Expression of the full-length form of gp2 of equine herpesvirus 1 (EHV-1) completely restores respiratory virulence to the attenuated EHV-1 strain KyA in CBA mice

Wild-type equine herpesvirus 1 (EHV-1) strains express a large (250-kDa) glycoprotein, gp2, that is encoded by EUs4 (gene 71) located within the unique short region of the genome. DNA sequence analysis revealed that EUs4 of the pathogenic EHV-1 strain RacL11 is an open reading frame of 2,376 bp that encodes a protein of 791 amino acids. The attenuated EHV-1 vaccine strain KyA harbors an in-frame deletion of 1,242 bp from bp 222 to 1461 and expresses a truncated gp2 of 383 amino acids. To determine the relative contribution of gp2 to EHV-1 pathogenesis, we compared the course of respiratory infection of CBA mice infected with either wild-type RacL11, attenuated KyA, or a recombinant KyA that expresses the full-length gp2 protein (KyARgp2F). Mice infected with KyA lost a negligible amount of body weight (0.18% total weight loss) on day 1 postinfection and regained weight thereafter, whereas mice infected with KyARgp2F or RacL11 steadily lost weight beginning on day 1 and experienced a 20 and 18% loss in body weight, respectively, by day 3. Immunohistochemical and flow cytometric analyses revealed higher numbers of T and B lymphocytes and an extensive consolidation consisting of large numbers of Mac-1-positive cells in the lungs of animals infected with KyARgp2F compared to animals infected with KyA. RNase protection analyses revealed increased expression of numerous cytokines and chemokines, including interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, MIP-2, interferon gamma-inducible protein, monocyte chemotactic protein 1, and T-cell activation gene 3 at 12 h postinfection with KyARgp2F. Three independent DNA array experiments confirmed these results and showed a 2- to 13-fold increase in the expression of 31 inflammatory genes at 8 and 12 h postinfection with KyARgp2F compared to infection with KyA. Taken together, the results indicate that expression of full-length gp2 is sufficient to restore full respiratory virulence to the attenuated KyA strain and raise caution concerning the inclusion of full-length gp2 in the development of EHV-1 vaccines.     

The truncated form of glycoprotein gp2 of equine herpesvirus 1 (EHV-1) vaccine strain KyA is not functionally equivalent to full-length gp2 encoded by EHV-1 wild-type strain RacL11

Most equine herpesvirus 1 (EHV-1) strains, including the naturally occurring virulent RacL11 isolate, encode a large glycoprotein, gp2 (250 kDa), which is expressed from gene 71. Besides other alterations in the viral genome, the avirulent strain KyA harbors an in-frame deletion of 1,242 nucleotides in gene 71. To examine the contributions of gp2 variation to virus growth and virulence, mutant RacL11 and KyA viruses expressing full-length or truncated gp2 were generated. Western blot analyses demonstrated expression of a 250-kDa gp2 in cells infected with RacL11 virus or a mutant KyA virus harboring full-length gene 71, whereas a 75- to 80-kDa gp2 was detected in cells infected with KyA or mutant RacL11 virus expressing KyA gp2. The RacL11 gp2 precursor of 250 kDa in size and its truncated KyA counterpart of 80 kDa, as well as the 42-kDa carboxy-terminal gp2 subunit, were incorporated into virus particles. Absence of gp2 in RacL11 resulted in a 6-fold reduction of extracellular virus titers and a 13% reduction of plaque diameters, whereas gp2-negative KyA exhibited a 55% reduction in plaque diameter and a 51-fold decrease in extracellular virus titers. The massive growth defects of gp2-negative KyA could be restored by reinsertion of the truncated but not the full-length gp2 gene. The virulence of the generated gp2 mutant viruses was compared to the virulence of KyA and RacL11 in a murine infection model. RacL11 lacking gp2 was apathogenic for BALB/c mice, and insertion of the truncated KyA gp2 gene into RacL11 was unable to restore virulence. Similarly, replacement in the KyA genome of the truncated with the full-length RacL11 gene 71 did not result in the generation of virulent virus. From the results we conclude that full-length and truncated EHV-1 gp2 are not functionally equivalent and cannot compensate for the action of their homologues in allogeneic virus backgrounds.     

Characterization of the Cytolytic T-Lymphocyte Response to a Candidate Vaccine Strain of Equine Herpesvirus 1 in CBA Mice

The cytolytic T-lymphocyte (CTL) response to respiratory infection with equine herpesvirus 1 (EHV-1) in CBA (H-2^k) mice was investigated. Intranasal (i.n.) inoculation of mice with the attenuated EHV-1 strain KyA resulted in the generation of a primary virus-specific CTL response in the draining mediastinal lymph nodes 5 days following infection. EHV-1-specific CTL could be restimulated from the spleen up to 26 weeks after the resolution of infection, indicating that a long-lived memory CTL population was generated. Depletion of CD8⁺ T cells by treatment with antibody and complement prior to assay eliminated CTL activity from both primary and memory populations, indicating that cytolytic activity in this model was mediated by class I major histocompatibility complex-restricted, CD8⁺ T cells. A single i.n. inoculation with KyA induced protective immunity against infection with the pathogenic EHV-1 strain, RacL11. The adoptive transfer of splenocytes from KyA-immune donors into sublethally irradiated recipients resulted in a greater than 250-fold reduction in RacL11 in the lung. The elimination of both CD4⁺ and CD8⁺ T cells from the transferred cells abrogated clearance of RacL11, while the selective depletion of either subpopulation alone had little effect. These results suggested that both lymphocyte subpopulations contribute to viral clearance, with either subpopulation alone being sufficient.Equine herpesvirus 1 (EHV-1) is a prevalent respiratory pathogen of horses worldwide (8, 17, 38). Infection of horses with EHV-1 results in fever, respiratory distress, abortagenic disease, and severe neurological sequelae (3, 13, 27, 35, 36). The highly contagious respiratory transmission of EHV-1 has resulted in disastrous outbreaks of disease in domestic horse populations and has had a significant economic impact on the equine industry. EHV-1 infection of the horse results in the generation of a short-lived humoral response but does not confer long-term protection, as disease often occurs following natural infection (10, 22). Although both live and inactivated vaccines are currently available for EHV-1, only relatively short-lived protection has been observed (11, 12, 24). Furthermore, it is not clear which immune functions are responsible for conferring the short-lived protection following vaccination. In addition to specific antibody responses, peripheral blood leukocytes from vaccinated horses produce gamma interferon in culture (19). EHV-1-specific, CD8⁺ class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) have been identified in peripheral blood mononuclear cells, reaching maximal levels 2 to 3 weeks postinfection (p.i.) (4, 20). However, the effectiveness of the current vaccines in stimulating EHV-1-specific CTL and their role in protective immunity in vivo are currently not known.Horses inoculated with the attenuated EHV-1 strain Kentucky A (KyA) exhibited a reduction in clinical signs following challenge with a pathogenic EHV-1 strain (33, 34). Although the attenuated strain induced a protective response, in terms of a reduced duration of viral shedding and viremia, the ability of this strain to induce an EHV-1-specific antibody response was weaker than that of the virulent strain. This finding suggested that immune functions other than the generation of specific antibodies might be critical in the resolution of infection. To generate a more effective EHV-1 vaccine, a better understanding of the precise immune functions associated with protection and resolution from EHV-1 infection is essential.A murine model of respiratory EHV-1 infection which closely mimicked EHV-1 infection in the natural host was established in various strains of mice (5). Common features included replication in the respiratory mucosae, the development of pneumonitis, cell-associated viremia, and abortion (5, 6). Specific immune responses are important for modulating infection. In the mouse, the passive transfer of hyperimmune polyclonal rabbit EHV-1-specific antibodies into infected mice significantly reduced the viremia following challenge with live EHV-1 (5). Subsequent studies demonstrated that various EHV-1 glycoproteins, including gB, gC, gD and gH, were capable of inducing the generation of neutralizing antibodies (9, 23, 40, 49, 52, 56). Furthermore, inoculation of mice with gB, gC, and/or gD elicited a protective response against subsequent challenge with pathogenic EHV-1 (39, 49, 50, 52, 56). However, each of these EHV-1 gene products is capable of eliciting both B- and T-cell responses; thus, the role of distinct immune functions conferring protection is not clearly defined.In the most extensively studied BALB/c mouse model of EHV-1 infection, adoptive transfer experiments demonstrated that immune spleen cells isolated from mice primed with live, but not heat-killed, EHV-1 reduced viral levels in both the lungs and nasal turbinates of infected recipient mice (5). Although the specific cell population responsible for protection was not determined, the data suggested that cell-mediated immune functions may be critical in the resolution of EHV-1 infection. The adoptive transfer of defined T-cell subpopulations demonstrated that both CD4⁺ and CD8⁺ T cells play a role in controlling EHV-1 respiratory infection. The CD8⁺ T-cell subpopulation appeared to play a more dominant role, although the functions by which protection was mediated were not defined (7). If the immune response was elicited by immunization with recombinant baculovirus-derived EHV-1 glycoproteins, the response was altered so that CD4⁺ T cells were predominantly associated with protection (52). Thus, either T-cell subpopulation is likely to play an important role in the optimal response to infection.While adoptive transfer studies have implicated an important role for CD8⁺ T cells in the control of EHV-1 infection in the lungs of BALB/c mice (7), there has been no direct assessment of CD8⁺ T-cell effector functions in this model. This is due predominantly to the lack of suitable class I MHC-compatible, H-2^d-expressing murine cells that are susceptible to infection with EHV-1. However, the attenuated KyA strain of EHV-1 has been propagated in our laboratory in suspension cultures of murine L-M fibroblasts that express the H-2^k haplotype. Therefore, an alternative mouse model, using mice expressing the H-2^k haplotype, was adopted principally to assess the activation of EHV-1-specific CTL responses. Initial studies by Awan et al. (5) demonstrated that CBA (H-2^k) mice were susceptible to infection with EHV-1 strain Ab4. In the present study, CBA mice were susceptible to respiratory tract infection by both the nonpathogenic KyA and the pathogenic RacL11 strains of EHV-1. Furthermore, infection of CBA mice with the attenuated KyA strain generated a vigorous CD8⁺, class I MHC-restricted, EHV-1-specific primary CTL response in the draining mediastinal lymph nodes (MLN) and a long-term memory CTL response in the spleen. These studies provide the basis for a model system to analyze the potential importance of class I MHC-restricted CTL activity in controlling EHV-1 infection in vivo.     

The equine herpesvirus 1 glycoprotein gp21/22a, the herpes simplex virus type 1 gM homolog, is involved in virus penetration and cell-to-cell spread of virions.

Experiments to analyze the function of the equine herpesvirus 1 (EHV-1) glycoprotein gM homolog were conducted. To this end, an Rk13 cell line (TCgM) that stably expressed EHV-1 gM was constructed. Proteins with apparent M(r)s of 46,000 to 48,000 and 50,000 to 55,000 were detected in TCgM cells with specific anti-gM antibodies, and the gM protein pattern was indistinguishable from that in cells infected with EHV-1 strain RacL11. A viral mutant (L11deltagM) bearing an Escherichia coli lacZ gene inserted into the EHV-1 strain RacL11 gM gene (open reading frame 52) was purified, and cells infected with L11deltagM did not contain detectable gM. L11deltagM exhibited approximately 100-fold lower titers and a more than 2-fold reduction in plaque size relative to wild-type EHV-1 when grown and titrated on noncomplementing cells. Viral titers were reduced only 10-fold when L11deltagM was grown on the complementing cell line TCgM and titrated on noncomplementing cells. L11deltagM also exhibited slower penetration kinetics compared with those of the parental EHV-1 RacL11. It is concluded that EHV-1 gM plays important roles in the penetration of virus into the target cell and in spread of EHV-1 from cell to cell.     

Antigen receptor engagement selectively induces macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta chemokine production in human B cells

We show herein that B cell Ag receptor (BCR) triggering, but not stimulation by CD40 mAb and/or IL-4, rapidly induced the coordinated expression of two closely related T cell chemoattractants, macrophage inflammatory protein-1 beta (MIP-1 beta) and MIP-1 alpha, by human B cells. Naive, memory, and germinal center B cells all produced MIP-1 alpha/beta in response to BCR triggering. In contrast to MIP-1 alpha/beta, IL-8, which is spontaneously produced by germinal center B cells but not by naive and memory B cells, was not regulated by BCR triggering. Culturing follicular dendritic cell-like HK cells with activated B cells did not regulate MIP-1 alpha/beta production, but it did induce production of IL-8 by HK cells. Microchemotaxis assays showed that CD4+CD45RO+ T cells of the effector/helper phenotype actively migrated along a chemotactic gradient formed by BCR-stimulated B cells. This effect was partially blocked by anti-MIP-1 beta and anti-CC chemokine receptor 5 Ab, but not by anti-MIP-1 alpha Ab suggesting that MIP-1 beta plays a major role in this chemoattraction. Since maturation of the B cell response to a peptide Ag is mostly dependent on the availability of T cell help, the ability of Ag-stimulated B cells to recruit T cells via MIP-1 alpha/beta, may represent one possible mechanism enabling cognate interactions between rare in vivo Ag-specific T and B cells.     

Nitric oxide regulates MIP-1alpha expression in primary macrophages and T lymphocytes: implications for anti-HIV-1 response

BACKGROUND: Chemokines and chemokine receptors have been shown to play a critical role in HIV infection. Chemokine receptors have been identified as coreceptors for viral entry into susceptible target cells, and several members of the beta chemokine subfamily of cytokines, MIP-1alpha, MIP-1beta, and RANTES, have been identified as the major human immunodeficiency virus (HIV)-suppressive factors produced by activated CD8+ T lymphocytes. In macrophages, HIV-1 infection itself was shown to upregulate the production of MIP-1alpha and MIP-1beta. In the present study, we address the mechanisms by which HIV-1 infection regulates beta chemokine responses in macrophages and lymphocytes. MATERIAL AND METHODS: To address whether nitric oxide (NO), generated as a consequence of HIV-1 infection, regulates beta chemokine responses in monocyte/macrophages and/or macrophage-depleted peripheral blood mononuclear cells (PBMCs) these two cell populations were isolated from HIV seronegative donors, placed in culture, and infected with HIV-1 in either the presence or absence of exogenous activators (e.g. lipopolysaccharide, phytohemagglutinin), inhibitors of nitric oxide synthase (NOS), or chemical donors of NO. Cultures were analyzed for beta chemokine responses by ELISA and RNase protection. RESULTS: LPS-induced MIP-1alpha release is enhanced in HIV-1-infected, as compared to uninfected, monocyte/macrophage cultures, and this enhancing effect is partially blocked by the addition of inhibitors of NOS, and can be reproduced by chemical generators of NO even in the absence of HIV-1 infection. A similar strategy was used to demonstrate a role for NO in HIV-1-mediated induction of MIP-1alpha in unstimulated macrophage cultures. NOS inhibitors also decreased MIP-1alpha and MIP-1beta production by phytohemagglutinin-stimulated monocyte-depleted PBMC cultures. CONCLUSIONS: These results indicate that NO amplifies MIP-1alpha responses in activated macrophages and lymphocytes, and suggests that this pleiotropic molecule might function as an enhancing signal that regulates secretion of beta chemokines during HIV-1 infection. These findings reveal a novel mechanism by which NO might regulate the anti-HIV activity of immune cells.     

The equine herpesvirus 1 Us2 homolog encodes a nonessential membrane-associated virion component

Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 Us2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 Us2 protein specifically detected a protein with an Mr of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-Mr Us2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-Mr Us2 polypeptide. Irrespective of its size, the Us2 protein was incorporated into virions. The EHV-1 Us2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 Us2 protein or to a truncated Us2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 Us2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the Us2 protein in the viral envelope and plasma membrane of infected cells, a Us2-negative RacL11 mutant (L11DeltaUs2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a Us2-repaired virus. After infection of BALB/c mice with L11DeltaUs2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 Us2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.     

Chemokine responses and accumulation of inflammatory cells in the lungs of mice infected with highly virulent Cryptococcus neoformans: effects of interleukin-12

We examined the mechanisms involved in the development of lung lesions after infection with Cryptococcus neoformans by comparing the histopathological findings and chemokine responses in the lungs of mice infected with C. neoformans and assessed the effect of interleukin (IL) 12 which protects mice from lethal infection. In mice infected intratracheally with a highly virulent strain of C. neoformans, the yeast cells multiplied quickly in the alveolar spaces but only a poor cellular inflammatory response was observed throughout the course of infection. Very little or no production of chemokines, including MCP-1, RANTES, MIP-1alpha, MIP-1beta and IP-10, was detected at the mRNA level using RT-PCR as well as at a protein level in MCP-1, RANTES and MIP-1alpha. In contrast, intraperitoneal administration of IL-12 induced the synthesis of these chemokines and a marked cellular inflammatory response involving histiocytes and lymphocytes in infected mice. Our findings were confirmed by flow cytometry of intraparenchymal leukocytes obtained from lung homogenates which showed IL-12-induced accumulation of inflammatory cells consisting mostly of macrophages and CD4+ alphabeta T cells. On the other hand, C-X-C chemokines including MIP-2 and KC, which attract neutrophils, were produced in infected and PBS-treated mice but treatment with IL-12 showed a marginal effect on their level, and neutrophil accumulation was similar in PBS- and IL-12-treated mice infected with C. neoforman. Our results demonstrate a close correlation between chemokine levels and development of lung lesions, and suggest that the induction of chemokine synthesis may be one of the mechanisms of IL-12-induced protection against cryptococcal infection.     

Identification of human macrophage inflammatory proteins 1alpha and 1beta as a native secreted heterodimer

Chemokines are secreted proteins that function as chemoattractants for leukocytes. The chemokines macrophage inflammatory protein 1alpha and 1beta (MIP-1alpha and MIP-1beta) now have been shown to be secreted from activated human monocytes and peripheral blood lymphocytes (PBLs) as a heterodimer. Immunoprecipitation and immunoblot analysis revealed that antibodies to either MIP-1alpha or MIP-1beta precipitated a protein complex containing both MIP-1alpha and MIP-1beta under normal conditions from culture supernatants and lysates of these cells. Mass spectrometry of the complexes, precipitated from the culture supernatants of monocytes and PBLs, revealed the presence of NH(2)-terminal truncated MIP-1alpha (residues 5-70) together with either intact MIP-1beta or NH(2)-terminal truncated MIP-1beta (residues 3-69), respectively. The secreted MIP-1alpha/beta heterodimers were dissociated into their component monomers under acidic conditions. Exposure of monocytes or PBLs to monensin induced the accumulation of heterodimers composed of NH(2)-terminal truncated MIP-1alpha and full-length MIP-1beta in the Golgi complex. The mixing of recombinant chemokines in vitro demonstrated that heterodimerization of MIP-1alpha and MIP-1beta is specific and that it occurs at physiological conditions, pH 7.4, and in the range of nanomolar concentrations. The data presented here provide the first biochemical evidence for the existence of chemokine heterodimers under natural conditions. Formation of heterodimers of MIP-1alpha/beta may have an impact on intracellular signaling events that contribute to CCR5 and possibly to other chemokine receptor functions.     

The Equine Herpesvirus 1 IR6 Protein That Colocalizes with Nuclear Lamins Is Involved in Nucleocapsid Egress and Migrates from Cell to Cell Independently of Virus Infection

The equine herpesvirus 1 (EHV-1) IR6 protein forms typical rod-like structures in infected cells, influences virus growth at elevated temperatures, and determines the virulence of EHV-1 Rac strains (Osterrieder et al., Virology 226:243–251, 1996). Experiments to further elucidate the functions and properties of the IR6 protein were conducted. It was shown that the IR6 protein of wild-type RacL11 virus colocalizes with nuclear lamins very late in infection as demonstrated by confocal laser scan microscopy and coimmunoprecipitation experiments. In contrast, the mutated IR6 protein encoded by the RacM24 strain did not colocalize with the lamin proteins at any time postinfection (p.i.). Electron microscopical examinations of ultrathin sections were performed on cells infected at 37 and 40°C, the latter being a temperature at which the IR6-negative RacH virus and the RacM24 virus are greatly impaired in virus replication. These analyses revealed that nucleocapsid formation is efficient at 40°C irrespective of the virus strain. However, whereas cytoplasmic virus particles were readily observed at 16 h p.i. in cells infected with the wild-type EHV-1 RacL11 or an IR6-recombinant RacH virus (HIR6-1) at 40°C, virtually no capsid translocation to the cytoplasm was obvious in RacH- or RacM24-infected cells at the elevated temperature, demonstrating that the IR6 protein is involved in nucleocapsid egress. Transient transfection assays using RacL11 or RacM24 IR6 plasmid DNA and COS7 or Rk₁₃ cells, infection studies using a gB-negative RacL11 mutant (L11ΔgB) which is deficient in direct cell-to-cell spread, and studies using lysates of IR6-transfected cells demonstrated that the wild-type IR6 protein is transported from cell to cell in the absence of virus infection and can enter cells by a yet unknown mechanism.     

CD8+ T cells are a biologically relevant source of macrophage inflammatory protein-1 alpha in vivo

Chemokines are small proteins that direct the migration of leukocytes to inflammatory foci. Many cell types, including macrophages, fibroblasts, endothelial cells, and lymphocytes, produce chemokines in vitro, but biologically relevant sources of chemokines in vivo have not been well characterized. To investigate the pertinent sources of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in vivo, we used MIP-1 alpha-deficient (MIP-1 alpha-/-) mice as donors and as recipients in adoptive transfer experiments after a lethal infection with Listeria monocytogenes (LM). Unexpectedly, we found that the production of MIP-1 alpha by CD8+ T cells was critical in this system, as the cells from MIP-1 alpha-/- mice primed with LM were significantly less effective in protecting naive mice against a lethal infection by LM than were the CD8+ T cells from wild-type (wt) mice. This requirement for donor T cell production of MIP-1 alpha was confirmed by the observation that wt donor T cells do not mediate protection when coadministered with an anti-MIP-1 alpha polyclonal antiserum. Production of MIP-1 alpha by the recipient mice was not required for protection, because wt and MIP-1 alpha-/- recipients were equally well protected by wt T cells. A 2- to 3-fold decrease in the number of transferred lymphocytes was seen in the spleens of mice receiving T cells from MIP-1 alpha-/- mice compared with those receiving wt T cells. In addition, CD8+ T cells from MIP-1 alpha-/- mice had a reduced ability to kill LM-infected target cells in vitro. These findings demonstrate that T cell production of MIP-1 alpha is required for clearance of an intracellular pathogen in vivo.     

Inducible expression of inflammatory chemokines in respiratory syncytial virus-infected mice: role of MIP-1alpha in lung pathology

Lower respiratory tract disease caused by respiratory syncytial virus (RSV) is characterized by profound airway mucosa inflammation, both in infants with naturally acquired infection and in experimentally inoculated animal models. Chemokines are central regulatory molecules in inflammatory, immune, and infectious processes of the lung. In this study, we demonstrate that intranasal infection of BALB/c mice with RSV A results in inducible expression of lung chemokines belonging to the CXC (MIP-2 and IP-10), CC (RANTES, eotaxin, MIP-1beta, MIP-1alpha, MCP-1, TCA-3) and C (lymphotactin) families. Chemokine mRNA expression occurred as early as 24 h following inoculation and persisted for at least 5 days in mice inoculated with the highest dose of virus (10(7) PFU). In general, levels of chemokine mRNA and protein were dependent on the dose of RSV inoculum and paralleled the intensity of lung cellular inflammation. Immunohisthochemical studies indicated that RSV-induced expression of MIP-1alpha, one of the most abundantly expressed chemokines, was primarily localized in epithelial cells of the alveoli and bronchioles, as well as in adjoining capillary endothelium. Genetically altered mice with a selective deletion of the MIP-1alpha gene (-/- mice) demonstrated a significant reduction in lung inflammation following RSV infection, compared to control littermates (+/+ mice). Despite the paucity of infiltrating cells, the peak RSV titer in the lung of -/- mice was not significantly different from that observed in +/+ mice. These results provide the first direct evidence that RSV infection may induce lung inflammation via the early production of inflammatory chemokines.     

The chemokine macrophage-inflammatory protein-1 alpha and its receptor CCR1 control pulmonary inflammation and antiviral host defense in paramyxovirus infection

In this work, we explore the responses of specific gene-deleted mice to infection with the paramyxovirus pneumonia virus of mice (PVM). We have shown previously that infection of wild type mice with PVM results in pulmonary neutrophilia and eosinophilia accompanied by local production of macrophage-inflammatory protein-1 alpha (MIP-1 alpha). Here we examine the role of MIP-1 alpha in the pathogenesis of this disease using mice deficient in MIP-1 alpha or its receptor, CCR1. The inflammatory response to PVM in MIP-1 alpha-deficient mice was minimal, with approximately 10-60 neutrophils/ml and no eosinophils detected in bronchoalveolar lavage fluid. Higher levels of infectious virus were recovered from lung tissue excised from MIP-1 alpha-deficient than from fully competent mice, suggesting that the inflammatory response limits the rate of virus replication in vivo. PVM infection of CCR1-deficient mice was also associated with attenuated inflammation, with enhanced recovery of infectious virus, and with accelerated mortality. These results suggest that the MIP-1 alpha/CCR1-mediated acute inflammatory response protects mice by delaying the lethal sequelae of infection.     

Soluble CD40 ligand induces beta-chemokine production by macrophages and resistance to HIV-1 entry

CD40 ligand (CD40L) is a cell surface molecule of CD4(+)T cells that interacts with its receptor CD40 on antigen presenting cells to mediate thymus-dependent humoral immunity and inflammatory reactions. We report here that treating monocyte-derived macrophages (MDM) with a trimeric soluble form of CD40L (CD40LT) induced them to secrete high levels of the beta-chemokines RANTES, MIP-1alpha and MIP-1beta that are ligands for CCR5 and able to inhibit HIV-1 entry. CD40LT inhibited the entry of M-tropic HIV-1 reporter viruses. Furthermore, supernatants obtained from CD40LT-stimulated macrophages protected CEMx174-CCR5 cells from infection by HIV-1(JRFL)reporter virus. The inhibitory activity appeared to be due to beta-chemokines present in the supernatant, since pretreating them with a cocktail of antibodies to RANTES, MIP-1alpha and MIP-1beta neutralized the inhibitory activity of the supernatants. In addition, treating monocytes with CD40LT caused CCR5 and CD4 to be downregulated from the cell surface. In vivo, macrophages activated through CD40 could interfere with HIV replication.     

The role of CD1d in the immune response against Listeria infection

To address the role of CD1d in mucosal immune regulation in bacterial infection, we infected CD1d KO mice with Listeria monocytogenes (Lm). A higher systemic bacterial burden associated with inflammatory lymphocytic infiltrations within the intestine was found in CD1d KO compared with wild type (WT) mice. Lm induced strong IFN-gamma mRNA expression in the liver of WT and the intestine of CD1d KO mice, thus demonstrating the dual, opposing immune activities of IFN-gamma in Lm infection that is dependent on CD1d and/or NKT cells. Analysis of hepatic T cell population demonstrated a reduction of NK1.1(+)TCRbeta+ cells in both mice, followed by recovery only in WT mice. Last, the proportion of alpha4beta1 integrin on lung lymphocytes from CD1d KO was dramatically increased compared with WT mice. Thus, the absence of CD1d resulted in increased susceptibility towards Listeria infection, induced changes in NKT cells, and increased trafficking of alpha4beta1 molecule to inflamed lung.     

Interleukin-1beta stimulates macrophage inflammatory protein-1alpha and -1beta expression in human neuronal cells (NT2-N)

Chemokines are important mediators in immune responses and inflammatory processes of neuroimmunologic and infectious diseases. Although chemokines are expressed predominantly by cells of the immune system, neurons also express chemokines and chemokine receptors. We report herein that human neuronal cells (NT2-N) produce macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha and MIP-1beta), which could be enhanced by interleukin (IL)-1beta at both mRNA and protein levels. The addition of supernatants from human peripheral blood monocyte-derived macrophage (MDM) cultures induced MIP-1beta mRNA expression in NT2-N cells. Anti-IL-1beta antibody removed most, but not all, of the MDM culture supernatant-induced MIP-1beta mRNA expression in NT2-N cells, suggesting that IL-1beta in the MDM culture supernatants is a major factor in the induction of MIP-1beta expression. Investigation of the mechanism(s) responsible for IL-1beta-induced MIP-1alpha and -1beta expression demonstrated that IL-1beta activated nuclear factor kappa B (NF-kappaB) promoter-directed luciferase activity in NT2-N cells. Caffeic acid phenethyl ester, a potent and specific inhibitor of activation of NF-kappaB, not only blocked IL-1beta-induced activation of the NF-kappaB promoter but also decreased IL-1beta-induced MIP-1alpha and -1beta expression in NT2-N cells. These data suggest that NF-kappaB is at least partially involved in the IL-1beta-mediated action on MIP-1alpha and -1beta in NT2-N cells. IL-1beta-mediated up-regulation of beta-chemokine expression may have important implications in the immunopathogenesis of inflammatory diseases in the CNS.     

Macrophage inflammatory protein 1alpha inhibits postentry steps of human immunodeficiency virus type 1 infection via suppression of intracellular cyclic AMP

Primary isolates of human immunodeficiency virus type 1 (HIV-1) predominantly use chemokine receptor CCR5 to enter target cells. The natural ligands of CCR5, the beta-chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES, interfere with HIV-1 binding to CCR5 receptors and decrease the amount of virions entering cells. Although the inhibition of HIV-1 entry by beta-chemokines is well documented, their effects on postentry steps of the viral life cycle and on host cell components that control the outcome of infection after viral entry are not well defined. Here, we show that all three beta-chemokines, and MIP-1alpha in particular, inhibit postentry steps of the HIV-1 life cycle in primary lymphocytes, presumably via suppression of intracellular levels of cyclic AMP (cAMP). Productive HIV-1 infection of primary lymphocytes requires cellular activation. Cell activation increases intracellular cAMP, which is required for efficient synthesis of proviral DNA during early steps of viral infection. Binding of MIP-1alpha to cognate receptors decreases activation-induced intracellular cAMP levels through the activation of inhibitory G proteins. Furthermore, inhibition of one of the downstream targets of cAMP, cAMP-dependent PKA, significantly inhibits synthesis of HIV-1-specific DNA without affecting virus entry. These data reveal that beta-chemokine-mediated inhibition of virus replication in primary lymphocytes combines inhibitory effects at the entry and postentry levels and imply the involvement of beta-chemokine-induced signaling in postentry inhibition of HIV-1 infection.     

Proteolysis of macrophage inflammatory protein-1alpha isoforms LD78beta and LD78alpha by neutrophil-derived serine proteases

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a chemokine that leads to leukocyte recruitment and activation at sites of infection. Controlling chemokine activity at sites of infection is important, since excess accumulation of leukocytes may contribute to localized tissue damage. Neutrophil-derived serine proteases modulate the bioactivity of chemokine and cytokine networks through proteolytic cleavage. Because MIP-1alpha is temporally expressed with neutrophils at sites of infection, we examined proteolysis of MIP-1alpha in vitro by the neutrophil-derived serine proteases: cathepsin G, elastase, and proteinase 3. Recombinant human MIP-1alpha isoforms LD78beta and LD78alpha were expressed and purified, and the protease cleavage sites were analyzed by mass spectrometry and peptide sequencing. Chemotactic activities of parent and cleavage molecules were also compared. Both LD78beta and LD78alpha were cleaved by neutrophil lysates at Thr16-Ser17, Phe24-Ile25, Tyr28-Phe29, and Thr31-Ser32. This degradation was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Incubation of the substrates with individual proteases revealed that cathepsin G preferentially cleaved at Phe24-Ile25 and Tyr28-Phe29, whereas elastase and proteinase 3 cleaved at Thr16-Ser17 and Thr31-Ser32. Proteolysis of LD78beta resulted in loss of chemotactic activity. The role of these proteases in LD78beta and LD78alpha degradation was confirmed by incubation with neutrophil lysates from Papillon-Lefevre syndrome patients, demonstrating that the cell lysates containing inactivated serine proteases could not degrade LD78beta and LD78alpha. These findings suggest that severe periodontal tissue destruction in Papillon-Lefevre syndrome may be related to excess accumulation of LD78beta and LD78alpha and dysregulation of the microbial-induced inflammatory response in the periodontium.