Increased interleukin-10 mRNA expression in tumor-bearing or persistently lymphocytotic animals infected with bovine leukemia virus.

Interleukin-10 (IL-10), produced by Th2 helper T cells, B cells, and macrophages, can inhibit cytokine production by Th1 cells and enhance B-cell proliferation and differentiation. Here, we show that peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus-infected animals with late-stage disease express considerably more IL-10 mRNA than animals that are not infected or that are in the early stages of disease. In contrast, the quantities of type 1 cytokines, IL-2 and gamma interferon, decrease with disease progression. In addition, we observed that IL-10 is expressed principally by monocytes/macrophages, not B lymphocytes, in persistently lymphocytotic animals. This observation supports a role for monocytes/macrophages in progression of bovine leukemia virus infection and, of importance, indicates that proliferating B cells are not the source of IL-10 expression. These findings suggest that IL-10 produced by monocytes/macrophages may influence the progression of bovine leukosis in animals that develop persistent lymphocytosis of B cells or B-cell lymphosarcoma.     

Bovine Leukemia Virus-Induced Persistent Lymphocytosis in Cattle Does Not Correlate with Increased Ex Vivo Survival of B Lymphocytes

Bovine leukemia virus (BLV) is an oncogenic retrovirus associated with B-cell lymphocytosis, leukemia, and lymphosarcoma in the ovine and bovine species. We have recently reported that in sheep, BLV protects the total population of peripheral blood mononuclear cells (PBMCs) from ex vivo spontaneous apoptosis. This global decrease in the apoptosis rates resulted from both direct and indirect mechanisms which allow extension of cell survival. Although sheep are not natural hosts for BLV, these animals are prone to develop virus-induced leukemia at very high frequencies. Most infected cattle, however, remain clinically healthy. This difference in the susceptibilities to development of leukemia in these two species might be related to alterations of the apoptotic processes. Therefore, we designed this study to unravel the mechanisms of programmed cell death in cattle. We have observed that PBMCs from persistently lymphocytotic BLV-infected cows were more susceptible to spontaneous ex vivo apoptosis than cells from uninfected or aleukemic animals. These higher apoptosis rates were the consequence of an increased proportion of B cells exhibiting lower survival abilities. About one-third of the BLV-expressing cells did not survive the ex vivo culture conditions, demonstrating that viral expression is not strictly associated with cell survival in cattle. Surprisingly, culture supernatants from persistently lymphocytotic cows exhibited efficient antiapoptotic properties on both uninfected bovine and uninfected ovine cells. It thus appears that indirect inhibition of cell death can occur even in the presence of high apoptosis rates. Together, these results demonstrate that the protection against spontaneous apoptosis associated with BLV is different in cattle and in sheep. The higher levels of ex vivo apoptosis occurring in cattle might indicate a decreased susceptibility to development of leukemia in vivo.     

The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection.

The bovine leukemia virus (BLV) is an oncogenic retrovirus that is associated with the development of persistent lymphocytosis (PL) and lymphoma in cattle. While B lymphocytes have been shown to be the primary cellular target of BLV, recent studies suggest that some T lymphocytes and monocytes may be infected by the virus. Because virally altered functions of monocytes and/or T cells could contribute to the development of lymphoproliferative disease, we sought to clarify the distribution of the BLV provirus in subpopulations of peripheral blood mononuclear cells in seropositive cows with and without PL. CD2+ T cells, monocytes, and CD5+ and CD5- B cells were sorted by flow cytometry and tested for the presence of BLV by single-cell PCR. We did not obtain convincing evidence that peripheral blood monocytes or T lymphocytes contain the BLV provirus in seropositive cows with or without PL. In seropositive cows without PL (n=14), BLV-infected CD5+ and CD5- B cells accounted for 9.2% +/- 19% and 0.1% +/- 1.8% of circulating B lymphocytes, respectively. In cows with PL (n=5), BLV-infected CD5+ and CD5- B cells accounted for 66% +/- 4.8% and 13.9% +/- 6.6% of circulating B lymphocytes, respectively. The increase in lymphocyte numbers in cows with PL was entirely attributable to the 45-fold and 99-fold expansions of infected CD5+ and CD5- B-cell populations, respectively. Our results demonstrate that B cells are the only mononuclear cells in peripheral blood that are significantly infected with BLV. On the basis of the absolute numbers of infected cells in seropositive, hematologically normal animals, there appear to be differences in susceptibility to viral spread in vivo that may be under the genetic control of the host.     

Association between BoLA and subclinical bovine leukemia virus infection in a herd of Holstein-Friesian cows

The role of the bovine major histocompatibility system (BoLA) in subclinical bovine leukemia virus (BLV) infection was investigated in a herd of Holstein-Friesian cows (n=240). The BoLA W8.1 allele was negatively associated with the presence of antibodies to the major BLV envelope glycoprotein, BLV-gp51 (corrected P<0.001, relative risk =0.31). These results suggest that a BoLA-linked gene(s) may influence the early spread of BLV infection. Since B cells are the primary target of BLV infection, we then determined the relationship between BoLA-A locus phenotypes and B-cell numbers in peripheral blood of seropositive and seronegative cows. There were no significant differences between BoLA-A alleles for any hematological parameter in seronegative cows. Seropositive cows with the W12.1 allele had significantly greater absolute numbers of lymphocytes per microliter and B cells per microliter than did seropositive cows with other BoLA-A phenotypes (P<0.01, respectively). The average effect associated with the W12.1 allele in BLV-infected cows was an increase of 2010 B cells per microliter of whole blood relative to BLV-infected cows with other BoLA-A phenotypes. These results demonstrate that susceptibility to the polyclonal expansion of BLV-infected B lymphocytes is associated with the W12.1 allele in Holstein-Friesian cattle. Compared with results of a previous study in a herd of Shorthorn cattle, it appears that resistance and susceptibility to subclinical progression of BLV infection are associated with different BoLA-A locus alleles in different cattle breeds.Abbreviations used in this paper AGID agar gel immunodiffusion - BLV bovine leukemia virus - BoLA bovine lymphocyte antigen - EBL enzootic bovine leukosis - HLA human leukocyte antigen - MHC major histocompatibility complex - PL persistent lymphocytosis     

Reduced bovine leukaemia virus proviral load in genetically resistant cattle

The bovine leukaemia virus (BLV) is an exogenous retrovirus that is closely related to the human T cell leukaemia viruses. Genetic resistance and susceptibility to persistent lymphocytosis (PL), an advanced subclinical stage of infection characterized by a polyclonal expansion of the infected B cell population, have been mapped to structural motifs in bovine MHC DRB3 (class II) alleles. To determine whether alleles of DRB3 influence the number of BLV-infected B cells in peripheral blood, seven pairs of Holstein cows naturally infected with BLV were matched on the basis of DRB3 genotype (resistance or susceptibility to PL), age, and year of seroconversion. Flow cytometry was used to separate B cell populations that then were tested for the presence of provirus by a single-cell PCR methodology. Animals with the PL-resistance associated DRB3.2*11 allele had significantly fewer BLV-infected B cells than did age- and seroconversion-matched cows with DRB3 alleles associated with susceptibility to PL. Our results demonstrate that DRB3 or a closely linked gene may play a direct role in controlling the number of BLV-infected peripheral B cells in vivo . Association of MHC class II alleles with resistance to disease progression in cattle naturally infected with BLV provides a unique immunogenetic model for the study of host resistance to human and other animal retroviral infections.     

Interleukin-12 p40 mRNA Expression in Bovine Leukemia Virus-Infected Animals: Increase in Alymphocytosis but Decrease in Persistent Lymphocytosis

Interleukin-12 (IL-12), a key cytokine in immune regulation, has an important role in activating the cell-mediated immune response in infectious diseases. Recently, a dichotomy between IL-12 and IL-10 regarding progression of a variety diseases has emerged. IL-12 activates type 1 cytokine production and has an antagonistic effect on type 2 cytokines. Here, by using quantitative competitive PCR, we show that peripheral blood mononuclear cells from bovine leukemia virus-infected animals in the alymphocytotic stage of disease express an increased amount of IL-12 p40 mRNA. In contrast, IL-12 p40 mRNA expression by cells from animals with late-stage disease, termed persistent lymphocytosis, was significantly decreased compared to that by normal and alymphocytotic animals. Interestingly, IL-12 p40 mRNA was also detected in tumor-bearing animals. IL-12 p40 expression occurred only in monocytes/macrophages, not B or T lymphocytes. The present study combined with previous findings suggest that IL-12 in bovine leukemia virus-infected animals may regulate production of other cytokines such as gamma interferon and IL-10 and the progression of bovine leukosis in animals that develop more advanced disease such as a persistent lymphocytosis of B cells or B-cell lymphosarcoma.     

Pathogenicity of molecularly cloned bovine leukemia virus.

To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.     

Co-expression of IL-12 receptors along with CXCR3 and CD25 on activated peripheral blood T lymphocytes

IL-12 receptors (IL-12R) play a critical role in maintaining IL-12 regulation of T helper-1 (Th1) type immune responses. We studied the expression of two IL-12R, beta1 and beta2 on peripheral blood mononuclear cells (PBMCs) from normal donors, stimulated with polyclonal activators in the presence or absence of exogenous rhIL-12. Unstimulated peripheral blood T lymphocytes (PBTs) expressed moderate levels of IL-12Rbeta1 and very low to undetectable levels of IL-12Rbeta2. Superantigens and anti-CD3+anti-CD28 induced higher expression of both IL-12R on PBTs than PHA-P stimulation. Exogenous rhIL-12 further enhanced the PHA-P or anti-CD3+anti-CD28 induced IL-12Rbeta2 expression. Only a fraction of mitogen activated IL-12Rbeta1+ or beta2+ T lymphocytes co-expressed CD25 (with further enhancement by exogenous rhIL-12), while a higher percentage of these cells were CXCR3+. The majority of superantigen or anti-CD3+anti-CD28-induced IL-12R+ PBTs were positive for both CD25 and CXCR3 markers. Our results indicated differential induction of IL-12R expression that correlated with up regulation of CD25 and CXCR3 expression on activated PBTs and provide a useful insight for monitoring these markers during treatment of Th1 type inflammatory diseases.     

Decreased Insulin-Like Growth Factor-I (IGF-I) Receptor Sites on Circulating Mononuclear Cells from Cows with Persistent Lymphocytosis

Abstract

Plasma IGF-I concentrations and IGF-I receptor binding on mononuclear cells have been studied on bovine leukemia virus (BLV)-negative (CO), BLV-infected aleukemic (AL) cows or cows with persistent lymphocytosis (PL). No significant differences in plasma IGF-I concentrations were demonstrated among the three groups of animals. However, a linear negative correlation existed between the number of circulating mononuclear cells and the number of IGF-I binding sites on these cells from control cows. In addition, mononuclear cells from PL cows had fewer IGF-I binding sites per cell when compared with control cows. These results suggest involvement of IGF-I in etiology of BLV infection and progression and warrant further studies to establish whether IGF-I plays a major physiological role in these conditions.     

Cutaneous lymphocyte antigen expression on activated lymphocytes and its association with IL-12R (beta1 and beta2), IL-2Ralpha, and CXCR3

The majority of T lymphocytes that infiltrate psoriatic lesions express cutaneous lymphocyte antigen (CLA), a skin homing receptor involved in the influx of memory T cells to cutaneous sites. We investigated CLA expression on normal human peripheral blood mononuclear cells (PBMCs) and evaluated its association with IL-12 receptors, chemokine receptor, CXCR3, and IL-2Ralpha. PBMCs were stimulated in vitro with or without polyclonal activators (mitogen, or superantigens, or anti-CD3+anti-CD28) in the presence or absence of exogenous rhIL-12. The percentage of CLA+ T lymphocytes increased significantly after superantigen stimulation compared to anti-CD3+anti-CD28 or mitogen activation. The majority of activation induced CLA+ T lymphocytes co-expressed IL-12Rbeta1, IL-12Rbeta2, CXCR3, and CD25 in the presence of rhIL-12. Our results indicate that CLA expression on activated T lymphocytes is IL-12 and activation dependent and correlates with the expression of IL-12 receptors, IL-2Ralpha, and CXCR3. Monitoring the levels of Th1 differentiation markers such as CXCR3 and IL-12Rbeta2 along with activation marker, CD25 on skin homing CLA+ T lymphocytes may provide insight into the mechanism of action of immunotherapies directed against Th1 type skin inflammatory diseases.     

Bovine leukemia virus transmembrane protein gp30 physically associates with the down-regulatory phosphatase SHP-1

In B lymphocytes, the down-regulatory phosphatase SHP-1 associates with CD22 and CD32b (also known as FcgammaRIIB) and acts as a critical negative regulator of B-cell receptor signaling. Bovine leukemia virus, a retrovirus of the HTLV/BLV group, causes persistently increased numbers of peripheral blood B lymphocytes, known as persistent lymphocytosis (PL) and, in some animals, progression to B-cell leukemia and/or lymphoma. Here, we show that SHP-1 associates with the bovine leukemia virus transmembrane protein, gp30. This interaction is either direct or indirect. The interaction is dependent on tyrosine phosphorylation, and the interaction increases after cell stimulation with sodium pervanadate. The gp30-SHP-1 interaction is seen in all of the BLV-infected, PL animals tested, but is not seen in uninfected animals or in most BLV-infected, non-PL animals, which do not express significant quantities of gp30. However, one BLV-infected, non-PL animal expressed large quantities of gp30, yet no gp30-SHP-1 interaction was detected, suggesting that there may be other factors in cells from the PL animals that facilitate the gp30-SHP-1 interaction. The association of gp30 and SHP-1 suggests the hypothesis that gp30 may act as a decoy to sequester SHP-1, resulting in up-regulation of B-cell receptor signaling. The implication of this could be a novel mechanism of viral activation of lymphocytes by removal of a down-regulatory phosphatase.     

Evidence for BoLA-linked resistance and susceptibility to subclinical progression of bovine leukaemia virus infection

The role of the bovine major histocompatibility complex in bovine leukaemia virus (BLV) infection and disease progression was investigated in a herd of Shorthorn cattle (n = 117). The frequency of cows that were seropositive to BLV-glycoprotein antigen was 51%. Twenty-three per cent of the seropositive cows were lymphocytotic. At the herd level, relative resistance to BLV-dependent B-cell proliferation and lymphocytosis among seropositive cows was associated with bovine lymphocyte antigen (BoLA)-DA7, whereas susceptibility was associated with BoLA-DA12.3. These associations were also confirmed at the family level, where BoLA phenotypes were used as haplotypic markers. Among the offspring of one BoLA-heterozygous sire (n = 33), resistance segregated with the DA7 haplotype and susceptibility with the DA12.3 haplotype. In this sire group, maternal transmission of the BoLA-w8 allele was associated with increased susceptibility to B-cell proliferation and lymphocytosis in w8/DA12.3 heterozygotes. These data provide the first evidence that subclinical progression of BLV infection is under the control of the BoLA complex, and suggest that the BoLA system can be used to select for resistance to B-cell proliferation and the development of lymphocytosis in BLV-infected herds.     

Recombinant human interleukin 6 (rhIL-6) promotes the terminal differentiation of in vivo-activated human B cells into antibody-secreting cells

Antigen-activated peripheral blood B cells were induced by parenteral immunization of healthy individuals with a polyvalent pneumococcal vaccine, or diphtheria toxoid. Seven to nine days after immunization, high frequencies of antigen-specific antibody-secreting cells, representing in vivo activated lymphoblastoid B cells, were detectable in peripheral blood or spleen. The B cell-enriched fractions were stimulated for 7 days with different concentrations of rhIL-6. Both the frequency of antibody-secreting cells and the secreted amount of antibody to the immunizing antigen were increased by rhIL-6 in a dose-dependent fashion. Stimulation with rhIL-6 did not alter the isotype distribution of antibody-secreting cells. A polyclonal anti-IL-6 serum completely abrogated the stimulatory effect of rhIL-6 on the in vitro antibody secretion. Fluorescence-activated cell sorter analysis revealed that 25-29% of cells in the large B cell fraction which presumably contained the in vivo activated cells bore the IL-6 receptor. Thus, rhIL-6 enhances the terminal differentiation of in vivo activated B cells into antibody-secreting plasma cells.     

Differential expression of interferon-gamma,IL-4 and IL-10 in peripheral blood mononuclear cells during early pregnancy of the bovine

Maternal systemic immune response is regulated by conceptus-derived signals through peripheral blood mononuclear cells (PBMCs) via blood circulation during early pregnancy in cattle. In this study, the PBMCs from day 18 in non-pregnant cows and days 14, 18 and 30 in pregnant cows were used to explore the expression of interferon-gamma (IFN-γ), interleukin 4 (IL-4) and IL-10, and the plasma progesterone (P4) concentration was also determined. The results showed that the expression levels of mRNA and the protein of IFN-γ were lower and that IL-4 and IL-10 were higher in the PBMCs from the pregnant cows than in those of non-pregnant cows. From this study, early pregnancy induced a lower Th1 immunity (IFN-γ) and a higher Th2 immunity (IL-4 and IL-10) in the PBMCs, which may be related to interferon-tau and P4, thereby contributing to successful pregnancy in cattle.     

Regulation of Bovine Leukemia Virus tax and pol mRNA Levels by Interleukin-2 and -10

Recently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLV tax and pol mRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLV tax and pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLV tax and pol mRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLV tax and pol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10 production in late disease stages suppresses BLV mRNA levels, while IL-2-activated immune responses stimulate BLV expression by BLV-infected B cells.     

Inhibition of T-lymphocyte mitogenic responses and effects on cell functions by bovine herpesvirus 1.

The mitogenic response of bovine peripheral blood mononuclear cells stimulated by concanavalin A (ConA) was suppressed by infectious bovine herpesvirus 1 (BHV-1). Proliferation in response to interleukin-2 (IL-2) by IL-2-dependent lymphocyte cultures was also inhibited by BHV-1. Although inhibition of mitogenesis approached 100%, less than 1 cell in 1,000 was productively infected by BHV-1 in ConA-stimulated cultures. Neither conditioned medium from mitogen-stimulated peripheral blood mononuclear cell cultures nor human recombinant IL-2 reversed suppression by the virus. Infection by BHV-1 did not influence the expression of IL-2 or IL-2 receptor mRNA in ConA-stimulated cultures, nor did it affect the cytolytic capabilities of lymphocytes. The data suggest that the inhibition of T-lymphocyte proliferation is the result of a nonproductive BHV-1 infection.     

Tuberculin response of lymphocytes from human skin test nonreactors; evidence for in vitro primary sensitization of T lymphocytes.

Tuberculin-purified protein derivative (PPD) is a B-lymphocyte mitogen in a variety of experimental animals. Although peripheral blood mononuclear cells (PB MNC) from healthy human tuberculin responders consistently responded to PPD by increased incorporation of [³H]thymidine, cell fractionation studies showed this to be due to T-lymphocyte rather than B-cell blastogenesis. Moreover, utilizing thymidine suicide experiments, the T-lymphocyte response could be categorized as antigenic rather than nonspecific mitogenic reactivity. Kinetic studies revealed a delayed peak of PPD-induced thymidine incorporation in PB MNC from tuberculin skin test-negative as compared to skin test-positive donors. This suggested in vitro primary sensitization of T lymphocytes to PPD, which was corroborated in experiments demonstrating tuberculin reactivity of human umbilical-cord blood lymphocytes.