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.     

Tropism of human herpesvirus 8 for peripheral blood lymphocytes in patients with Castleman's disease

Multicentric Castleman's disease (MCD), also called multicentric angiofollicular lymphoid hyperplasia, is a systemic lymphoproliferative disorder causing fever, lymphadenopathy and splenomegaly. Recently, Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) DNA sequences have been detected in cases of MCD. We examined HHV-8 DNA sequences in the peripheral blood mononuclear cells (PBMCs) of two HIV-negative patients with MCD and in PBMCs and the lymph node of a HIV-negative patient with localized Castleman's disease (LCD) by the polymerase chain reaction. The novel sequences were detected in all DNA samples. Furthermore, the sequences were detected in only the CD19+ B-lymphocyte fraction of the patient with LCD as previously reported. However, the sequences were detected in CD19+ B-lymphocyte and CD2+ T-lymphocyte fractions of two patients with MCD. These results suggest that HHV-8 has tropisms for both B lymphocytes and T lymphocytes in Castleman's disease.     

Cytokine gene expression after allogeneic bone marrow transplantation

Cytokines produced by T lymphocytes, monocytes/macrophages, and fibroblasts play a central role in the immune response and in the development of graft-versus-host disease (GVHD). Also, it has been reported that dysregulated production of cytokines maybe the primary mediator of clinical manifestation of acute GVHD. Regarding cytokine gene expression after human allogeneic bone marrow transplantation (allo BMT), we have demonstrated increased IL-1 beta, IL-6, and TNF-alpha mRNA expression in peripheral blood mononuclear cells during the development of acute and chronic GVHD and that the degree of the increase was dependent on the severity of the disease. Furthermore, overexpression of these cytokine mRNAs could be detected before the clinical manifestations of GVHD developed. In contrast, IL-2 mRNA expression was not detected in peripheral blood mononuclear cells in GVHD patients. On the other hand, we have reported that increased mRNA expression and protein product of IL-2 and IFN-gamma were evident in the mixed lymphocyte culture of the cases who developed severe lethal transplantation-related complications. Therefore, the detection of increased IL-2 and IFN-gamma gene expression in MLC appeared to be useful for predicting transplantation-related complications in BMT patients. Furthermore, we found increased IL-2 receptor alpha subunit mRNA expression in the peripheral blood mononuclear cells during GVHD. These findings may indicate the important role of inflammatory cytokines such as IL-1 beta, IL-6 and TNF-alpha in the development of the clinical manifestation of GVHD and also may be indicative of the important role of IL-2 and the IL-2 receptor in allo response perhaps mainly as an autocrine effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of cytokine genes in CD27-positive and -negative CD4 lymphocyte subsets from healthy humans and rheumatoid arthritis patients

Immunofluorescence analysis of CD27 expression by CD4 lymphocytes from the peripheral blood of healthy humans or rheumatoid arthritis (RA) patients and from the synovial fluid (SF) of RA patients was carried out, along with the estimation of cytokine gene [interleukin (IL) 2, IL-3, IL-4, IL-5, IL-6, IL-6R, IL-10 and interferon-gamma (IFN-gamma)] expression in these lymphocyte subsets by RT-PCR. Although no differences in CD27-positive and -negative peripheral blood CD4 cell subset distribution were revealed, marked differences in IL-3, IL-4, IL-5 and IFN-gamma mRNA expression were detected between these lymphocyte subsets and between control and disease states. These results showed that phenotyping of different cell subsets in disease cannot provide adequate information about lymphocyte functional status. To estimate differences in cytokine gene expression, CD4 lymphocytes from the peripheral blood and SF of RA patients were compared. In both cases, mRNAs for IL-2, IL-4, IL-10 and IFN-gamma were detected, but CD4 cells from SF failed to express detectable levels of IL-5 mRNA despite our findings of a CD27-cell accumulation within the synovial population of CD4 lymphocytes. These are the first data to demonstrate that expression of the IL-5 gene in RA SF CD27- lymphocytes is down-regulated and that IL-5 disregulation in RA cannot be ruled out.     

Regulation of IL-17, IFN-gamma and IL-10 in human CD8(+) T cells by cyclic AMP-dependent signal transduction pathway

In the present study, the expression of interleukin 17 (IL-17) by human CD8(+) T lymphocytes and its regulation following PKA activation was determined and compared with that of interferon gamma (IFN-gamma) and IL-10. IL-17 mRNA was highly expressed in human CD8(+) T lymphocytes at least at the same level than in CD4(+) T cells that were isolated from peripheral blood mononuclear cells (PBMC). Expression of IL-17 mRNA in CD8(+) T cell was induced by prior activation of PBMC for 18 h with Ca2+ ionophore and phorbol myristate acetate (PMA). Furthermore, our results clearly showed that CD8(+) T cells are sensitive to elevation of cAMP and PKA activation pathway. Data demonstrated a significant inhibition of IL-17 as well as of IFN-gamma mRNA expression in CD8(+) T cells isolated from activated PBMC cultured in the presence of either dibutyryl cAMP (db-cAMP) or PGE2. In contrast, IL-10 mRNA expression was strongly enhanced in the same experimental conditions. The differential expression of IL-10 and IFN-gamma production in CD8(+) T cells was also observed at the protein level as it was measured by a double immunofluorescence technique and flow cytometry analysis. Taken together, these results provide evidence that human CD8(+) T cells are also the source of massive expression of IL-17, and that PKA plays a prominent role in the switch of CD8(+) T cells to a Th2 like profile and an inhibition of IL-17 expression, thus suggesting that the activation of cAMP signal transduction pathway may have consequences for the relative role of CD8(+) T cells in the immune and inflammatory process.     

IL-6 functions in cynomolgus monkeys blocked by a humanized antibody to human IL-6 receptor

A humanized antibody to the human interleukin-6 receptor (IL-6R), hPM-1, blocked the interleukin-6 (IL-6) functions in normal cynomolgus monkey lymphocytes in vitro. The binding activity of hPM-1 to non-human primate IL-6R was examined in peripheral blood lymphocytes by flow cytometry. PM-1 recognized the IL-6R on T lymphocytes of cynomolgus and rhesus monkeys, but did not on those of marmosets. The homology between human IL-6R and its cynomolgus monkey counterpart was 97.3% in the extracellular domain of the amino acid sequence, as determined by DNA sequencing of the PCR product from peripheral blood mononuclear cells. PM-1 inhibited two functional parameters in vitro in cynomolgus monkeys: (1), T-cell proliferation stimulated by phytohemaglutinin and human IL-6; (2), Immunoglobulin G-production evoked by Staphylococcus aureus Cowan-1- and human IL-6-stimulated B lymphocytes. These data show that hPM-1 binds to and functionally blocks the cynomolgus monkey IL-6 receptors.     

Esophageal intramural pseudodiverticulosis

Cows that develop a persistent lymphocytosis (PL) as a result of bovine leukemia virus (BLV) infection develop massive proliferation of B-lymphocytes expressing both IgM and CD5 markers. The association of these two markers on peripheral blood mononuclear cells (PBMC) derived from BLV-infected cows and also expressing BLV-gp51 antigen marker on these cells was determined by three-color cytometric analysis. After in vitro cultivation of PBMC in the presence of PHA for 24 h, the mean percentages of marker-reactive cells of five PL+ cows were as follows; 43% +/- 4.5 of the PBMC expressed BLV-gp51 antigen; 90% +/- 1.6 of these cells expressed both IgM and CD5 at the same time, whereas but 7.5% +/- 1.9 expressed only IgM and 2.9% +/- 0.4 expressed only CD5. The PBMC, IgM positive cells accounted for 77.8% +/- 6.8, while both CD5 and BLV-gp51 were detected simultaneously on 52.0% +/- 2.4 of the IgM+ cells, while the CD5 marker and BLV-gp51 antigen were detected independently on 35.0% +/- 1.9 and in 9.0% +/- 3.1, respectively of the IgM+ cells. Of the CD5+ cells (equivalent to 75.5% +/- 9.0 of the PBMC), 54.7% +/- 4.7 expressed simultaneously IgM and BLV-gp51, while BLV-gp51 and IgM were expressed separately by 3.0% +/- 0.5 and 37.8% +/- 3.3, respectively. An association between the B-cell phenotype and BLV tropism might exist. It is also possible that cells bearing both IgM and CD5 markers are the main target cells for BLV infection.     

Chronic expression of P-selectin on endothelial cells stimulated by the T-cell cytokine, interleukin-3

P-selectin expressed on the surface of endothelium mediates leukocyte adhesion in vitro and rolling in vivo. Several inducers of cell-surface P-selectin expression on endothelial cells (EC) have previously been identified, all of which yield transient cell-surface expression of P-selectin lasting minutes to a few hours. We now show that a T-lymphocyte product, interleukin-3 (IL-3), stimulates the long-term endothelial cells (HUVEC). IL-3 induced cell-surface P-selectin expression in two phases. An initial peak at 10 minutes was followed by a prolonged upregulation beginning 16 hours after IL-3 addition and lasting at least 4 days. The level of P-selectin expression induced by IL-3 added for 48 hours was similar to that induced by treatment of HUVEC for 10 minutes with thrombin, and the effect of adding IL-3 for 48 hours followed by thrombin for 10 minutes was additive. Induction of cell-surface P-selectin expression by IL-3 was blocked by pretreatment of EC with a blocking monoclonal antibody against the IL-3 receptor alpha-chain. Lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) and a mutant form of IL-3 with decreased potency did not induce cell-surface P-selectin expression after 48 hours' incubation with HUVEC, suggesting that the effect was specific to IL-3. The increase in cell-surface P-selectin expression occurring after 16 hours of incubation with IL-3 was accompanied by a similar prolonged increase in the steady-state mRNA level that was not observed at 10 minutes after IL-3 addition. As T-lymphocyte infiltration is a hallmark of chronic inflammation, our observations suggest that the secretion of IL-3 by T lymphocytes may serve to maintain the inflammatory state during chronic inflammation.     

Induction of T-cell hyporesponsiveness by intrahepatic modulation of donor antigen-presenting cells

In this study, we examined the ability of varying populations of donor cells from B6 mice to induce hyporesponsiveness in T lymphocytes from C3H mice in vitro and in vivo. Small, resting B lymphocytes were inefficient stimulators of T-lymphocyte proliferation compared to splenic mononuclear cells (SMNC) and lipopolysaccharide (LPS)-induced B-cell blasts in vitro (P < 0.05). Pretreatment of SMNC with anti-B7-1 or anti-intracellular adhesion molecule-1 (ICAM-1) monoclonal antibodies (mAb) similarly resulted in inefficient stimulation of T-cell proliferation in vitro (P < 0.05). However, in vivo, only intrahepatic, but not intravenous, injection of donor cells into C3H mice resulted in decreased T-lymphocyte proliferation in response to restimulation by alloantigen. This effect was most pronounced following intrahepatic injection of resting B lymphocytes or SMNC pretreated with anti-ICAM-1 mAb compared to uninjected or intravenously injected mice (P < 0.05). The hyporesponsiveness was associated with an increased production of interleukin-4 (IL-4) by the responder T lymphocytes and correlated with enhanced skin allograft survival. These data demonstrate that intrahepatic injection of donor-derived cells induces T-lymphocyte hyporesponsiveness. The mechanism appears to be modulated by an ICAM-1-mediated signal resulting in expansion of an IL-4-producing T-lymphocyte population.     

Immunomodulating properties of the antibiotic novobiocin in human monocytes

We show that the coumeromycin antibiotic novobiocin, a potent inhibitor of ADP ribosylation, prevents lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, and IL-10 secretion in human peripheral blood mononuclear cells. It shares these cytokine-suppressing properties with other inhibitors of ADP ribosylation. We found that novobiocin prevents TNF-alpha production by inhibiting translation of the TNF-alpha mRNA. Elevated TNF-alpha levels in mice treated with D-galactosamine (GalN)-LPS or GalN-TNF were not reduced by novobiocin; however, the drug exhibited hepatoprotective properties. Novobiocin causes downregulation of the surface molecules on monocytes, among which CD14 was the most affected. The diminished expression of surface molecules was not observed on T and B lymphocytes. Similar to other inhibitors of ADP ribosylation, novobiocin prevents LPS-induced phosphate labelling of gamma-actins.     

Possible role of interleukin-10 (IL-10) and CD40 ligand expression in the pathogenesis of hypergammaglobulinemia in human immunodeficiency virus infection: modulation of IL-10 and Ig production after intravenous Ig infusion

The mechanisms leading to polyclonal hypergammaglobulinemia in patients with human immunodeficiency virus (HIV) infection are not well understood. In light of the important role of interleukin-10 (IL-10) and the interaction between CD40 and CD40 ligand in the normal regulation of B-lymphocyte function and Ig production, we examined these parameters in 24 HIV-infected patients. Both plasma IL-10 levels and the percentage of CD4(+) and CD8(+) lymphocytes expressing CD40 ligand were significantly higher in the patients than in the 10 blood donor controls. Serum IgG correlated positively with circulating IL-10 levels and the percentage of CD4(+) lymphocytes expressing CD40 ligand. Furthermore, a single bolus infusion of intravenous Ig (0.4 g/kg) in 8 HIV-infected patients caused a further increase in IL-10 levels in plasma and an increase in both IL-10 and IgG production in peripheral blood mononuclear cell cultures. In another patient group (Wegener's granulomatosis) receiving a single bolus infusion of intravenous Ig, a similar increase in plasma IL-10 levels was found, suggesting that this may be a general effect of intravenous Ig. In patients with HIV infection, our data suggest that a vicious cycle may be operative where high endogenous Ig levels may enhance IL-10 production that, in turn, leads to higher Ig production.     

Vascular endothelial growth factor tightly regulates in vivo development of murine hepatocellular carcinoma cells

Cytokines from peripheral blood mononuclear cells (PBMC) from human T lymphotropic virus (HTLV)-II-infected persons were studied to delineate the mechanism(s) of spontaneous lymphocyte proliferation (SLP). Culturing HTLV-II-infected PBMC that spontaneously proliferate (SLP+) resulted in greater mRNA expression and production of interferon-gamma, interleukin (IL)-4, and IL-5, with a concomitant decrease in IL-10, than was seen with nonproliferating (SLP ) and normal PBMC. While IL-2 mRNA expression was higher, production was lower in SLP+ PBMC than in SLP and normal PBMC, implying that the proliferating cells are utilizing IL-2. Neutralization of IL-2 resulted in partial inhibition, suggesting that other cytokines also affect SLP. Addition of recombinant IL-10 inhibited the proliferation of SLP+ PBMC. Further, blocking costimulatory signals with monoclonal antibodies against CD80/CD86 resulted in increased IL-10 production with concomitant inhibition of SLP. The mechanism(s) underlying HTLV-II-associated SLP in vitro involve increased utilization of IL-2 and down-regulation of IL-10.