In mice, intravenous injections are commonly administered in the lateral tail vein. This technique is sometimes difficult to carry out and may cause stress to mice. Though injection through the retro-orbital venous sinus can provide certain advantages over lateral tail vein injection, this method is poorly defined and infrequently used. To compare the efficacy of these two routes of drug delivery, the authors injected MAFIA transgenic mice with the depletion agent AP20187, which selectively induces apoptosis in macrophages. Each mouse received five consecutive daily injections through either the lateral tail vein or the retro-orbital venous sinus. The authors then compared macrophage depletion in different tissues (lung, spleen, bone marrow and peritoneal exudate cells). Both routes of injection were similarly effective. A separate experiment using BALB/c mice indicated that retro-orbital venous sinus injection was the less stressful of the two methods.

Intranasal application of vesicular stomatitis virus (VSV) causes acute infection of the central nervous system (CNS). However, VSV encephalitis is not invariably fatal, suggesting that the CNS may contain a professional antigen-presenting cell (APC) capable of inducing or propagating a protective antiviral immune response. To examine this possibility, we first characterized the cellular elements that infiltrate the brain as well as the activation status of resident microglia in the brains of normal and transgenic mice acutely ablated of peripheral dendritic cells (DCs) in vivo. VSV encephalitis was characterized by a pronounced infiltrate of myeloid cells (CD45highCD11b+) and CD8+ T cells containing a subset that was specific for the immunodominant VSV nuclear protein epitope. This T cell response correlated temporally with a rapid and sustained upregulation of MHC class I expression on microglia, whereas class II expression was markedly delayed. Ablation of peripheral DCs profoundly inhibited the inflammatory response as well as infiltration of virus-specific CD8+ T cells. Unexpectedly, the VSV-induced interferon-gamma (IFN-γ) response in the CNS remained intact in DC-deficient mice. Thus, both the inflammatory and certain components of the adaptive primary antiviral immune response in the CNS are dependent on peripheral DCs in vivo.

We report herein that vesicular stomatitis virus (VSV) induced a concurrent primary Th1 (T helper 1) and Th2 cytokine response detectable ex vivo. Liposome-encapsulated clodronate-mediated elimination of CD8- marginal dendritic cells (DCs) and splenic macrophages (m Phi), but not CD8+ interdigitating DCs, prior to infection resulted in a markedly diminished chemokine and Th1 (IL-2, interferon-gamma) cytokine response, although the Th2 response (IL-4) remained relatively intact. Repopulation with marginal DCs and marginal metallophilic macrophages (MMM) restored Th1 cytokine profiles but did not restore chemokine responsiveness or reduce VSV-induced morbidity/mortality. Chemokine competency returned approximately 4 weeks post-depletion, which correlated temporally with repopulation of the spleen with marginal zone macrophages (MZM) and red pulp macrophages (RPM). Unexpectedly, virus-induced morbidity persisted for over 1 month post-depletion and was associated with virus dissemination and distinctive histological lesions in the liver. Depletion of interferon-producing plasmacytoid dendritic cells did not account for virus-induced morbidity because serum levels of type I interferon were not diminished in Cl2MBP-liposome-treated mice. Thus, distinct m Phi subsets are critical for chemokine production and viral clearance, and, in their absence, VSV disseminates even in the presence of high titers of interferon.

We have examined the role of dendritic cells (DCs) in the antiviral immune response and viral clearance using a transgenic mouse model (CD11c-diphtheria toxin (DT) receptor GFP) that allows for their conditional ablation in vivo. DT administration systemically ablated conventional and IFN-producing plasmacytoid DCs (pDCs) in transgenic, but not nontransgenic littermates, without elimination of splenic macrophages. Unexpectedly, early (12 and 48 h postinfection) viral clearance of vesicular stomatitis virus was normal in DC-depleted mice despite markedly reduced serum titers of type I IFN. DC-depleted mice remained virus-free with the exception of a subset (∼30%) that developed overwhelming and fatal brain infections 6 days postinfection. However, DT treatment profoundly inhibited clonal expansion of naive CD8+ vesicular stomatitis virus-specific T cells without altering the primary Th1 and Th2 cytokine response. Optimal clonal expansion required pDCs because selective elimination of these cells in vivo with a depleting Ab also suppressed expansion of tetramer+ cells, although Th1/Th2 cytokine production remained unaltered. Collectively, these data indicate that conventional DCs and to a lesser extent pDCs are critical for proliferation of naive antiviral T cells. However, other components of the primary adaptive immune response (Th1/Th2 cytokines) are essentially normal in the absence of DCs, which may account for the efficient viral clearance seen in DC-depleted mice. Thus, sufficient redundancy exists in the immune system to sustain efficient viral clearance despite loss of an APC considered essential for induction of a primary antiviral immune response.

H-2d-encoded gene products were analyzed as restriction antigens for anti-vesicular stomatitis virus (VSV) cytotoxic T lymphocytes (CTL). Cold target competition experiments revealed that VSV recognition was H-2D region-restricted; H-2K-end-restricted recognition of VSV could not be demonstrated. That VSV is not recognized in the context of K-region-encoded gene products is also supported by the observation that H-2dm1 and H-2dm2 mice, strains that contain H-2Kd but have an alteration in H-2L and/or H-2D/L, are nonresponders in the CTL assay. Two different lines of evidence eliminated H-2Dd, H-2Md, and H-2Rd as the restriction antigens: (a) H-2dm2-VSV inhibitors that express H-2Dd and H-2Md did not block the lysis of P815-VSV targets by Balb/c anti-VSV killer cells, and (b) a hybridoma specific for H-2Dd failed to inhibit killer cell activity in this same effector/target combination. However, two monoclonal antibodies specific for H-2Ld but not H-2Rd completely blocked anti-VSV cytotoxic activity. Taken together, in the H-2d haplotype, anti-VSV CTL recognize VSV solely in the context of the H-2Ld molecule. This is the first demonstration of the exclusive use by a mouse stain of the H-2L molecule only for H-2-restricted recognition, and thus supports the notion that H-2L plays a major role in restricting antigen specific recognition. Finally, the fact that an anti-H-2Ld monoclone completely blocked an H-2dm2 anti-BALB/c CTL response indicates that H-2R, a molecule absent in H-2dm2 anti-BALB/c CTL response indicates that H-2R, a molecule absent in H-2dm2 but not BALB/c, does not sensitize H-2 alloreactive CTL.

Intranasal application of vesicular stomatitis virus (VSV) induces acute encephalitis characterized by a pronounced myeloid and T cell infiltrate. The role of distinct phagocytic populations on VSV encephalitis was therefore examined in this study. Ablation of peripheral macrophages did not impair VSV encephalitis or viral clearance from the brain, whereas, depletion of splenic marginal dendritic cells impaired this response and enhanced morbidity/mortality. Selective depletion of brain perivascular macrophages also suppressed this response without altering viral clearance. Thus, two anatomically distinct phagocytic populations regulate VSV encephalitis in a nonredundant fashion although neither population is essential for viral clearance in the CNS.

Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as “filters” in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection.

We previously demonstrated that in murine T cells thermotolerance correlated with heat shock protein 70 (hsp7O) synthesis and protection of nuclear ype I topoisomerase (topo 1). Topo I activity returned to normal levels following heat stress even in cells not rendered thermotolerant by a prior heat shock. Recovery of topo I activity was not dependent on de novo protein synthesis, suggesting that the cell possesses a pathway(s) for refolding this nuclear protein.In this report we demonstrate that topo I and hsc70, the constitutively produced member of the hsp70 family, associated in vivo during heat stress. That this association may play a physiologically important role in protecting topo I activity from heat stress was suggested by the observation that hsc70 protected topo I from heat inactivation in vitro. hsc70 but not actin also reactivated previously heat-denatured topo I in a dose-dependent fashion.However, refolding of heat-denatured topo I by purified hsc70 was inefficient relative to a hsc70-containing cell lysate. Protection from heat inactivation as well as reactivation by hsc70 did not require exogenous ATP. Similarly, reactivation by the cell lysate was not inhibited by ADP or a nonhydrolyzable analogue of ATP. Thus, our studies suggest that nuclear topo I complexes with hsc70 during heat stress, which may explain, at least in part, why hsp70 proteins accumulate in the nucleus, particularly the nucleolus. This interaction may limit heatinduced protein damage and/or accelerate restoration of protein function in an ATP-independent reaction. Elevated temperatures induce a transient resistance to subsequent heat exposure in cells from various organisms. This phenomenon, termed acquired thermotolerance, is associated with the rapid and preferential synthesis of a small set of highly conserved proteins, the heat shock proteins (hsps; reviewed in ref. 1). Some hsps, such as the 70-kDa cognate hsp (hsc70), are constitutively produced in nonstressed cells and their synthesis is only moderately enhanced following heat shock. In contrast, hsp70, a distinct but closely related protein to hsc70, is not constitutively produced in rodent cells but can rapidly become an abundantly synthesized protein following heat stress. A number of studies suggest that hsps, particularly members of the hsp70 family, play a role in the acquisition of thermotolerance; however, this evidence is largely circumstantial and rests on a correlation of cellular levels of hsp70 with thermotolerance (2-4). During heat stress hsc70 and hsp70 both leave the cytoplasm and nucleoplasm and rapidly accumulate in large amounts in the nucleolus (5). This migration is transient because removal of the stress results in redistribution back to the nucleoplasm and cytoplasm. Understanding why members of the hsp70 family accumulate in the nucleolus during heat stress will undoubtedly provide insights on hsp7O function and its role in thermotolerance.Previous studies from our laboratory demonstrated that heat stress initially inhibited DNA replica...