Diana M. Stone

Clearance of Virulent but Not Avirulent Rhodococcus equi from the Lungs of Adult Horses Is Associated with Intracytoplasmic Gamma Interferon Production by CD4+ and CD8+ T Lymphocytes

ABSTRACT Rhodococcus equi is a gram-positive bacterium that infects alveolar macrophages and causes rhodococcal pneumonia in horses and humans. The virulence plasmid of R. equi appears to be required for both pathogenicity in the horse and the induction of protective immunity. An understanding of the mechanisms by which virulent R. equi circumvents protective host responses and by which bacteria are ultimately cleared is important for development of an effective vaccine. Six adult horses were challenged with either virulent R. equi or an avirulent, plasmid-cured derivative. By using a flow cytometric method for intracytoplasmic detection of gamma interferon (IFN-γ) in equine bronchoalveolar lavage fluid (BALF) cells, clearance of the virulent strain was shown to be associated with increased numbers of pulmonary CD4+ and CD8+ T lymphocytes producing IFN-γ. There was no change in IFN-γ-positive cells in peripheral blood, suggesting that a type 1 recall response at the site of challenge was protective. The plasmid-cured strain of R. equi was cleared in horses without a significant increase in IFN-γ-producing T lymphocytes in BALF. In contrast to these data, a previous report in foals suggested an immunomodulating role for R. equi virulence plasmid-encoded products in downregulating IFN-γ expression by equine CD4+ T lymphocytes. Intracytoplasmic detection of IFN-γ provides a method to better determine whether modulation of macrophage-activating cytokines by virulent strains occurs uniquely in neonates and contributes to their susceptibility to rhodococcal pneumonia.

Up-regulation of IL-2 receptor α and MHC class II expression on lymphocyte subpopulations from bovine leukemia virus infected lymphocytotic cows

Infection with bovine leukemia virus (BLV) leads to a persistent lymphocytosis (PL) characterized by a marked increase in circulating B lymphocytes that express the orthologue of CD5. To gain insight into the factors accounting for lymphocytosis, experiments were conducted to determine the functional activation status of lymphocytes from BLV seronegative and BLV infected aleukemic cows with PL. Stimulation with the B lymphocyte mitogen Staphylococcus aureus Cowan strain I (SAC), recombinant human interleukin-2 (rIL-2), or pokeweed mitogen (PWM), a T lymphocyte-dependent B lymphocyte mitogen, revealed differences in the pattern of expression of IL-2 receptor alpha (IL-2R alpha) and major histocompatibility (MHC) class II molecules on B and T lymphocytes from uninfected and BLV infected PL cows. rIL-2 induced expression of IL-R alpha on B lymphocytes from PL cows but not B lymphocytes from BLV seronegative cows. SAC alone, or in combination with rIL-2, had no effect on B lymphocytes from BLV seronegative cows. However, rIL-2 alone or in combination with SAC induced expression of IL-2R alpha on B lymphocytes from PL cows. PWM stimulated expression of IL-2R alpha on bovine B lymphocytes regardless of BLV status, and induced a significantly higher level of expression on B lymphocytes from PL cows. Mitogens and rIL-2 had a similar stimulatory effect on induction of IL-2R alpha expression on CD4 T lymphocytes regardless of BLV status. Only PWM induced expression of IL-2R alpha on bovine CD8 T lymphocytes and induced a significantly higher level of expression on this T lymphocyte subset from PL cows. Examination of freshly isolated B lymphocytes from PL cows revealed increased spontaneous expression of the MHC class II molecule compared to B lymphocytes from control cows. None of the culture conditions examined induced MHC-II expression on CD4 and CD8 T lymphocytes from BLV seronegative cows. In contrast, SAC+IL-2 and PWM induced MHC-II expression on CD4 and CD8 T lymphocytes from BLV infected PL cows, resulting in a significantly greater proportions of these lymphocyte subsets expressing this molecule compared to CD4 and CD8 T lymphocytes from control cows. The data indicate that infection with BLV affects the response of B and T lymphocytes to signals of activation, up-regulating the expression of surface molecules involved in both direct contact and cytokine-mediated T lymphocyte-dependent B lymphocyte activation.

Rhodococcus equi secreted antigens are immunogenic and stimulate a type 1 recall response in the lungs of horses immune to R. equi infection

ABSTRACT Rhodococcus equi is an opportunistic pathogen in immunocompromised humans and an important primary pathogen in young horses. Although R. equi infection can produce life-threatening pyogranulomatous pneumonia, most foals develop a protective immune response that lasts throughout life. The antigen targets of this protective response are currently unknown; however, Mycobacterium tuberculosis is a closely related intracellular pathogen and provides a model system. Based on previous studies of M. tuberculosis protective antigens released into culture filtrate supernatant (CFS), a bacterial growth system was developed for obtaining R. equi CFS antigens. Potential immunogens for prevention of equine rhodococcal pneumonia were identified by using immunoblots. The 48-h CFS contained five virulence-associated protein bands that migrated between 12 and 24 kDa and were recognized by sera from R. equi-infected foals and immune adult horses. Notably, the CFS contained the previously characterized proteins VapC, VapD, and VapE, which are encoded by genes on the R. equi virulence plasmid. R. equi CFS was also examined for the ability to stimulate a type 1-like memory response in immune horses. Three adult horses were challenged with virulent R. equi, and cells from the bronchoalveolar lavage fluid were recovered before and 1 week after challenge. In vitro stimulation of pulmonary T-lymphocytes with R. equi CFS resulted in significant proliferation and a significant increase in gamma interferon mRNA expression 1 week after challenge. These results were consistent with a memory effector response in immune adult horses and provide evidence that R. equi CFS proteins are antigen targets in the immunoprotective response against R. equi infection.

Spontaneously proliferating lymphocytes from bovine leukaemia virus-infected, lymphocytotic cattle are not the virus-expressing lymphocytes, as these cells are delayed in G0/G1 of the cell cycle and are spared from apoptosis.

Bovine leukaemia virus (BLV) is in the family of oncogenic retroviruses which includes human T cell leukaemia virus (HTLV). BLV infects B lymphocytes and induces a non-neoplastic persistent lymphocytosis (PL) of B lymphocytes in cattle. A characteristic of BLV- and HTLV-induced disease is spontaneous lymphocyte proliferation of cultured peripheral blood mononuclear cells (PBMC). To investigate the role of virus expression on lymphocyte survival and proliferation, we evaluated cell cycle position, apoptosis and virus expression on a single-cell basis of cultured PBMC from BLV-infected PL cattle, BLV-infected non-PL cattle and uninfected cattle. Results demonstrated that the majority of bovine B lymphocytes spontaneously entered G(2)/M of the cell cycle and died by apoptosis by 24 h post-culture, regardless of BLV infection or PL status. The spontaneous proliferation that characterizes PL cattle was primarily due to a small population of surviving B lymphocytes, but T lymphocytes also contributed. Viral protein expression was detectable in only 5-15% of cultured PBMC from PL cattle and the majority of these lymphocytes were delayed in cell cycle and spared from apoptosis. Unexpectedly, we determined that only 3% of the spontaneously proliferating lymphocytes expressed viral proteins. Previous reports show that spontaneous proliferation decreases when virus expression is suppressed. Together with our results, this suggests that virus expression by one population of B lymphocytes promotes proliferation of another population of B lymphocytes that does not express virus. This may be due to an effect of virus on CD4 T lymphocytes, as depletion of CD4 T lymphocytes significantly decreased spontaneous proliferation.

Folate-sensitive and aphidicolin-inducible fragile sites are expressed in the genome of the domestic cat.

Peripheral blood lymphocytes from three clinically normal domestic cats were cultured for folate-sensitive and aphidicolin-inducible fragile site expression. Induction of folate-sensitive fragile sites was accomplished by culturing cells with trimethoprim plus caffeine. Chromosomes from all cats expressed both folate-sensitive and aphidicolin-inducible breaks and gaps. There were no significant differences between the two methods of fragile site induction in the percentage of cells expressing chromosome breaks and gaps or the mean number of breaks and gaps per cell. All three cats expressed specific chromosome breaks resembling fragile sites at A1q21-22, A1p22, and B1q32. All three sites were induced by aphidicolin. The sites at A1q21-22 and B1q32 were also induced in folate-deficient medium. This is the first report of the induction of chromosomal fragile sites in a feline species.

Antiviral activity of shiga toxin requires enzymatic activity and is associated with increased permeability of the target cells.

ABSTRACT This study expanded our earlier finding that Shiga toxin type 1 (Stx1) has activity against bovine leukemia virus (BLV) (W. A. Ferens and C. J. Hovde, Infect. Immun. 68:4462-4469, 2000). The Stx molecular motifs required for antiviral activity were identified, and a mechanism of Stx action on virally infected cells is suggested. Using inhibition of BLV-dependent spontaneous lymphocyte proliferation as a measure of antiviral activity, we showed that Stx2 had antiviral activity similar to that of Stx1. Enzymatic and antiviral activities of three StxA1 chain mutants deficient in enzymatic activity or aspects of receptor-mediated cytotoxicity were compared. Using protein synthesis inhibition to measure enzymatic activity, the mutant E167D was 300-fold less catalytically active than wild-type StxA1, was minimally active in antiviral assays, and did not inhibit synthesis of viral proteins. Two StxA1 mutants, A231D-G234E and StxA11 (enzymatically active but unable to kill cells via the classical receptor-mediated route), had undiminished antiviral activity. Although binding of radiolabeled StxA1 to bovine blood cells or to free virus was not detected, flow cytometric analysis showed that the number of BLV-expressing cells were specifically reduced in cultures treated with Stx. These unique and rare lymphocytes were highly permeable to 40- and 70-kDa fluorescent dextrans, indicating that direct absorption of toxins by virus-expressing cells is a potential mechanism of target cell intoxication. These results support the hypothesis that Stx-producing Escherichia coli colonization of the gastrointestinal tract may benefit ruminant hosts by the ability of Stxs to exert antiviral activity.

Bovine leukemia virus gp30 transmembrane (TM) protein is not tyrosine phosphorylated: examining potential interactions with host tyrosine-mediated signaling.

Bovine leukemia virus (BLV) causes persistent lymphocytosis, a preneoplastic, polyclonal expansion of B lymphocytes. The expansion increases viral transmission to new hosts, but the mechanisms of this expansion have not been determined. We hypothesized that BLV infection contributes to B-cell expansion by signaling initiated via viral transmembrane protein motifs undergoing tyrosine phosphorylation. Viral mimicry of host cell proteins is a well-demonstrated mechanism by which viruses may increase propagation or decrease recognition by the host immune system. The cytoplasmic tail of BLV transmembrane protein gp30 (TM) has multiple areas of homology to motifs of host cell signaling proteins, including two immunoreceptor tyrosine-based activation motifs (ITAMs) and two immunoreceptor tyrosine-based inhibition motifs (ITIMs), which are homologous to B-cell receptor and inhibitory co-receptor motifs. Signaling by these motifs in B cells typically relies on tyrosine phosphorylation, followed by interactions with Src-homology-2 (SH2) domains of nonreceptor protein tyrosine kinases or phosphatases. Phosphorylation of tyrosine residues in the cytoplasmic tail of TM was tested in four systems including ex vivo cultured peripheral blood mononuclear cells from BLV infected cows, BLV-expressing fetal lamb kidney cell and bat lung cell lines, and DT40 B cells transfected with a fusion of mouse extracellular CD8alpha and cytoplasmic TM. No phosphorylation of TM was detected in our experiments in any of the cell types utilized, or with various stimulation methods. Detection was attempted by immunoblotting for phosphotyrosines, or by metabolic labeling of cells. Thus BLV TM is not likely to modify host signal pathways through interactions between phosphorylated tyrosines of the ITAM or ITIM motifs and host-cell tyrosine kinases or phosphatases.

Translocation of the B cell receptor to lipid rafts is inhibited in B cells from BLV-infected, persistent lymphocytosis cattle.

Bovine leukemia virus (BLV) infection causes a significant polyclonal expansion of CD5(+), IgM+ B lymphocytes known as persistent lymphocytosis (PL) in approximately 30% of infected cattle. There is evidence that this expanded B cell population has altered signaling, and resistance to apoptosis has been proposed as one mechanism of B cell expansion. In human and murine B cells, antigen binding initiates movement of the B cell receptor (BCR) into membrane microdomains enriched in sphingolipids and cholesterol, termed lipid rafts. Lipid rafts include members of the Src-family kinases and exclude certain phosphatases. Inclusion of the BCR into lipid rafts plays an important role in regulation of early signaling events and subsequent antigen internalization. Viral proteins may also influence signaling events in lipid rafts. Here we demonstrate that the largely CD5(+) B cell population in PL cattle has different mobilization and internalization of the BCR when compared to the largely CD5-negative B cells in BLV-negative cattle. Unlike B cells from BLV-negative cattle, the BCR in B cells of BLV-infected, PL cattle resists movement into lipid rafts upon stimulation and is only weakly internalized. Expression of viral proteins as determined by detection of the BLV transmembrane (TM) envelope glycoprotein gp30 did not alter these events in cells from PL cattle. This exclusion of the BCR from lipid rafts may, in part, explain signaling differences seen between B cells of BLV-infected, PL, and BLV-negative cattle and the resistance to apoptosis speculated to contribute to persistent lymphocytosis.

CYTOGENETIC EVALUATION OF FOUR CANINE MAST CELL TUMORS

We evaluated four canine cutaneous mast cell tumors cytogenetically. All four tumors contained both hypodiploid and hyperdiploid cells, an increase in the number of metacentric chromosomes, exchange configurations, and cells showing loss of an X chromosome. All tumors contained metaphases with chromosome gaps and breaks at frequencies greater than observed spontaneous chromosome breaks in normal cultured canine peripheral blood lymphocytes. Three of the four tumors had a normal modal chromosome number of 78. The fourth tumor had a modal chromosome number of 93, which represented 15% of the cells evaluated from this tumor.