Lymphocyte phenotype subsets in the cerebrospinal fluid of normal horses and horses with equine protozoal myeloencephalitis.

The percentages of T-lymphocytes, lymphocyte subsets CD4+ and CD8+ T-cells, and lymphocyte adhesion molecule CD11a/CD18 were determined in the peripheral blood and cerebrospinal fluid (CSF) of seven normal horses and four horses with equine protozoal myeloencephalitis (EPM) using flow cytometry. There was a greater percentage of CD5+ cells in the CSF (79.0%) than in peripheral blood (67.0%), although this did not achieve statistical significance. Furthermore, the lymphocyte population in CSF comprises a significantly greater (P = .01) percentage of CD8+ T-cells, resulting in a decrease of the CD4/CD8 ratio. Lymphocyte phenotype subsets in peripheral blood or CSF from horses affected with EPM did not differ from normal horses, although CD5+ T-lymphocytes were seen in significantly greater numbers in the CSF of EPM-affected horses (93.2%) than in normal horses (79.0%).

Transforming growth factor beta concentrations and interferon gamma responses in cerebrospinal fluid of horses with equine protozoal myeloencephalitis.

The following experiment was performed to test the hypothesis that transforming growth factor beta (TGF-beta) concentration varies in the cerebrospinal fluid and serum of horses with EPM and to determine if cerebrospinal fluid (CSF) alters the interferon-gamma (IFN-gamma) rersponse of equine peripheral blood mononuclear cells (PBMCs). The concentration of transforming growth factor-beta (TGF-beta2) was investigated in the serum and cerebrospinal fluid (CSF) of 18 horses (9 normal, 9 affected with equine protozoal myeloencephalitis [EPM]). The TGF-beta2 assay was validated in a group of 6 normal horses. Intra-assay variability was 4.7%, and interassay variability was 10.7%. The slope of the curve of the unknown samples of various volumes demonstrated parallelism with a curve developed using equal volumes of assay kit standard. Assay of normal and EPM-affected horses found that TGF-beta2 was present in both the serum and CSF of all animals. However, the concentration of TGF-beta2 in the CSF was less (P = 0.03) in EPM-affected horses (144 pg/ml) than in normal horses (256 pg/ml). In addition, the effect of CSF from normal and EPM-affected horses on the production of interferon-gamma (IFN-gamma) by PHA-P stimulated PBMCs from normal horses was investigated using a bioassay. It was found that CSF from normal and EPM-affected horses enhanced IFN-gamma activity from PHA-P stimulated peripheral blood mononuclear cells (P < or = 0.05); however, the response to CSF from EPM-affected horses was no different than the response to CSF from normal horses. Treatment of cells with anti-TGF-beta2 monoclonal antibodies slightly increased the response when co-incubated with CSF from normal horses, and slightly decreased it when co-incubated with CSF from EPM-affected horses. These differences, however, did not achieve statistical significance (P > 0.05). Results of this study indicated that production of TGF-beta2 is altered in horses with EPM, and that CSF appears to contain substances which alter the inflammatory reaction to plant lectins. These findings confirm the immunomodulatory properties of CSF and suggest new techniques for future research regarding the pathophysiology of EPM.

Loss of interferon alpha and interferon tau-induced antiviral protection in interferon regulatory factor-2 DNA-binding domain dominant negative mutants.

The role of the interferon regulatory factory (IRF) family of transcription factors in regulation of interferon alpha and interferon tau antiviral activity was investigated using a dominant negative mutant of IRF-2. The IRF-2 DNA binding domain (DBD), without the C-terminal regulatory region, was stably transfected into myeloid U937 cells. Expression of the IRF-2 DBD resulted in an increase in constitutive 2'5' oligoadenylate synthetase (OAS) levels, indicative of an active repressive mechanism, but was not sufficient to protect cells from challenge with vesicular stomatitis virus. Treatment of the DBD clones with interferons alpha A and tau failed to upregulate 2'5' OAS expression and did not elicit an antiviral response. While interferon alpha A was more sensitive than interferon tau to the inhibitory effects of the IRF-2 DBD, IRF-mediated gene induction is involved in successful interferon alpha and tau-induced anti-VSV activity.

Regulation of macrophage interleukin-6 (IL-6) and IL-10 expression by prostaglandin E2: the role of p38 mitogen-activated protein kinase.

Prostaglandin E2 (PGE2) regulates production of a wide array of cytokines. We have found that PGE2 can upregulate the levels of both interleukin-10 (IL-10) and IL-6 produced by activated murine macrophages, but the molecular pathways leading to their augmentation differ. Synthesis of IL-10 in response to PGE2 is dependent on p38 MAP kinase activity, whereas synthesis of IL-6 is not. Evidence to support this derives from two experimental approaches. First, we established that PGE2 is effective in elevating IL-10 levels only when it is added to cells in which p38 kinase has been activated. In contrast, PGE2 can augment IL-6 levels regardless of whether or not p38 kinase is active. Second, we showed that inhibitors that are selective for p38 kinase prevent the IL-10 response to PGE2 but not the IL-6 response. We found that p38 kinase inhibitors are able to inhibit IL-6 production in activated macrophages, but this occurs primarily as a result of their concurrent inhibition of cyclooxygenase-2 and endogenous PGE2 synthesis. These results indicate that macrophage IL-10 and IL-6 expression is differentially regulated by PGE2 and p38 MAP kinase in murine inflammatory macrophages.

An IFN regulatory factor-2 DNA-binding domain dominant negative mutant exhibits altered cell growth and gene expression.

In order to study interferon regulatory factor (IRF) family mediation of cell growth regulation, we established U937 cell lines stably transfected with a truncated form of IRF-2 lacking the transcriptional repressor domain. The truncated IRF-2 contained the DNA binding domain (DBD) and bound the ISRE. Phenotypically, the IRF-2 DBD transfectants exhibited reduced cell growth, altered morphology and increased cell death. Consistent with alterations in growth characteristics, the IRF-2 DBD transfectants constitutively expressed higher levels of the cyclin dependent kinase inhibitor p21WAF1/Cip1 than did control clones. The level of p21WAF1/Cip1 expression was positively correlated with the level of DBD expressed, as well as with the level of growth inhibition in these clones. DBD expression also correlated with expression of other members of the growth regulatory complex, cyclin dependent kinase 2 and cyclin A, but not proliferating cell nuclear antigen. These results imply active repression by IRF-2 to keep p21WAF1/Cip1 transcriptionally silent.

Increased T cell cytotoxicity by Betathine-induced upregulation of TNFalpha.

Betathine (BT) is a low molecular weight disulfide that has previously been shown to exhibit in vivo antitumor activity in murine myeloma and melanoma models. We have shown that BT treatment of both human T cells and monocytes is associated with an increase in surface tumor necrosis alpha (TNFalpha) expression. Further, in T cells and monocytes that have been stimulated with PMA and ionomycin, the addition of BT results in a dose and time dependent increase in the percentage of high TNFalpha-expressing cells. Unlike TNFalpha upregulation produced by the commonly used thiol antioxidant N-acetyl-L-cysteine (NAC), the BT-induced increase in TNFalpha is observed consistently in different donors. This increase in surface TNFalpha is associated with elevated levels of TNFalpha mRNA. In addition, expression of TNFalpha receptor I is also significantly enhanced by BT treatment. The upregulation of surface TNFalpha by BT has functional consequences, in that, BT-treated T cells exhibit enhanced cytotoxic activity. Thus, increased TNFalpha expression may be one mechanism responsible for the antineoplastic activity of BT.

Functional characterization of monoclonal antibodies to interferon-tau.

Interferon tau (IFNtau) produces an array of biological effects, including antiluteolytic, antiviral, antiproliferative and immunomodulatory activities, without the consequent cytotoxicity associated with other type I IFNs. Four anti-IFNtau monoclonal antibodies (MAbs) have been characterized by determining regional epitopes and observation of their effects on IFNtau binding, antiviral and antiproliferative activity. Using an enzyme-linked immunoadsorbent assay (ELISA) developed against six overlapping synthetic peptides representing the entire linear sequence of IFNtau, three antibodies, HL-98, HL-100 and HL-127, were found to react with the carboxy terminal peptide, while HL-129 bound the penultimate amino terminal peptide. Binding studies indicated that MAbs directed against either region could effectively inhibit the binding of alkaline phosphatase labeled IFNtau to cells expressing type I IFN receptors. While only two of the MAbs significantly reversed IFNtau-induced growth inhibition, the antiviral activity of IFNtau was significantly inhibited by MAbs that bound the amino and carboxy termini, confirming the functional importance of these domains in the binding and subsequent activity of IFNtau.

Potent anti-feline immunodeficiency virus and anti-human immunodeficiency virus effect of IFN-tau.

Ovine IFN-tau is a newly described protein related to IFN-alpha that is responsible for maternal recognition of pregnancy in sheep. It has been shown to exhibit potent antiviral and antiproliferative activity. To determine its antiviral activity against feline immunodeficiency virus (FIV) and HIV, the activity of the RNA-dependent DNA polymerase, reverse transcriptase, was assayed in FIV- and HIV-infected feline and human PBL treated with IFN-tau. Significant dose-dependent inhibition of reverse transcriptase activity by IFN-tau was detected by day 6 of culture and was maintained through the peak of virus replication. In addition, production of the FIV core protein, p25, was blocked by IFN-tau. Both the amino- and carboxyl-terminal regions of IFN-tau, as identified by synthetic peptides, appear to be involved in its antiretroviral activity. Comparison of the anti-HIV activities of IFN-tau and recombinant human IFN-alpha2 (rHuIFN-alpha2) indicated that while rHuIFN-alpha2 was toxic to cells at 10,000 U/ml, IFN-tau antiretroviral activity was not associated with a decrease in either cell viability or immunologic reactivity. Thus, IFN-tau displayed potent anti-FIV and anti-HIV activity without the cytotoxicity associated with high concentrations of rHuIFN-alpha2.

Differential recognition of the type I interferon receptor by interferons tau and alpha is responsible for their disparate cytotoxicities.

Interferon tau (IFN tau), originally identified as a pregnancy recognition hormone, is a type I interferon that is related to the various IFN alpha species (IFN alpha s). Ovine IFN tau has antiviral activity similar to that of human IFN alpha A on the Madin-Darby bovine kidney (MDBK) cell line and is equally effective in inhibiting cell proliferation. In this study, IFN tau was found to differ from IFN alpha A in that is was > 30-fold less toxic to MDBK cells at high concentrations. Excess IFN tau did not block the cytotoxicity of IFN alpha A on MDBK cells, suggesting that these two type I IFNs recognize the type I IFN receptor differently on these cells. In direct binding studies, 125I-IFN tau had a Kd of 3.90 x 10(-10) M for receptor on MDBK cells, whereas that of 125I-IFN alpha A was 4.45 x 10(-11) M. Consistent with the higher binding affinity, IFN alpha A was severalfold more effective than IFN tau in competitive binding against 125I-IFN tau to receptor on MDBK cells. Paradoxically, the two IFNs had similar specific antiviral activities on MDBK cells. However, maximal IFN antiviral activity required only fractional occupancy of receptors, whereas toxicity was associated with maximal receptor occupancy. Hence, IFN alpha A, with the higher binding affinity, was more toxic than IFN tau. The IFNs were similar in inducing the specific phosphorylation of the type I receptor-associated tyrosine kinase Tyk2, and the transcription factors Stat1 alpha and Stat2, suggesting that phosphorylation of these signal transduction proteins is not involved in the cellular toxicity associated with type I IFNs. Experiments using synthetic peptides suggest that differences in the interaction at the N terminal of IFN tau and IFN alpha with the type I receptor complex contribute significantly to differences in high-affinity equilibrium binding of these molecules. It is postulated that such a differential recognition of the receptor is responsible for the similar antiviral but different cytotoxic effects of these IFNs. Moreover, these data imply that receptors are "spare'' with respect to certain biological properties, and we speculate that IFNs may induce a concentration-dependent selective association of receptor subunits.

Predicted structural motif of IFN tau.

Ovine interferon tau (IFN tau) is a type I interferon that was originally identified as ovine trophoblast protein and is associated with the maternal recognition of pregnancy in sheep. Additionally, IFN tau possesses potent antiviral and antiproliferative activity without the corresponding toxicity found in known IFN alpha s. Structure-function studies with synthetic peptides have identified three discontinuous functional sites on the protein that are involved in receptor interaction and biological activity. However, the structural relationship of these regions is unknown. Therefore, a model relationship of these regions is unknown. Therefore, a model of the 3-D structure of IFN tau would be useful in interpretation of existing data and the design of future structure-function studies. Combining information from circular dichroism (CD) of both the full length recombinant IFN tau and synthetic peptides representing regions of the IFN tau molecule, with sequence homology of IFN tau to IFN beta, a protein of known 3-D structure, we have constructed a model of IFN tau using distance geometry and energy minimization methods. The most striking feature of this model is that functionally active domains of IFN tau, discontinuous in the primary structure, are localized to one side of the molecule and found to be spatially contiguous. This observation is consistent with multiple binding sites on IFN tau interacting simultaneously with the IFN tau receptor.

Structure/function studies with interferon tau: evidence for multiple active sites.

A novel interferon (IFN), called IFN-tau (IFN-tau), has recently been discovered and has been shown to be a pregnancy recognition hormone. Unlike known IFNs, however, IFN-tau exhibits high antiviral and antiproliferative activity without cytotoxicity. The structural basis for IFN-tau function has been examined using six overlapping synthetic peptides corresponding to the entire ovine (Ov) IFN-tau sequence. Four peptides representing amino acids 1-37, 62-92, 119-150, and 139-172 inhibited OvIFN-tau antiviral activity in a dose-dependent manner. Polyclonal antipeptide antisera directed against the same four peptides blocked OvIFN-tau binding and antiviral activity, confirming the specificity of the peptide competitions. Because IFN-tau and IFN-alpha both interact with the type I IFN receptor, peptide inhibition of bovine and human IFN alpha activity was also determined. Of importance, only three peptides, OvIFN-tau (62-92), (119-150), and (139-172) inhibited IFN-alpha antiviral activity. The amino-terminal IFN-tau peptide, OvIFN-tau(1-37), was not inhibitory. These data suggest that the internal and carboxy-terminal reactive domains of IFN-tau may interact with a common type I IFN site on the receptor, while the amino terminus interacts with a site that elicits activity unique to OvIFN-tau. Finally, the antiproliferative activity of OvIFN-tau was localized primarily to the broad carboxy-terminal region, with OvIFN-tau(119-150) being the most effective inhibitor of OvIFN-tau-induced reduction of cell proliferation. Thus, multiple domains of IFN-tau have functional significance.(ABSTRACT TRUNCATED AT 250 WORDS)

Opsonization of Anaplasma marginale mediated by bovine antibody against surface protein MSP-1.

Antibody from cattle immunized with purified major surface protein-1 (MSP-1) was demonstrated to significantly enhance phagocytosis of Florida strain Anaplasma marginale by bovine macrophages in vitro. Serum immunoglobulin from individual MSP-1 immunized, protected cattle varied in ability to promote phagocytosis, however all sera were significantly opsonic as compared with sera from sham immunized control cattle.

Agonist properties of a microbial superantigen peptide.

Staphylococcal enterotoxin A (SEA) binds to class II major histocompatibility complex (MHC) molecules and stimulates monocytes to produce tumor necrosis factor alpha (TNF alpha) and interleukin one (IL-1). We have examined the monocyte stimulatory activity of individual synthetic peptides encompassing the entire sequence of the SEA molecule. Only one peptide, SEA(121-149), induced both TNF alpha and IL-1 production at a concentration as low as 30 microM. Consistent with its effects on monocyte function, SEA(121-149) was shown to bind directly to class II MHC molecules on the surface of both monocytes and B cells, and its binding was inhibited specifically by native SEA. Further, polyclonal antibody to SEA(121-149) inhibited induction of TNF alpha by both SEA and toxic shock syndrome toxin one. Thus, we have identified SEA(121-149) as a peptide agonist of SEA monocyte stimulatory activity.

Identification of binding domains on the superantigen, toxic shock syndrome-1, for class II MHC molecules.

Toxic shock syndrome toxin-1 (TSST-1) is a member of the staphylococcal enterotoxin superantigen family. In order to determine the regions on the TSST-1 molecule involved in binding to class II MHC, seven overlapping peptides of the entire TSST-1 molecule were synthesized and tested for their ability to compete with 125I-TSST-1 for binding to class II MHC on murine A20 cells and HLA on Raji cells. Peptides corresponding to N-terminal amino acid residues 39 through 68 and C-terminal residues 155 through 194 competed with 125I-TSST-1 for binding to class II MHC. Also, binding studies with class II MHC beta-chain peptides indicate that regions encompassed by I-A beta b(30-60) and I-A beta b(60-90) are binding regions for TSST-1. Thus, we have identified binding domains on the TSST-1 molecule for class II MHC molecule receptors on antigen presenting cells.

T-cell antigen receptor binding sites for the microbial superantigen staphylococcal enterotoxin A.

We have examined the interaction of the microbial superantigen staphylococcal enterotoxin A (SEA) with peptides corresponding to overlapping regions of the T-cell antigen receptor beta chain variable region V beta 3. SEA is known to stimulate murine T cells bearing certain V beta elements, among them V beta 3. Five peptides were synthesized representing amino acids 1-24, 20-44, 39-60, 57-77, and 74-95 of V beta 3. We demonstrate here that soluble V beta 3-bearing beta chains can bind to a complex of SEA and major histocompatibility complex class II and that the synthetic peptide V beta 3-(57-77) blocked this interaction. The peptide V beta 3-(57-77) also inhibited SEA-induced interferon-gamma production and SEA-induced proliferation of B10.BR spleen cells. Conversely, the peptide corresponding to amino acids 57-77 of V beta 8.2, a V beta element that is not recognized by SEA, decreased staphylococcal enterotoxin C-2-induced proliferation but did not affect SEA-induced proliferation. The peptide inhibition of SEA-induced function was due at least in part to inhibition of V beta 3-bearing T-cell activity, since the percentage of T cells reactive with an anti-V beta 3 monoclonal antibody was significantly reduced by V beta 3-(57-77). These data suggest that the region of V beta 3 encompassing amino acids 57-77 is an area that displays the appropriate sequence and conformation for binding of the SEA molecule and blocking of the resultant interaction with the T-cell antigen receptor.

Mapping of multiple binding domains of the superantigen staphylococcal enterotoxin A for HLA.

Multiple binding sites on the staphylococcal enterotoxin A (SEA) molecule which interact with class II MHC Ag have been suggested by previous studies comparing SEA binding with that of another superantigen, toxic shock syndrome toxin-1. Using the synthetic peptide approach we have identified multiple regions of the SEA molecule which are responsible for binding to HLA Ag on Raji cells. Overlapping peptides were synthesized corresponding to the complete amino acid sequence of SEA: SEA(1-45), SEA(39-66), SEA(62-86), SEA(83-104), SEA(102-124), SEA(121-149), SEA(146-173), SEA(166-193), SEA(187-217), and SEA(211-233). Like the native SEA molecule, all of the peptides exhibited relatively high beta-sheet and low alpha-helical structure as determined by circular dichroism spectroscopy. A direct competition assay was employed with peptide blockage of 125I-SEA binding to MHC Ag. SEA(1-45), SEA(39-66), SEA(62-86), and SEA(121-149) but none of the other peptides blocked binding to Raji cells. The relative potency of the peptides in blocking SEA binding was determined with SEA(39-66) much greater than SEA(1-45) = SEA(62-86) = SEA(121-149). Peptide competition was seen at concentrations as low as 55 microM. Further, antibodies were produced to all of the peptides and tested for their ability to bind to SEA and inhibit SEA binding to HLA. Consistent with the direct inhibition of binding, antisera to SEA(1-45), SEA(39-66), and SEA(62-86) reduced the ability of SEA to bind Raji cells, whereas, antisera to the remaining peptides failed to block binding. The data suggest that the binding of the superantigen SEA to MHC molecules involves several N-terminal regions on SEA as well as an additional internal domain. This allows for the presence of multiple binding sites in an extended N-terminal region of the SEA molecule or a discontinuous binding epitope.

Staphylococcal enterotoxin superantigens.

Staphylococcal enterotoxins (SE) are a family of structurally related proteins that are produced by Staphylococcus aureus. They play a role in the pathogenesis of food poisoning and are the most potent activators of T lymphocytes known. The receptors for SE on antigen-presenting cells are major histocompatibility complex class II molecules. Recent studies have shown that a complex of SE and major histocompatibility complex class II molecules is required for binding to the variable region of the T cell antigen receptor beta-chain. SE mitogenic activity is dependent on induction of interleukin 2, which may be intimately involved in the mechanism of SE toxicity. The minor lymphocyte-stimulating "endogenous" self-superantigen has recently been shown to be a retroviral gene product, so that this too is apparently a microbial superantigen. An understanding of the mechanism of action of these microbial superantigens has implications for normal and pathological immune functions.