Immune regulation of coronavirus-induced demyelinating encephalomyelitis.
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Adoptive Transfer↗
Biomedical subjects
S A Stohlman
Publications and source records attributed to S A Stohlman.
Exposure to T helper 2 cytokines in vivo before encounter with antigen selects for T helper subsets via alterations in antigen-presenting cell function.
The Th1 subset of CD4+ T cells mediate both delayed-type hypersensitivity (DTH) responses and experimental allergic encephalomyelitis (EAE). Th1 cells are induced by immunization of young adult female and older (> or = 10 wk of age) male SJL mice. By contrast, young adult (< or = 8 wk of age) male mice are characterized by the inability of immunization to induce either a DTH response or EAE, demonstrating a clear sex and age dependence to these Th1-mediated responses in SJL mice. T cell activation in age-matched female and male SJL mice was compared to understand the mechanism(s) of these differential responses. Here, we report that immunization of DTH responder female mice primes for Ag-specific secretion of IFN-gamma but not IL-4 and IL-10. In contrast, immunization of DTH nonresponder male mice primes Ag-specific T cells that secrete IL-4 and IL-10, but not IFN-gamma. Depletion of either IL-4 or IL-10 recovers DTH responsiveness in young adult male mice, demonstrating expansion of Th1 cells in these mice when Th2 cytokines are suppressed. The age- and sex-dependent inability to prime Th1 cells in young male mice is due to the functional absence of a macrophage APC population defined by co-expression of Mac-1 and Mac-3. To determine whether Th2 cytokines directly affect the APC's ability to support the priming of Th1 cells, Mac-3+ APC isolated from naive young male donors, which had been depleted of either IL-4 or IL-10, were transferred into DTH nonresponder males. Induction of DTH responses in these recipients demonstrates that in vivo suppression of Th2 cytokines enables the male-derived Mac-3+ APC to support priming of Th1 responses. These data indicate that, in addition to their regulatory roles in controlling preferential T cell subset expansion, exposure of APC to cytokines in vivo before the initial encounter with Ag may regulate induction of CD4+ T cell subsets.
Aging↗
The JHM strain of mouse hepatitis virus induces a spike protein-specific Db-restricted cytotoxic T cell response.
Cytotoxic T lymphocyte (CTL) activity specific for mouse hepatitis virus (MHV) JHM strain (JHMV or MHV-4) was examined using in vitro stimulated spleen cells derived from immunized C57BL/6 (H-2b) mice. Target cells infected with JHMV were specifically recognized; however, analysis of target cells expressing the virus structural proteins via recombinant vaccinia viruses showed no recognition of the viral nucleocapsid (N), membrane (M), small membrane (sM) or haemagglutinin-esterase (HE) proteins. Only target cells expressing the virus spike (S) protein were recognized. Furthermore, the majority of CTL activity was restricted to target cells expressing the MHC class I Db molecules. Analysis of truncations and deletions of the S protein expressed by recombinant vaccinia viruses and peptide coated targets identified a single antigenic epitope, aa 510-518, conforming to the Db binding motif. These amino acids are contained within a domain deleted from a number of strains of mouse hepatitis virus, suggesting a role for immune pressure. To determine the potential for CTL specific for an epitope(s) within a non-structural protein, 24 CTL lines were established and characterized. No evidence for the induction of non-specific CTL activity or virus-specific CTL restricted to an epitope in a non-structural protein was obtained. These data indicate that the predominant CTL activity in JHMV-infected C57BL/6 mice is Db restricted and specific for a single epitope contained within aa 510-518 of the S protein.
Amino Acid Sequence↗
Specificity of the H-2 L(d)-restricted cytotoxic T-lymphocyte response to the mouse hepatitis virus nucleocapsid protein.
Cytotoxic T lymphocytes provide protection against persistent infection of the central nervous system by the JHM strain of mouse hepatitis virus. In BALB/c (H-2d) mice, the dominant response is directed against an Ld-restricted peptide in the nucleocapsid protein (APTAGAFFF). Characterization of the fine specificity of this response revealed that the predicted anchor residues at positions 2 and 9 were the most critical for class I binding. Amino acids at positions 7 and 8 were identified as T-cell receptor contact residues. Virus-induced cytotoxic T lymphocytes to other Ld motif-containing nucleocapsid peptides were not detected, despite the identification of two epitopes with reduced Ld affinity. These data suggest that mutations within four residues of the dominant epitope could contribute to the persistence of the JHM strain of mouse hepatitis virus.
Amino Acid Sequence↗
Self-antigen-induced Th2 responses in experimental allergic encephalomyelitis (EAE)-resistant mice. Th2-mediated suppression of autoimmune disease.
Immunization of a limited number of rodent strains with central nervous system-derived Ags induces experimental allergic encephalomyelitis (EAE). In contrast to susceptible female SJL mice, age-matched males are resistant to actively induced EAE. The ability of immunization with neuroAg to induce Ag-specific T cell activation in resistant male mice was examined. Ag-specific T cell proliferation was found following immunization of both male and female SJL mice. Draining lymph node cytokine mRNA patterns demonstrated that immunization of EAE-resistant male mice resulted in a Th2-type pattern. By contrast, immunization of EAE-susceptible female mice resulted in a Th1-type pattern. Priming of Th1- and Th2-type responses was confirmed by analysis of cytokines secreted following Ag-specific proliferation. In contrast to the transfer of myelin basic protein (MBP)-specific Th1-type T cells derived from female mice, which induced acute and relapse EAE, transfer of MBP-specific Th2-type T cells derived from male mice resulted in no clinical or histologic evidence of EAE. A mixture of MBP-specific Th1 and Th2 type cells was transferred to naive recipients to determine if the neuroAg-specific Th2-type cells exerted a regulatory influence on EAE. Acute disease was partially eliminated and relapses were completely eliminated in these recipients. Analysis of spinal cords showed the presence of both Th1 and Th2 cytokine mRNAs. These data are consistent with both the ability of Th2-type cells to suppress autoimmunity and a homeostatic mechanism of T cell regulation based on the cross-regulation of Th1 and Th2 cells in the maintenance of peripheral tolerance.
Animals↗
Macrophages regulate induction of delayed-type hypersensitivity and experimental allergic encephalomyelitis in SJL mice.
The relationship between the delayed-type hypersensitivity (DTH) response and susceptibility to experimental allergic encephalomyelitis (EAE) was examined using a unique age-dependent defect in the DTH response in an EAE-susceptible mouse strain. Young adult male SJL mice ( < 10 weeks of age) are defective in DTH responses following immunization with a variety of soluble antigens. By contrast, they respond to antigens applied to the skin, demonstrating a normal contact sensitivity response. In this report, we show that the non-responder male SJL are also unable to mount a DTH response to soluble neuroantigens or neuroantigens emulsified in complete adjuvant, and are additionally resistant to actively induced EAE. This contrasts with the DTH response in older males ( > 10 weeks of age) and young adult females (6 weeks of age), which are both DTH responders and susceptible to EAE. By contrast, all three groups are susceptible to EAE mediated by the transfer of activated effector T cells, suggesting that the defect in young adult males is in the induction of effectors. Furthermore, transfer of a macrophage population from female responders to young male non-responders mediates the induction of both DTH responsiveness and EAE susceptibility. The phenotype of this antigen-presenting cell is (I-A+, Mac-1+, Mac-2-, Mac-3+), identical to the phenotype of the macrophage regulating DTH responsiveness in this strain of mice. These data are consistent with the hypothesis that a defect in this cell inhibits induction of both CD4+Th1 DTH and EAE effector T cells.
Adjuvants, Immunologic↗
Transcription and translation of proinflammatory cytokines following JHMV infection.
Infection with JHMV results in the transcriptional activation of two host cell genes encoding proinflammatory cytokines, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta. Analysis of irradiated mice showed that IL-1 beta mRNA accumulation in the central nervous system was predominantly derived from the mononuclear infiltrate. By contrast, accumulation of TNF-alpha mRNA was unaffected by immunosuppression, suggesting that resident cells were the source of this cytokine. Infected mice were treated with anti-TNF antibody to determine if TNF-alpha contributed to either the encephalomyelitis or demyelination associated with JHMV infection. Surprisingly, neither the cellular infiltrate nor demyelination were affected. In vitro analysis showed that IL-1 beta but not TNF was secreted from JHMV infected macrophages. The absence of TNF secretion is due to a block in translation of the TNF mRNA which accumulates during infection.
Animals↗
Mouse hepatitis virus-specific cytotoxic T lymphocytes protect from lethal infection without eliminating virus from the central nervous system.
Acute infection of the central nervous system by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces nucleocapsid protein specific cytotoxic T lymphocytes (CTL) not found in the periphery (S. Stohlman, S. Kyuwa, J. Polo, D. Brady, M. Lai, and C. Bergmann, J. Virol. 67:7050-7059, 1993). Peripheral induction of CTL specific for the nucleocapsid protein of JHMV by vaccination with recombinant vaccinia viruses was unable to provide significant protection to a subsequent lethal virus challenge. By contrast, the transfer of nucleoprotein-specific CTL protected mice from a subsequent lethal challenge by reducing virus replication within the central nervous system, demonstrating the importance of the CTL response to this epitope in JHMV infection. Transfer of these CTL directly into the central nervous system was at least 10-fold more effective than peripheral transfer. Histological analysis indicated that the CTL reduced virus replication in ependymal cells, astrocytes, and microglia. Although the CTL were relatively ineffective at reducing virus replication in oligodendroglia, survivors showed minimal evidence of virus persistence within the central nervous system and no evidence of chronic ongoing demyelination.
Animals↗
Tumor necrosis factor expression during mouse hepatitis virus-induced demyelinating encephalomyelitis.
Neutralizing anti-tumor necrosis factor alpha (TNF-alpha) antibody treatment of mice infected with the neurotropic JHMV strain of mouse hepatitis virus showed no reduction of either virus-induced encephalomyelitis or central nervous system demyelination. TNF-alpha-positive cells were present in the central nervous system during infection; however, TNF-alpha could not be colocalized with JHMV-infected cells. In vitro, TNF-alpha mRNA rapidly accumulated following JHMV infection; however, no TNF-alpha was secreted because of inhibition of translation. Both live and UV-inactivated virus inhibited TNF-alpha secretion induced by lipopolysaccharide. These data show that TNF-alpha is not secreted from infected cells and indicate that if contributes to either JHMV-induced acute encephalomyelitis nor primary demyelination.
Animals↗
Neuropathogenicity of mouse hepatitis virus JHM isolates differing in hemagglutinin-esterase protein expression.
The hemagglutinin-esterase (HE) protein of mouse hepatitis virus (MHV) is an optional envelope protein present in only some MHV isolates. Its expression is regulated by the copy number of a UCUAA pentanucleotide sequence present in the leader sequence of the viral genomic RNA. The functional significance of this viral protein so far is not clear. In this report, we compared the neuropathogenicity of two MHV isolates, JHM(2) and JHM(3), which express different amounts of HE protein. Intracerebral inoculation of these two viruses into C57BL/6 mice showed that JHM(2), which expresses an abundant amount of HE protein, was more neurovirulent than JHM(3), which expresses very little HE. Histopathology showed that early in infection, JHM(2) infected primarily neurons, while JHM(3) infected mainly glial cells. JHM(3) eventually infected neurons and caused a delayed death relative to JHM(2)-infected mice, suggesting that the progression of JHM(3) infection in the central nervous system was slower than JHM(2). In vitro infection of JHM(3) in primary mixed glial cell cultures of astrocyte-enriched cultures yielded higher virus titers than JHM(2), mimicking the preferential growth of JHM(3) in glial cells in vivo. These findings suggest that the reduced neuropathogenicity of JHM(3) may correlate with its preferential growth in glial cells. Sequence analysis showed that the S genes of these two viruses are identical, thus ruling out the S gene as the cause of the difference in neuropathogenicity between these two viruses. We conclude that the HE protein contributes to viral neuropathogenicity by influencing either the rate of virus spread, viral cell tropism or both.
Acetylesterase↗
Coronavirus translational regulation: leader affects mRNA efficiency.
Cells infected with the murine coronavirus, mouse hepatitis virus (MHV), show decreased host protein synthesis concomitant with an increase in viral protein synthesis. We examined the in vitro translation property of the conserved MHV 5'-leader RNA sequence by constructing chimeric mRNAs in which the 72-nt 5'-leader of M protein mRNA (A59 strain) was positioned upstream of the human alpha-globin coding region in a T7 expression vector. Synthetic 5'-capped transcripts of these mRNA constructs were translated in cell-free extracts prepared from uninfected and MHV-infected murine DBT cells. Nonviral mRNAs translated readily in both uninfected and infected cell-free extracts. By contrast, replacement of the human alpha-globin 5'-untranslated region (UR) with the MHV 5'-leader increased translation ca. three- to fourfold in cell-free extracts from MHV-infected cells versus translation in extracts from uninfected cells. Chimeric globin mRNA containing the reverse complementary sequence of the viral leader RNA in the 5'-UR showed no such increase in translation, indicating sequence specificity for the effect. A 13-nt region (-UCUAAUCCAAACA-) immediately proximal to the start codon was found to be important for the increased translation of the MHV leader-containing mRNAs. These data indicate that the apparent down-regulation of host translation is not primarily due to an inhibition of host translation but also involves a significant stimulation of viral translation in cis by a structural feature of the MHV 5'-leader RNA sequence in conjunction with a virus-specified or virus-induced factor.
Animals↗
Cytotoxic T cell repertoire selection. A single amino acid determines alternative class I restriction.
CTL responses are governed by intracellular Ag processing, affinity of peptides for MHC class I molecules, and the T cell repertoire. In this report we demonstrate that a class I Dd-restricted 10-mer CTL epitope within the gp160 envelope glycoprotein of HIV-1 strain IIIB (residues 318-327) contains a 9-amino acid peptide (residues 319-327), which efficiently binds to both the Dd and Ld class I molecules in vitro. The potential for broadening the naturally limited CTL response to include presentation on the Ld class I molecules in vivo was examined using a minigene-based vaccine strategy to insure cytosolic expression of "preprocessed" forms of the gp160 epitope. Immunization with recombinant vaccinia viruses (vac) expressing either the gp160 10 mer or 9 mer, both including an initiation methionine (M318-327 and M319-327, respectively), induced predominantly Dd-restricted CTL specific for native gp160. By contrast, recombinant vac expressing eight gp160 amino acids (M320-327) generated predominantly Ld-restricted CTL which are specific for synthetic gp160 peptides but not native gp160. The ability to induce Ld-restricted CTL suggests that the absence of an Ld-restricted response to native gp160 cannot be attributed to a limited T cell repertoire, but to inefficient processing of gp160 for presentation on Ld. The switch in class I restriction, controlled by a single amino acid within one epitope, demonstrates that nonanchor residues have a profound effect on differential MHC restriction and CTL induction. Thus, minigene-based vaccines expressing minimal epitopes may be useful in inducing a more heterogeneous CTL response than previously appreciated.
Amino Acid Sequence↗
TCR cross-linking induces CTL death via internal action of TNF.
TCR-mediated stimulation can result in either activation or apoptosis, raising the question of what the mechanisms of these opposing effects might be. Here we demonstrate that microinjection of Ab specific for TNF inhibits activation-induced cell death, implicating TNF in anti-CD3-induced apoptosis. Induction of CTL in the presence of antisense TNF oligodeoxynucleotides inhibits TNF secretion and cell death after stimulation with anti-CD3. Incubation of cells with IL-2 for 48 h before stimulation protects from anti-CD3-induced death, whereas the presence of IL-2 during anti-CD3 treatment is without effect.
Animals↗
Location of antibody epitopes within the mouse hepatitis virus nucleocapsid protein.
Thirteen monoclonal antibodies (Mab) specific for the nucleocapsid (N) protein of mouse hepatitis virus were mapped using a panel of carboxy-terminal N protein truncations expressed by recombinant vaccinia viruses. All of the Mab recognized both native protein and full-length N protein expressed in this vector by both Western blot and enzyme-linked immunoabsorbent assays (ELISA), indicating that they recognized linear epitopes. The results obtained by both Western blot and ELISA for binding to the truncated N proteins coincide for seven of the Mab tested. The linear epitopes recognized localize to four domains dispersed between amino acids 171 and 196, 231 and 277, and 374 and 455. The epitopes for six Mab were localized to domains comprising 29 amino acids or less as determined by ELISA. Seven Mab showed different reactivity patterns in Western blot versus ELISA, suggesting binding may be influenced by local conformation. Therefore, the fine specificity of these Mab could not be determined with certainty. These data represent the first determination of antibody binding domains within the mouse hepatitis virus N protein which forms the viral helical nucleocapsids and appears to perform a number of regulatory functions during virus replication.
Animals↗
Modulation of cellular macromolecular synthesis by coronavirus: implication for pathogenesis.
Infection with the murine coronavirus strain JHM decreases cell surface expression of major histocompatibility complex class I antigens. Northern blots showed that JHM virus infection rapidly reduced the level of actin mRNA, whereas the levels of major histocompatibility complex class I and tubulin mRNAs were reduced only slightly. By contrast, the mRNA levels of interleukin 1 beta, colony-stimulating factor 1 receptor, and tumor necrosis factor alpha increased following infection.
Actins↗
The detection and characterization of multiple hemagglutinin-esterase (HE)-defective viruses in the mouse brain during subacute demyelination induced by mouse hepatitis virus.
It has previously been shown that passive immunization with antibodies specific for the hemagglutinin-esterase (HE) protein of mouse hepatitis virus (MHV) prevents acute lethal encephalitis, resulting in a subacute and chronic demyelination in mice infected with JHM(2), an isolate of the neurotropic JHM strain of MHV. To determine possible genetic changes occurring during infection, viruses were isolated from the brain of infected mice at various time points after infection and examined for the patterns of their structural proteins. The results showed that the sizes and expression levels of the viral spike, membrane, and nucleocapsid proteins were constant among 161 virus isolates throughout the infection. In contrast, most of the viruses isolated later in infection did not synthesize HE protein. This finding suggests that the HE gene expression is extremely variable and is preferentially lost during prolonged viral infections. In contrast, when viruses were passaged in tissue culture, no significant accumulation of HE protein-defective mutants was observed, suggesting that the accumulation of HE protein-defective mutants in infected animals was most likely the result of the positive selection for these mutants during the subacute and chronic infection. The genetic defects of HE gene in these mutants were characterized by cloning, sequencing, and in vitro translation of HE genes. Most of the mutations in the HE protein-defective mutants consisted of deletions of various lengths at different sites within the HE-coding region, resulting in the change of the reading frame and early termination. However, most of the truncated HE proteins were not detected in the infected cells. Since viruses from different mice exhibited different types of defects, the HE mutations probably occurred de novo in the brain. These results demonstrated the exceptionally rapid selection of HE-defective mutants during viral infection of mice and suggest that their selection may be related to the neuropathogenicity or persistence of MHV.
Animals↗
A spike protein-dependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry.
Previous studies have shown that some mouse strains are resistant to mouse hepatitis virus (MHV) infection despite the presence of functional viral receptors (K. Yokomori and M. M. C. Lai, J. Virol. 66, 6931-6938, 1992). To determine the molecular requirement for MHV infection, several cell lines derived from both susceptible and resistant mouse strains were tested for their ability to support infection by two different MHV strains, JHM and A59. Most of the cell lines tested, including ones from susceptible mouse strains, exhibited selective resistance to JHM, but were susceptible to A59, suggesting that there is an additional cellular factor(s) discriminating JHM from A59 infection. Both RNA and protein syntheses of JHM were inhibited in the resistant cells; however, transfection of JHM genomic RNA into these cells led to the production of infectious virus, suggesting that the restriction step(s) is during an early stage of viral replication cycle. The mRNA for the MHV receptor (the murine homolog of the carcinoembryonic antigen) is expressed in all cell lines, and expression in COS cells of the receptor isolated from the resistant murine cell lines rendered the COS cells susceptible to both A59 and JHM infections. Furthermore, the transfection of additional MHV receptors into the resistant cells did not overcome the resistance to JHM virus infection. These results suggested that the viral receptor is functional; nevertheless, the JHM infection is restricted at an early step of infection in these cells. The study of the growth properties of the various recombinant viruses between A59 and JHM revealed that one of the viral genes determining viral replication in these cell lines is the S protein gene; thus, the second factor required for viral infection may interact directly or indirectly with the S protein at an early step of infection. Taken together, these studies suggest that expression of a functional viral receptor is not sufficient to establish MHV infection, and that an additional factor(s) is required for an early step of viral infection, possibly during virus entry.
Animals↗
Establishment of MHV-A59 S protein specific cytotoxic T lymphocyte clones.
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Animals↗