequentially mediated by effector CD8⫹ and CD4⫹ T cells recruited into the inflammatory sites of virus replication in the footpad (48), so this finding implies that VCAM-1 ablation may preferentially affect the initial phase of footpad swelling mediated by CD8⫹ T cells. Reduction of the footpad swelling reaction indicates that VCAM-1 expression on hematopoietic cells (for example, monocytes, DCs, and granulocytes) or tissue endothelial cells (especially vascular endothelial cells) is important for the migration and perhaps stimulation of inflammatory cytokine secretion at sites of virus replication and inflammatory reaction in the footpad. To examine these possibilities more closely, we measured the footpad swelling reaction in VCAM-1⌬/⌬ or B6 control mice that were infected with LCMV i.f. and transfused with immune splenocytes from infected (day 9) VCAM-1⌬/⌬ or B6 mice. Both B6 controls and VCAM-1⌬/⌬ recipient mice transfused with immune cells from B6-infected mice elicited footpad swelling of similar magnitudes and kinetics (Fig. 5B). This indicates that ablation of VCAM-1 from hematopoietic as well as tissue endothelial cells in the recipient mice did not significantly affect the swelling reaction elicited by donor immune

FIG. 5. VCAM-1 ablation affects the kinetics of virus-induced DTH reaction. (A) Footpad swelling reaction in VCAM-1⌬/⌬ (Œ) or control VCAM-1flox//flox (F) mice following LCMV infection with 104 PFU i.f. was monitored by measuring the increase in thickness of the infected compared to the uninfected foot. Data points represent means ⫾ standard errors of the means of five mice. (B) Kinetics of footpad swelling in B6 (F) versus VCAM-1⌬/⌬ (Œ) recipient mice transfused with immune splenocytes isolated from B6 controls (left panel) or VCAM-1⌬/⌬ donor mice (right panel) on day 9 after infection with 102 PFU of LCMV. Data points represent means ⫾ standard errors of the means of five mice.

splenocytes with normal VCAM-1 expression. However, a more pronounced and statistically significant difference in the kinetics of footpad swelling was observed when we compared B6 controls and VCAM-1⌬/⌬ recipients transfused with immune cells from VCAM-1⌬/⌬ mice (P ⬍ 0.005). In experiments where B6 recipient mice were reconstituted with VCAM-1deficient immune splenocytes and no suppression of footpad swelling was observed, the participation of recipient host-derived hematopoietic cells with intact VCAM-1 expression cannot be excluded and is very likely. Consistent with this notion, the loss of VCAM-1 expression on splenocytes (donor cells) as well as recipient hematopoietic and tissue endothelial cells suppressed the DTH response. Since VCAM-1 expression is also lost from recipient endothelial cells, however, the contribution of hematopoietic versus tissue endothelial cells to footpad swelling cannot be precisely defined. In conclusion, despite this limitation of the adoptive transfer model used, the data suggest that VCAM-1 expression on hematopoietic cells may play a more critical role in regulating the virus-specific, subdermal inflammatory response, although a contribution of tissue-specific endothelial cells is possible. Susceptibility of VCAM-1⌬/⌬ mice to LCMV-induced T-cellmediated CNS pathology. Another model for virus-induced

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FIG. 6. VCAM-1 ablation enhances resistance to fatal LCM. Survival of VCAM-1⌬/⌬ (Œ) or control VCAM-1flox//flox (F) mice infected with 102 or 104 PFU of LCMV i.c. Data represent a cohort of 10 mice for each mouse genotype.

T-cell-mediated inflammation and pathology is the fatal CNS disease that develops following i.c. inoculation of a relatively low dose of LCMV (11, 12, 22, 26, 47). As shown in Fig. 6, VCAM-1⌬/⌬ mice infected i.c. with 102 or 104 PFU of LCMV showed increased resistance to fatal virus-induced meningitis compared to control mice. Quantitative analysis of antiviral T-cell response in mice infected with 102 PFU i.c. did not reveal significant differences in virus-specific CD8⫹ T-cell induction and accumulation in lymphoid tissues or peripheral organs, including the brain (Fig. 7). Note that VCAM-1⌬/⌬ mice that survive i.c. infection developed memory CD8⫹ T cells at stable levels, but control mice died around day 9 after infection, excluding further analysis beyond this time period. The enhanced resistance of VCAM-1⌬/⌬ mice to virally induced meningitis thus did not reflect impaired antigen-specific CD8⫹ T-cell induction and expansion. To further investigate the functional relevance of VCAM-1 expression in the CNS, we studied antigen-specific CD4⫹ and CD8⫹ T-cell recruitment and localization in the virally infected brain. VCAM-1⌬/⌬ or control mice were infected i.c. with 102 PFU of LCMV and subsequently received a relatively low number (2 ⫻ 103) of LCMV-specific CD8⫹ (P14-Thy1.1) or CD4⫹ (SMARTA-Thy1.1) transgenic cells by adoptive transfer. The overall kinetics of transgenic cell proliferation in the spleen and homing into BM or brain tissues prior to fatal inflammatory disease in control mice (days 3 to 7 after infection) did not reveal significant differences between the genotypes, confirming our observations with polyclonal virus-specific CD8⫹ T cells (Fig. 8 and 9). These results suggest that the vascular transmigration and overall influx of antigen-specific T cells into the virus-infected CNS may not be significantly delayed in VCAM-1⌬/⌬ mice. However, VCAM-1 ablation did not eliminate, but rather reduced, the incidence of fatal CNS disease (Fig. 6), suggesting that additional parameters, such as the localization of infiltrating T cells at critical cellular targets within the CNS, may limit pathology in VCAM-1⌬/⌬ mice. To examine this possibility more closely, we evaluated the localization of transgenic CD8⫹ T cells (CD90.1; Thy1.1) in infected VCAM-1⌬/⌬ or control (CD90.2; Thy1.2) recipient mice by immunohistochemistry. In particular, brain sections from i.c. infected VCAM-1⌬/⌬ or control mice that were transfused with transgenic CD8⫹ T cells from VCAM-1-sufficient donors were analyzed at day 6 after infection. As shown in Fig. 8C

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(upper panels), transfused transgenic cells were localized in the meningeal vascular system at this early stage of CNS disease. Interestingly, we noted in the meningeal infiltrates a slightly increased accumulation of virus-specific CD8⫹ reactive T cells in control mice compared to VCAM-1⌬/⌬ recipient mice (Fig. 8C, middle panels). However this difference in antigenspecific CD8⫹ T-cell recruitment in the CNS was time limited, because we observed, as the disease progressed, in addition to the formation of prominent meningeal infiltrates, a wide distribution of immunoreactive CD8⫹ T cells in the brain parenchyma, including white matter regions, such as the corpus callosum, the internal capsule, and the white matter of the cerebellum. However, we did not observe at this advanced stage of disease significant differences in their extents in VCAM-1⌬/⌬ or B6 control recipient mice. Immunostaining for viral antigen in the brains of recipient mice did not reveal significant differences in either the extents or distributions of virus-infected cells between mouse strains, making it unlikely that viral spread in the brain could explain the difference in lethality (Fig. 8C, lower panels). To gain a better mechanistic insight into VCAM-mediated regulation of virus-induced inflammatory processes and associated CNS disease, we further analyzed the composition of the inflammatory cell populations in the brains of i.c. infected (102 PFU) VCAM-1⌬/⌬ versus B6 control mice. In agreement with the viral antigen immunostaining data, the kinetics of virus clearance from brain and other tissues did not differ significantly between mice (Fig. 10A1). In addition, the kinetics of accumulation in the brain of T cells (CD8⫹ or CD4⫹), B cells (B220⫹), and granulocytes/neutrophils (Gr-1⫹) were similar in both mouse strains (Fig. 10B). In contrast, we observed a substantial and statistically significant attenuation in the accumulation of macrophages (CD45high CD11b⫹) and DCs (CD45high CD11c⫹) in the brains of VCAM-1⌬/⌬ mice compared to B6 controls. This was seen at days 5, 6, and 7 after i.c. infection. This finding is important, as it indicates that VCAM-1 expression on brain endothelial cells and/or hematopoietic cells may be critically involved in facilitating the infiltration of professional APCs into the brain. Such cells possess the ability, either directly or via the stimulation of virus-specific T cells recruited into the CNS, to perpetuate and intensify the inflammatory process and thus associated pathology. In addition, to study the accumulation of accessory cells in the brain more directly, we used immunohistochemical analysis to evaluate the localization of infiltrating CD11b⫹ cells in brain sections from VCAM-1⌬/⌬ and B6 mice on days 5, 6, and 7 after i.c. infection.We observed significantly reduced numbers of CD11b⫹ cells in the brain of VCAM-1⌬/⌬ compared to B6 controls (shown for day 6 after infection in Fig. 10C), consistent with the flow cytometric analysis data. DISCUSSION A critical aspect in the pathophysiology of viral diseases is the regulation of inflammation and the antiviral response at the level of leukocyte extravasation and homing to the infected organs. This is a complex dynamic process involving a series of overlapping sequential steps mediated by distinct adhesion molecules, integrins, and chemokine receptors and their ligands. The data presented here add significant information to

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FIG. 7. Virus-specific CD8⫹ T-cell induction and recruitment in mice with conditional VCAM-1 ablation infected i.c. with LCMV. VCAM-1⌬/⌬ or control VCAM-1flox//flox mice were infected with 102 PFU of LCMV-WE i.c. (A) Total numbers of virus-specific CD8⫹ T cells (sum of GP133–41, GP2276–286, and NP396–404 peptide-specific T cells) were determined by staining with MHC I tetramers (Œ and F) or intracellular IFN-␥ (‚, E) following stimulation with virus-infected DC2.4 cells. Data shown are means ⫾ standard errors of the means (log10) of virus-specific T cells per tissue of three to five mice. (B) The percentages of virus-specific CD8⫹ T cells in different tissues of infected VCAM-1⌬/⌬ (Œ) or control (F) mice were determined by staining with MHC I tetramers. Note that for control mice, these analyses were confined to a period of 9 days after infection due to the deaths of animals (indicated by ⫹). (C) Flow cytometry profiles illustrate total virus-specific CD8⫹ T-cell populations at day 8 after infection (102 PFU i.c.) in different tissues. Percentages of CD8⫹ T cells staining positive for IFN-␥ following stimulation with virally infected APCs (DC-IFN-␥) are indicated in the lower corners. Plots shown are gated for live cells.

better understand this complex process, making several points. The first point is that VCAM-1 ablation on the surface of hematopoietic cells, including professional APCs (macrophages/DCs) as well as endothelial cells, does not significantly

affect the ability of the infected host to mount an antiviral T-cell or B-cell response. Thus, VCAM-1 expression was found to be redundant for the ability of virus-specific T cells to eliminate virus in mouse tissues despite the fact that the majority of

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FIG. 8. Unimpaired recruitment of virus-specific transgenic CD8⫹ T cells to the CNSs of VCAM-1⌬/⌬ mice infected i.c. (A) VCAM-1⌬/⌬ (Œ, ‚) or control B6 (F, E) mice were transfused with P14-Thy1.1 CD8⫹ TCR transgenic cells and infected 24 h later with 102 PFU of LCMV-WE i.c. The induction and recruitment of P14 TCR transgenic cells in brain, spleen, and BM was monitored until the preterminal stage of CNS diseases

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FIG. 9. Unimpaired recruitment of virus-specific transgenic CD4⫹ T cells to CNSs of VCAM-1⌬/⌬ mice infected i.c. (A) VCAM-1⌬/⌬ (Œ, ‚) or control B6 (F, E) mice were transfused with SMARTA-Thy1.1 CD4⫹ TCR transgenic cells and infected 24 h later with 102 PFU of LCMV-WE i.c. The induction and recruitment of CD4⫹ TCR transgenic cells in brain, spleen, and BM were monitored until the preterminal stage of CNS disease in control mice (day 8 after infection). Virus-specific CD4⫹ TCR transgenic cells were determined by triple staining with CD8, V␣2, and Thy1.1 antibody (F, Œ) or intracellular IFN-␥ staining (E, ‚) following stimulation of the cells with peptide I-Ab/GP60–80 epitope peptide. Data shown are means ⫾ standard errors of the means (log10) of virus-specific T cells per tissue of three to five mice. (B) Flow cytometry profiles illustrate IFN-␥-positive CD4⫹ TCR transgenic T cells at day 8 after infection (102 PFU i.c.). The percentages of CD4⫹-V␣2⫹-Thy1.1⫹ T cells staining positive for IFN-␥ are indicated in the lower corners. Plots shown are gated for live cells.

DCs (myeloid and lymphoid) in normal or infected animals express this adhesion molecule. Such VCAM-1-independent induction of antiviral immunity is in contrast to parasitic infections in which VCAM-1 plays a role in parasite control during Toxoplasma encephalitis by modulating B-cell and Tcell-mediated immunity (13). These divergent roles in different infection models demonstrate overlapping and redundant roles for adhesion molecules in immune regulation.

A second point of our model is that VCAM-1 expression provides a mechanism for the recruitment of leukocytes and antigen-specific T cells to certain infected target organs but not to others. Thus, VCAM-1 deletion impaired the T-cell-mediated inflammatory response in the skin, leukocyte influx into the lung, and more importantly, migration of accessory cells into the CNS at the onset of viral infection, although no significant role for VCAM-1 in cell trafficking of antigen-specific

in control mice (day 8 after infection). Virus-specific CD8⫹ TCR transgenic cells were determined by triple staining with H-2Db tetramers, CD8 and Thy1.1 antibody (F, Œ), or intracellular IFN-␥ (E, ‚) staining following the stimulation of cells with GP133–41 peptide. Data shown are means ⫾ standard errors of the means (log10) of virus-specific T cells per tissue of three to five mice. (B) Flow cytometry profiles illustrate IFN-␥-positive P14 TCR transgenic T cells at day 8 after infection (102 PFU i.c.). The percentages of CD8⫹-Thy1.1⫹ T cells staining positive for IFN-␥ following peptide stimulation in vitro are indicated in the lower corners. Plots shown are gated for live cells. (C) Representative sagittal brain sections from day 6 virus-infected (102 PFU i.c.) VCAM-1⌬/⌬ or control B6 mice that were transfused with P14 TCR transgenic cells. Staining of sections for CD31 (marker for endothelial cells) (red) and Thy1.1 (P14 TCR transgenic cells) (green) (upper panels), viral antigen (red) and Thy1.1 (P14 TCR transgenic cells) (green) (middle panels), or viral antigen (red) (lower panels).

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FIG. 10. Delayed accumulation of macrophages and DCs in CNSs of mice with conditional VCAM-1 deletion. Virus titers were determined in tissues of VCAM-1⌬/⌬ (filled columns) or control VCAM-1flox//flox (open columns) mice infected with 102 PFU of LCMV-WE i.v. (A) or i.c. (A1). Data shown are means ⫾ standard errors of the means (log10) of virus-specific T cells per tissue of three to five mice. (B) Kinetics of leukocyte recruitment into CNSs of LCMV-infected (102 PFU i.c.) VCAM-1⌬/⌬ (filled columns) or control VCAM-1flox//flox (open columns) mice. Distinct cell populations extracted from the brains of infected mice were quantified using multiparameter flow cytometry as described in Materials and Methods. Data shown are mean ⫾ standard errors of the means (log10) of leukocytes per brain of three experiments (two mice pooled/group). Vertical arrows indicate significant (P ⬍ 0.05) differences in cell counts between genotypes. (C) Immunohistochemical analysis of brain sections from VCAM-1⌬/⌬ or control VCAM-1flox//flox mice infected with 102 PFU i.c. and examined on day 6 after infection. Representative staining of CD11⫹ cells (red) in ventricular areas (left panels), brain parenchyma (middle panels), and meningeal areas (right panels) is presented. DAPI (4⬘,6⬘-diamidino-2-phenylindole) staining (blue) indicates cell nuclei. Original magnification, ⫻200.

CD8⫹ T cells within the inflamed CNS was observed. This was despite the fact that VCAM-1⌬/⌬ mice showed increased resistance to lethal LCMV i.c. infection. Although leukocytes in the mutant mice may utilize alternative mechanisms to migrate to infected sites, which may partly explain the lack of detectable cell homing inhibition in other tissues of infected mice, the strongly reduced footpad swelling reaction observed in this study confirms a pivotal role for VCAM-1 in supporting cell

migration in a physiological model of viral infection. Interestingly, similar defects in LCMV-induced footpad swelling have been reported in studies conducted with fucosyltransferase VII⫺/⫺ (lacking functional E-, P-, and L-selectin ligands) or P-selectin/ICAM-1⫺/⫺ mice, while no significant differences in the magnitude of footpad swelling were reported for E/Pselectin⫺/⫺ mice (17). An essential role for integrins and especially VLA-1 (␣1␤1), VLA-2 (␣2␤1), or VLA-4 (␣4␤1) in

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local inflammation by regulating cell trafficking has also been suggested in previous studies using an adoptive transfer model to show that treatment of virus-primed donor cells with antibody to VLA-1, VLA-2, or VLA-4 blocked the ability to transfer LCMV-specific DTH when donor cells were given i.v. but not when the cells were transferred directly into the foot (1, 10). However, a more recent study using VLA-1⫺/⫺ mice revealed that VLA-1 plays only a marginal role in LCMV-specific T-cell homing to different organs and inflammatory reactions (30). Interestingly, reduced numbers of memory CD8⫹ T cells in nonlymphoid tissues were found in VLA-1⫺/⫺ mice infected with influenza virus (57). Since VLA-4 is the ligand for VCAM-1, these findings and our own collectively illustrate the importance of integrin-ligand interactions for the migration of antigen-specific T cells to the skin. Furthermore, our adoptive transfer experiments suggest a more critical role of VCAM-1 expression on hematopoietic, rather than vascular endothelial cells, to virus-specific inflammatory processes. This leads us to speculate that VCAM-1 interactions with its ligands may play a significant role in controlling the extravasation of antigenpresenting cells, in particular DCs, at the site of virus replication in the footpad to induce and maintain antigen-specific T-cell activation, and its ablation in VCAM-1⌬/⌬ mice may negatively impact the virus-induced DTH reaction. This issue cannot be definitively addressed based on the T-cell adoptive transfer approaches widely used to study cell migration, because many aspects of virus-specific T-cell induction in situ are not precisely reproduced. One interesting avenue for future study of this issue could be based on analyses of mice in which VCAM-1 deletion on DCs is achieved by cell-specific Cre recombinase expression. Another point concerns the initiation of virus-induced inflammation in the lung. The VLA-4-VCAM-1 interaction has been suggested to constitute a critical component in the pulmonary immune response regulating lymphocyte homing to bronchus-associated lymphoid tissue and lung parenchyma (73), and our data support such a role. The fact that VCAM-1 ablation did not reveal specific defects in virus-specific T-cell recruitment to the lung, but rather showed an overall inhibition of leukocyte accumulation, suggests a broad function for VCAM-1 in regulating the overall pulmonary inflammatory process. However, this does not necessarily contradict the notion of antigen-specific T-cell-mediated initiation of cell recruitment because of the inherent limitations of tetramer or IFN-␥ assays in detecting a limited number of specific cells in the lung that are able to initiate the inflammatory process. The next step of amplification would result in the relatively nonspecific recruitment of other cells to assist with the virus clearance process. Finally, upregulation of VCAM-1 expression in blood vessels of the CNS under inflammatory conditions, such as in experimental autoimmune encephalitis, is critical for the recruitment of inflammatory cells, especially effector CD4⫹ T cells, across the blood-brain barrier. Antibody-mediated inhibition of the ␣4␤1-VCAM-1 interaction inhibits the entry of T cells into the CNS (16, 74) (71), contributing to the therapeutic effect of this treatment regimen. In addition, VLA-4 antibody treatment revealed a significant role in the development of inflammation in the CNS of viral infection (29, 59, 64). Ablation of VCAM-1 seems to have little effect on virus-specific

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T-cell recruitment into the CNS during LCMV infection, but interestingly, accumulation of macrophages and DCs early in the infection is substantially impaired. This deficit of accessory cell location in the brain can explain the marked differences in lethality between the genotypes, based on the prediction that these may enhance the inflammatory process via secretion of proinflammatory cytokines or by activating virus-specific T cells arriving in the brain. This scenario is also further supported by gene array experiments that we have performed (data not shown). That is, to evaluate the CNS inflammatory response in more detail, we quantified the transcripts of 114 cytokine genes in the brains of VCAM-1⌬/⌬ and B6 control mice on days 5 and 7 after i.c. infection (102 PFU of LCMVWE) using a mouse common cytokine array (SuperArray; Bioscience). We determined significant expression (normalized to standard GAPDH [glyceraldehyde-3-phosphate dehydrogenase] control) of 25 versus 10 genes (on day 5) and 12 versus 13 genes (on day 7) in B6 controls and VCAM-1⌬/⌬ mice, respectively. Among the differentially expressed genes, we observed prominent downregulation of IL-12a (14-fold), IL-12b (44-fold), IL-18 (11-fold), and glucose phosphatase isomerase 1 (19-fold) as well as upregulation of inhibin beta-A (4-fold) in VCAM-1⌬/⌬ versus B6 mice on day 5 after infection. On day 7 after infection, we found differential upregulation of inhibin alpha (38-fold) in the brains of VCAM-1⌬/⌬ versus B6 mice. Both IL-12 and IL-18 are known to possess proinflammatory properties and are preferentially produced by monocytes/macrophages and DCs (14, 28). Thus, downregulation of these cytokines early during the infection (day 5) could explain the decreased mortality in VCAM-1⌬/⌬ mice. The function of the differential expression of glucose phosphatase isomerase 1, inhibin alpha, and inhibin beta-A in the CNS pathology of infected mice is unclear. Since several studies have established that VCAM-1 expression is upregulated in the brains of virusinfected mice, it is likely that VCAM-1, in combination with other adhesion molecules, plays a role during the multistep process of inflammatory cell migration across the blood-brain barrier and helps this process progress efficiently. In this scenario, consistent with our experimental findings, VCAM-1 ablation may exert subtle effects in diminishing the initial inflammatory cell influx into the CNS, while still allowing efficient virus clearance by effector cells that successfully penetrate the blood-brain barrier. This explanation is consistent with a wealth of literature demonstrating that the fatal outcome of LCMV-induced meningitis is solely dependent on the rapid recruitment of CD8⫹ effector T cells to sites of virus replication in the CNS. Thus, partial suppression of the virus-specific CD8⫹ T-cell response by pharmacological inhibitors or the inoculation of a relatively high virus dose usually does not result in fatal meningitis but rather slows the recruitment of CD8⫹ T cells to the CNS, allowing viral clearance to proceed normally (22, 25). In conclusion, our results illustrate that VCAM-1 plays a significant role in regulating virus-induced inflammation in different organs during a viral infection. Under conditions where immune-mediated pathology associated with excessive inflammation is a dominant feature of virus infection or autoimmune conditions, inhibition of the ␣4␤1-VCAM-1 interaction alone or in combination with other members of the adhesion cascade may be beneficial in ameliorating disease symptoms. However,

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a challenge for such intervention is that reduced effector cell influx may compromise protection or can result in the establishment of persistent infection in the treated host. This consideration is understandable in light of observations that the pharmacological block of integrins (including ␣4␤1/VLA-4) in humans has powerful anti-inflammatory effects that seem to be highly effective in the drug treatment of multiple sclerosis. However, a potential limitation of such intervention is the possibility of drug-induced inhibition of normal lymphocyte trafficking in the CNS for effective immunosurveillance of JC virus (60). A challenge for future studies will be to define optimal conditions of treatment to avoid tissue pathology associated with excessive inflammation while allowing efficient immune-mediated viral clearance from the host (23, 40, 61). ACKNOWLEDGMENTS This work was supported by NIH grants AI42114 to D.M. and AI47379 to P.A.K. R.A.F. is an investigator of the Howard Hughes Medical Institute.

18.

19. 20.

21. 22.

23. 24.

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