Distinct role of neonatal and adult monocytes in the regulation of the in vitro antigen-induced plaque-forming cell response in man

Mononuclear cells from human cord blood (CBMC) are able to mount an antigen-specific IgM plaque-forming cell (PFC) response after primary in vitro stimulation with the T cell-dependent antigen ovalbumin (OA). The antigen dose-response relationship for the induction of PFC in cultures of CBMC is represented by a bell-shaped curve comparable to that found for mononuclear cells from adult peripheral blood (adult PBMC). The dose of OA optimal for the induction of a response in cultures of CBMC consistently, however, is 100-fold lower than the antigen dose optimal for adult PBMC (0.03 microgram OA/ml vs 3.0 micrograms OA/ml). Results obtained from co-culture experiments in which semiallogeneic combinations of parental/neonatal lymphocytes and monocytes were stimulated with a variable dose of OA indicate that the adherent cell (AC) plays a pivotal role in the establishment of the optimum antigen dose. From experiments using antigen-pulsed AC, it was concluded that neonatal and adult AC differ in their antigen handling capacity. In the presence of the prostaglandin synthetase inhibitor indomethacin the antigen dose-response relationship for the induction of PFC in cultures of CBMC shifts to an "adult type" of curve. From pulsing experiments it emerges that indomethacin affects the interaction between antigen and monocytes. Indomethacin causes an enhancement of the expression of HLA-DR at the surface of neonatal as well as adult AC; this can be down regulated by the addition of prostaglandin E2 (PGE2). The addition of PGE2 to cultures of adult PBMC leads to a shift of the optimal antigen dose for induction of PFC toward lower concentrations. Although higher levels of PGE2 were measured in the supernatant of cultured neonatal AC compared with adult AC, it seems unlikely that this observation can explain the distinct antigen dose-response relationship for the induction of a PFC response in cultures of CBMC.     

Regulation of NK cell activity by prostaglandin E2: the role of T cells

The influence of T cells on the production of prostaglandins (PGE2) and on PGE2-mediated regulation of natural killer (NK) activity was studied. Supernatants from peripheral blood mononuclear cells (PBMC) and from PBMC depleted of T cells ((PBMC)-T), both of which had been incubated in plastic petri dishes overnight, contained similar amounts of PGE2, as detected by radioimmunoassay and by their potential to inhibit NK activity of peripheral blood mononuclear cells in a 51Cr release assay with K 562 cells as the target population. However, the NK activity of PBMC was inhibited significantly more strongly (P less than 0.005) by PGE2-containing supernatants than was the NK activity of (PBMC)-T. In further assays, in which synthetic PGE2 in concentrations of 10(-4) and 10(-5)M was added, a significant inhibition of NK activity was observed in PBMC populations (P less than 0.05), but not in (PBMC)-T. Thus, T cells did not seem to be involved in the control of PGE2 production, but their presence was necessary for PGE2-mediated inhibition of NK activity.     

In vitro CD4+ lymphocyte transformation and infection in a rabbit model with a molecular clone of human T-cell lymphotrophic virus type 1.

We transfected human and rabbit peripheral blood mononuclear cells (PBMC) with the ACH molecular clone of human T-cell lymphotropic virus type 1 (HTLV-1) to study its in vitro and in vivo properties. PBMC transfected with ACH were shown to transfer infection to naive PBMC. ACH transformed rabbit PBMC, as indicated by interleukin-2-independent proliferation of a transfectant culture. This transformant culture was shown by flow cytometric analysis to be a CD4+ CD25+ T-lymphocyte population containing, as determined by Southern blot analysis, at least three integrated HTLV-1 proviral copies. HTLV-1 infection was produced in rabbits inoculated with ACH-transfected, irradiated PBMC. Inoculated rabbits seroconverted to positivity for antibodies against HTLV-1 and had steady or rising HTLV-1 enzyme-linked immunosorbent assay antibody titers. Western blot (immunoblot) analysis revealed sustained seroconversion of rabbits to positivity for antibodies against all major viral antigenic determinants. Infection of rabbits was further demonstrated by antigen capture assay of p24 in PBMC and lymph node cultures and PCR amplification of proviral sequences from PBMC. These data suggest that ACH, like wild-type HTLV-1, infects and transforms primary CD4+ T lymphocytes and is infectious in vivo. This clone will facilitate investigations into the role of viral genes on biological properties of HTLV-1 in vitro and in vivo.     

Immune activation suppresses initiation of lytic Epstein-Barr virus infection

Primary infection with Epstein-Barr virus (EBV) is asymptomatic in children with immature immune systems but may manifest as infectious mononucleosis, a vigorous immune activation, in adolescents or adults with mature immune systems. Infectious mononucleosis and chronic immune activation are linked to increased risk for EBV-associated lymphoma. Here we show that EBV initiates progressive lytic infection by expression of BZLF-1 and the late lytic genes gp85 and gp350/220 in cord blood mononuclear cells (CBMC) but not in peripheral blood mononuclear cells (PBMC) from EBV-naive adults after EBV infection ex vivo. Lower levels of proinflammatory cytokines in CBMC, used to model a state of minimal immune activation and immature immunity, than in PBMC were associated with lytic EBV infection. Triggering the innate immunity specifically via Toll-like receptor-9 of B cells substantially suppressed BZLF-1 mRNA expression in acute EBV infection ex vivo and in anti-IgG-stimulated chronically latently EBV-infected Akata Burkitt lymphoma cells. This was mediated in part by IL-12 and IFN-gamma. These results identify immune activation as critical factor for the suppression of initiation of lytic EBV infection. We hypothesize that immune activation contributes to EBV-associated lymphomagenesis by suppressing lytic EBV and in turn promotes latent EBV with transformation potential.     

Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13(II) is required for viral infectivity in vivo

Human T-lymphotropic virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia, encodes unique regulatory and accessory proteins in the pX region of the provirus, including the open reading frame II product p13(II). p13(II) localizes to mitochondria, binds farnesyl pyrophosphate synthetase, an enzyme involved in posttranslational farnesylation of Ras, and alters Ras-dependent cell signaling and control of apoptosis. The role of p13(II) in virus infection in vivo remains undetermined. Herein, we analyzed the functional significance of p13(II) in HTLV-1 infection. We compared the infectivity of a human B-cell line that harbors an infectious molecular clone of HTLV-1 with a selective mutation that prevents the translation of p13(II) (729.ACH.p13) to the infectivity of a wild-type HTLV-1-expressing cell line (729.ACH). 729.ACH and 729.ACH.p13 producer lines had comparable infectivities for cultured rabbit peripheral blood mononuclear cells (PBMC), and the fidelity of the start codon mutation in ACH.p13 was maintained after PBMC passage. In contrast, zero of six rabbits inoculated with 729.ACH.p13 cells failed to establish viral infection, whereas six of six rabbits inoculated with wild-type HTLV-1-expressing cells (729.ACH) were infected as measured by antibody responses, proviral load, and HTLV-1 p19 matrix antigen production from ex vivo-cultured PBMC. Our data are the first to indicate that the HTLV-1 mitochondrion-localizing protein p13(II) has an essential biological role during the early phase of virus infection in vivo.     

Effect of prostaglandins against alloxan-induced cytotoxicity to insulin secreting insulinoma RIN cells in vitro

In the present study, we studied the effect of various prostaglandins (PGs) on alloxan-induced cytotoxicity to rat insulinoma (RIN) cells. Of all the PGs tested, PGE(1), PGE(2), PGI(2), PGF(1 alpha), and PGF(3 alpha) protected RIN cells from alloxan-induced cytotoxicity (P<0.05 compared to alloxan), whereas thromboxane B(2) and 6-keto-PGF(1 alpha) were not effective. PGE(1) induces a statistically significant increase in the activities of superoxide dismutase and glutathione peroxidase and decrease in lipid peroxides in alloxan-treated RIN cells (P<0.001). PGE(1) restored nitric oxide/lipid peroxide ratio to normalcy, suggesting that PGE(1) suppresses oxidant stress induced by alloxan in RIN cells in vitro. Furthermore, PGE(1) prevented DNA damage and apoptosis induced by alloxan. These results indicate that PGE(1) prevents alloxan-induced cytotoxicity to RIN cells in vitro.     

Lipopolysaccharide and silica-stimulated mononuclear cell prostaglandin production in ulcerative colitis

Basal, lipopolysaccharide (LPS) and silica-stimulated prostaglandin (PG) production were compared between peripheral blood mononuclear cells (PBMNC) from UC patients and healthy subjects (HS). Basal and LPS-stimulated PBMNC PGI2, but not PGE2, production was greater in UC. LPS stimulated both PGE2 and PGI2 by PBMNC from HS and UC patients. Silica stimulated production of both PGs by cells from HS but only PGE2 by cells from UC patients. The differences in responses to silica and LPS may result from differences in activation of NFkappaB or, alternatively, prior sensitisation to one of these agents. That PBMNC PGE2 production is not increased in UC, as it is in Crohn's disease, suggests that there are differences in PBMNC behaviour between these two diseases.     

1,25-Dihydroxyvitamin D(3) and prostaglandin E(2) act directly on circulating human osteoclast precursors.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and prostaglandin E(2) (PGE(2)) are known to influence osteoclast formation indirectly through their effects on osteoblasts. To determine whether 1, 25(OH)(2)D(3) and PGE(2) also have a direct effect on circulating osteoclast precursors, these factors were added to long-term cultures of human peripheral blood mononuclear cells (PBMCs) in the presence of osteoprotegerin ligand and macrophage colony-stimulating factor (M-CSF) (+/-dexamethasone). The number of TRAP(+) and VNR(+) multinucleated cells and the area of lacunar resorption were decreased when 1,25(OH)(2)D(3) alone was added. A marked increase in resorption pit formation was noted when the combination of 1, 25(OH)(2)D(3) and dexamethasone was added to PBMC cultures. Dose-dependent inhibition of osteoclast formation and lacunar resorption was seen when PGE(2) was added to PBMC cultures in both the presence and the absence of dexamethasone. Thus, 1,25(OH)(2)D(3) and PGE(2) not only influence osteoclast formation in the presence of bone stromal cells but also act directly on circulating osteoclast precursors to influence osteoclast differentiation.     

Prostaglandin E2 inhibits human T-cell proliferation after crosslinking of the CD3-Ti complex by directly affecting T cells at an early step of the activation process

We have studied the effects of prostaglandin E2 (PGE2) on in vitro human T-cell activation induced by crosslinking of the CD3-Ti complex with the monoclonal anti-CD3 antibodies OKT3 and UCHT-1. PGE2 (greater than or equal to 3 X 10(-9) M) when added simultaneously with anti-CD3 to cultures of peripheral blood mononuclear cells (PBMC), significantly suppressed, in a dose-dependent way, T-cell proliferation (P less than 0.002). However, when T cells were first preactivated with OKT3 for 3 days, subsequent proliferation driven by recombinant interleukin 2 (IL-2) was not inhibited by addition of PGE2. This indicates that PGE2 affects the activation step resulting from crosslinking of CD3-Ti, but not the IL-2-driven proliferative phase. Other manifestations of T-cell activation were therefore examined. Both IL-2 production and the expression of receptors for IL-2 (as detected with the anti-Tac monoclonal antibody) were inhibited by PGE2. The addition of purified interleukin 1 (IL-1) or recombinant IL-2 to the cultures did not reverse the inhibiting effect of PGE2 on IL-2-receptor expression. PGE2, added at the time of culture initiation, also inhibited T-cell proliferation in cultures which were supplemented with exogenous IL-1 or IL-2. Proof for a direct effect of PGE2 on T cells was obtained in experiments in which monocyte-depleted T cells were stimulated, in the presence of IL-1, with solid-phase-bound anti-CD3 antibody. Proliferation of T cells in this system is accessory cell independent and still was strongly inhibited by PGE2. Finally, preincubation of PBMC with PGE2 (3 X 10(-6) M) for 48 hr did not result in the generation of suppressor cells for anti-CD3-induced T-cell proliferation or for IL-2 production. Our results demonstrate that PGE2 has a direct inhibitory effect on an early step of T-cell activation, resulting in decreased IL-2 production, decreased IL-2-receptor expression, decreased responsiveness to exogenous IL-2, and decreased proliferation. However, PGE2 does not affect IL-2-driven proliferation of activated T cells. The inhibitory effect on T-cell activation is not mediated through suppressor T cells, nor through inhibition of accessory cell function.     

Receptors and signaling mechanisms required for prostaglandin E2-mediated regulation of mast cell degranulation and IL-6 production

Mast cells are implicated in the pathogenesis of a broad spectrum of immunological disorders. These cells release inflammatory mediators in response to a number of stimuli, including IgE-Ag complexes. The degranulation of mast cells is modified by PGs. To begin to delineate the pathway(s) used by PGs to regulate mast cell function, we examined bone marrow-derived mast cells (BMMC) cultured from mice deficient in the EP(1), EP(2), EP(3), and EP(4) receptors for PGE(2). Although BMMCs express all four of these PGE(2) receptors, potentiation of Ag-stimulated degranulation and IL-6 cytokine production by PGE(2) is dependent on the EP(3) receptor. Consistent with the coupling of this receptor to G(alphai), PGE(2) activation of the EP(3) receptor leads to both inhibition of adenylate cyclase and increased intracellular Ca(2+). The magnitude of increase in intracellular Ca(2+) induced by EP(3) activation is similar to that observed after activation of cells with IgE and Ag. Although PGE alone is not sufficient to initiate BMMC degranulation, stimulation of cells with PGE along with PMA induces degranulation. These actions are mediated by the EP(3) receptor through signals involving Ca(2+) mobilization and/or decreased cAMP levels. Accordingly, these studies identify PGE(2)/EP(3) as a proinflammatory signaling pathway that promotes mast cell activation.     

Mechanism of effector cell blockade. III. The increased sensitivity of neonatal PFC to effector cell blockade

The response to staphylococcal protein A (SPA) of unfractionated, T-cell-enriched (TCE), and T-cell-depleted (TCD) populations of both adult mononuclear cells (AMC) and cord blood mononuclear cells (CBMC) was studied. Adult unfractionated and TCE populations formed more T-lymphocyte colonies than did similar populations of CBMC. However, TCD populations of CBMC formed B-lymphocyte colonies at least as well as did the TCD population of AMC. These results suggest that B cells from CBMC may be more mature than CBMC T-cell populations.     

Prostaglandin E2 induces degranulation-independent production of vascular endothelial growth factor by human mast cells

Mast cells accumulate in large numbers at angiogenic sites, where they have been shown to express a number of proangiogenic factors, including vascular endothelial growth factor (VEGF-A). PGE(2) is known to strongly promote angiogenesis and is found in increased levels at sites of chronic inflammation and around solid tumors. The expression pattern of VEGF and the regulation of VEGF-A by PGE(2) were examined in cord blood-derived human mast cells (CBMC). CBMC expressed mRNA for five isoforms of VEGF-A and other members of the VEGF family (VEGF-B, VEGF-C, and VEGF-D) with strong expression of the most potent secretory isoforms. PGE(2) was a very strong inducer of VEGF-A(121/165) production by CBMC and also elevated VEGF-A mRNA expression. The amount of VEGF-A(121/165) protein production induced by PGE(2) was 4-fold greater than that induced by IgE-mediated activation of CBMC. Moreover, the response to PGE(2) as well as to other cAMP-elevating agents such as forskolin and salbutamol was observed under conditions that were not associated with mast cell degranulation. CBMC expressed substantial levels of the EP(2) receptor, but not the EP(4) receptor, when examined by flow cytometry. In contrast to other reported PGE(2)-mediated effects on mast cells, VEGF-A(121/165) production occurred via activation of the EP(2) receptor. These data suggest a role for human mast cells as a potent source of VEGF(121/165) in the absence of degranulation, and may provide new opportunities to regulate angiogenesis at mast cell-rich sites.     

Nitric oxide-mediated immune complex-induced prostaglandin E(2) production by peripheral blood mononuclear cells of humans infected with Schistosoma mansoni.

Granuloma reaction around Schistosoma mansoni eggs is the prominent lesion in human schistosomiasis. Studies have suggested the involvement of a series of suppressive mechanisms in the control of this reaction. Using an in vitro model of granuloma formation, we have shown that immune complexes (IC) isolated from sera of chronic intestinal schistosomiasis patients were able to reduce granulomatous reaction developed against soluble egg antigen-conjugated polyacrylamide beads. In this system, the role of the l-arginine-nitric oxide (NO) pathway in the formation of prostaglandin E(2) (PGE(2)) by human peripheral blood mononuclear cells (PBMC) of patients infected with schistosomiasis was investigated using IC. Preincubation of PBMC with IC produced a significant increase of both nitrite and PGE(2) levels in the cell supernatant. This effect was inhibited by coincubation of cells with Nomega-nitro-l-arginine methyl ester (L-NAME), a NO synthase inhibitor, showing that the release of PGE(2) subsequent to IC stimulation was driven by NO. The inhibitory effect of L-NAME on PGE(2) release by IC-treated PBMC was reversed by sodium nitroprusside, a known NO donor. Our results indicate that NO could be an important second signal for the stimulation of PGE(2) production induced by IC in PBMC from human schistosomiasis patients.     

Interferon γ stimulates prostaglandin E2 production by mouse Kupffer cells

When mononuclear phagocytes, including Kupffer cells, are activated by various agents, they synthesize and release arachidonic acid metabolites, prostaglandins (PGs) and leukotrienes (LTs). In this study, we examined the effect of in vitro Kupffer cell activation with recombinant murine IFN gamma on PGE2 and LTB4 secretion. IFN gamma enhanced PGE2 secretion, and this effect of IFN gamma was stronger than that of IL-1 or TNF. Moreover, IFN gamma promoted LTB4 release especially in the absence of PGs. On the other hand, dexamethasone and indomethacin inhibited and, EGTA and TMB-8, which reduce intracellular Ca++ Levels, blocked IFN gamma induced PGE2 production, which suggested that the activation of phospholipase A2 and cyclooxygenase in Kupffer cells requires the elevation of intracellular Ca++ levels.     

Human T-Lymphotropic Virus Type 2 (HTLV-2) Provirus in Circulating Cells of the Monocyte/Macrophage Lineage in Patients Dually Infected with Human Immunodeficiency Virus Type 1 and HTLV-2 and Having Predominantly Sensory Polyneuropathy

We investigated the presence of human T-lymphotropic virus type 2 (HTLV-2) DNA in the peripheral blood mononuclear cell subsets obtained from 18 patients coinfected with human immunodeficiency virus type 1 and HTLV-2, 6 of whom also had predominantly sensory polyneuropathy (PSP). HTLV-2 DNA and RNA were found in CD8- and CD19-positive cells, and, for patients with PSP, in CD14-positive cells as well. Furthermore, the patients with PSP had higher proviral loads than those without PSP.