Intranuclear rodlets in woodchuck thyroid follicular cells

Summary Intranuclear rodlets are seen in thyroid follicular cells of woodchucks fixed during summer and fall. They are not observed in winter and spring animals. A functional significance of these structures is suggested.Supported in part by PHS 05429-13-5 (P.P. Krupp, Department of Anatomy) and PHS R01 10254 (E.A. Sims, Metabolic Unit, Department of Medicine)     

Association of B-1 B cells with follicular dendritic cells in spleen

Although CD5(+) B-1 B cells have been recognized as an infrequent B cell subset in mice for many years, attempts to identify their histologic location in normal mouse spleen have proven difficult due to both their paucity and low level expression of CD5. In this study we have studied V(H)11/D(H)/J(H) gene-targeted mice, V(H)11t, that develop elevated numbers of CD5(+) V(H)11/V(k)9 B cells with an anti-phosphatidylcholine (anti-PtC) autoreactive specificity, allowing B-1 B cell detection by anti-PtC Id-specific Abs in spleen section staining. Using this approach we found that anti-PtC B-1 cells first appear within the white pulp in neonates, expand in association with follicular dendritic cells (FDC), and localize more centrally than other (non-B-1) IgD(high) follicular B cells in adults. Among neonatal B cells, CD5(+) B-1 cells in both normal and V(H)11t mouse spleen and peritoneal cavity express the highest levels of CXCR5, which is important for FDC development. Injection of purified spleen or peritoneal B-1 cells into RAG knockout mice resulted in B-1 cell follicle formation in spleen, inducing FDC development and plasma cell generation. These results indicate that B-1 B cells are the first B cells to express fully mature levels of CXCR5, thereby promoting the development of FDC.     

The function and origin of follicular dendritic cells

Follicular dendritic cells (dendritic reticular cells) in germinal centres bind antigen-antibody complexes via C3 receptors and retain the complexes at their surface for long periods of time. The follicular dendritic cells (FDC) are distinct from macrophages and from dendritic cells found in T-dependent areas, and are not derived from bone marrow stem cells. On histological evidence it has been proposed that they are derived from reticulum cells. Complexes are probably transported to FDC by a subpopulation of B cells in the marginal zone. Binding of complexes to FDC causes germinal centre enlargement and is a very efficient, and possibly essential stimulus to the generation of B memory cells which recognize epitopes on antigen or antibody in the complexes. An hypothesis is discussed which draws together these observations and suggests that antigen on FDC plays a central role in control of humoral immunity.     

Thyroglobulin may affect telomerase activity in thyroid follicular cells

Telomerase (TA) activity is known to be present in malignant tumor cells, but not in most somatic differentiated cells. TA shows relatively high activity in thyroid cancer cells, but reports vary. This fact prompted us to elucidate whether cell component inhibitors of TA in the thyroid follicles can modulate its activity. The activity of TA extracted from Hela cells was inhibited by mixing with the supernatant fraction of human thyroid tissue extract. To examine the effect of iodine, thyroid hormones (l-T3 and l-T4) and human thyroglobulin (hTg) contained in the thyroid follicles, l-T3, l-T4 and hTg were added to the TRAP assay system in vitro, using TA from Hela cells. Iodine, l-T3 and l-T4 did not affect TA activity, but hTg inhibited the TA activity in a dose-dependent manner (IC(50) of hTg: ca 0.45 microM: inhibiting concentration of hTg was from 0.15 microM to 3.0 microM). The hTg inhibition was not evident in the RT-PCR system, suggesting no effect of hTg on Taq DNA polymerase activity. The hTg inhibition of TA activity was attenuated by dNTP but not significantly by TS primer. These data suggest that hTg contained in thyroid follicular cells of various thyroid diseases may affect the TA activity measured in biopsied thyroid specimens, and that the reduction of the TA activity by hTg may induce slow progression and growth, and low grade malignancy of thyroid cancer, particularly differentiated carcinoma.     

Steroid metabolism by avian ovarian cells during follicular maturation

The profiles of steroid hormones produced by ovarian cells from the domestic hen were examined. Theca cells from the immature, small white follicles (SWFT), the third largest (T3), and largest (T1) preovulatory follicles, and the ruptured, postovulatory follicle (POFT) were incubated for 3 h at 37 degrees with [3H] progesterone (Prog) or [3H] pregnenolone (Preg). Granulosa cells from the largest preovulatory follicle were incubated with [3H] Preg or were coincubated with theca cells and [3H] Preg. The production of specific steroid metabolites was determined on the basis of coelution of radioactivity with known standard compounds, using an isocratic high-pressure liquid chromatography (HPLC) technique. Granulosa cells converted 93% of [3H] Preg substrate to Prog. More Prog was utilized by T3 cells than by T1 and SWFT cells, either when [3H] Prog was the substrate or when coincubated with granulosa cells and [3H] Preg. The major metabolites of Prog were androstenedione, 17-hydroxyprogesterone, and an unidentified compound with an elution time of 53 min. The POFT cells metabolized [3H] Prog to the same extent as T3 cells did, but their profile of steroidogenesis favored production of the unidentified 53 min metabolite. SWFT cells utilized the least amount of [3H] Preg substrate. The results point to marked changes in enzyme activities in theca cells during maturation and following ovulation.     

Emperipolesis of lymphoid cells by human follicular dendritic cells in vitro.

Isolated follicular dendritic cells (FDCs) showed true and pseudoemperipolesis of fresh tonsillar lymphocytes, even after long-term (50-day) cultivation. Emperipolesis by FDCs was not restricted by allotype specificity, nor was it inhibited by the addition of antibodies against MHC-I & II antigens. Follicular dendritic cells predominantly engulfed B-cells; monocytes and macrophages were not found between FDC cytoplasmic extensions. When highly purified T-cell populations were added to FDC cultures emperipolesis of T-cells occurred, particularly those of the CD4-positive phenotype. Mitoses appeared within 6 h in the emperipolesed lymphocytes and, after an additional 18 h, some lymphocytes exhibited apoptosis.     

Precise localization of antigens on follicular dendritic cells

Summary Horse-spleen ferritin or bovine serum albumin conjugated to colloidal gold (BSA-gold) were injected subcutaneously in preimmunized mice. In draining lymph nodes both antigens were located in macrophages or between the cytoplasmic processes of follicular dendritic cells (FDC). Some of the antigens remained trapped on FDC until day 31 after injection. Simultaneous injection of both antigens showed that they were located between the infoldings of the same FDC. These cells are thus able to retain at least two different antigens on their surface. The peculiar arrangement of ferritin between the cytoplasmic infoldings suggests that this antigen is fixed on both cell membranes by specific antibodies. The trapped immune complexes could thus stabilize the FDC membrane system.The antigen retention requires the presence of specific antibodies since BSA-gold or ferritin injected without preimmunization were not found between FDC processes. Nonantigenic materials, such as colloidal gold or carbon particles, are not trapped by FDC, except when injected in large amounts.The antigens were trapped on the surface of FDC, however unfrequently in close contact with lymphocytes. FDC might protect lymphocytes against an excess of immune complexes and act as regulators of contacts between lymphocytes and immune complexes.Abbreviations BSA bovine serum albumin - BSA-gold BSA conjugated to colloidal gold particles - FDC follicular dendritic cells     

Thyroglobulin may affect telomerase activity in thyroid follicular cells

Telomerase (TA) activity is known to be present in malignant tumor cells, but not in most somatic differentiated cells. TA shows relatively high activity in thyroid cancer cells, but reports vary. This fact prompted us to elucidate whether cell component inhibitors of TA in the thyroid follicles can modulate its activity. The activity of TA extracted from Hela cells was inhibited by mixing with the supernatant fraction of human thyroid tissue extract. To examine the effect of iodine, thyroid hormones (?-T3 and ?-T4) and human thyroglobulin (hTg) contained in the thyroid follicles, ?-T3, ?-T4 and hTg were added to the TRAP assay system in vitro, using TA from Hela cells. Iodine, ?-T3 and ?-T4 did not affect TA activity, but hTg inhibited the TA activity in a dose-dependent manner (IC₅₀ of hTg: ca 0.45 μM: inhibiting concentration of hTg was from 0.15 μM to 3.0 μM). The hTg inhibition was not evident in the RT-PCR system, suggesting no effect of hTg on Taq DNA polymerase activity. The hTg inhibition of TA activity was attenuated by dNTP but not significantly by TS primer. These data suggest that hTg contained in thyroid follicular cells of various thyroid diseases may affect the TA activity measured in biopsied thyroid specimens, and that the reduction of the TA activity by hTg may induce slow progression and growth, and low grade malignancy of thyroid cancer, particularly differentiated carcinoma.     

Cell population kinetics of thyroid follicular cells in infant rats

Abstract. T cell population kinetics of thyroid follicular cells in rats were studied by means of autoradiography and a statmokinetic technique. During the first fortnight after birth no significant changes in the mitotic index (MI) and labelling index (LI) were found. In the next 2 weeks a constant decrease in the number of proliferating cells occurs. In 10-day old animals 40% of the follicular cells were in the cell cycle (GF); 3.25 ± 0.77 (SEM) % in the S phase and 0.18 ± 0.04% in mitoses (MI). Day–night changes in the LI and mitotic rate (MR) indicated a peak value at 13.30 hours with a lowest value at 22.30 hours. The mean LI and MR averaged over the whole 24 hr were 3.1 ± 0.1% and 122.2 ± 18.1%, respectively. In 10-day old animals, using the fraction of labelled mitoses (FLM) method the median cell cycle time ( T _C) was 79 hr and the phase durations were T _G1—64.6 hr, T _s—8.2 hr and T _G2—5.1 hr. The decrease in the number of proliferating cells with the age of the animals is considered to be a result of both cell cycle prolongation and in growth fraction reduction.     

Developmental potential of mouse follicular epithelial cells and cumulus cells after nuclear transfer.

The developmental potential after nuclear transfer of mouse follicular epithelial cells cultured in vitro was examined. Follicular epithelial cells surrounding growing oocytes (type 5, diameter of oocytes, 62.6 +/- 5.9 microm; n = 14) were obtained from ovaries of adult mice. Before nuclear transfer, cells were cultured for several passages and subjected to serum starvation for several days. When the nuclear-transfer oocytes were at the 2-cell stage, serial nuclear transfer was performed. Additionally, cumulus cells surrounding ovulated oocytes were used as nuclear donors, with or without thermal stimulation (from -25 degrees C to 60 degrees C for 10 min) before nuclear transfer. Nuclear-transfer oocytes with follicular epithelial cells developed into blastocysts (34%) after serial nuclear transfer, and 4 living fetuses on Day 10.5 (25%, 16 transferred) and 1 dead fetus on Day 19.5 of pregnancy (3%, 30 transferred) were obtained after transfer to recipients. Although blastocysts (20%) were obtained after serial nuclear transfer of cumulus cells, only one implantation site without a fetus was observed on Day 10.5 of pregnancy. Thermal stimulation of cumulus cells before nuclear transfer did not enhance the ability to develop into fetuses or blastocysts.     

Persistence of infectious HIV on follicular dendritic cells

Follicular dendritic cells (FDCs) trap Ags and retain them in their native state for many months. Shortly after infection, HIV particles are trapped on FDCs and can be observed until the follicular network is destroyed. We sought to determine whether FDCs could maintain trapped virus in an infectious state for long periods of time. Because virus replication would replenish the HIV reservoir and thus falsely prolong recovery of infectious virus, we used a nonpermissive murine model to examine maintenance of HIV infectivity in vivo. We also examined human FDCs in vitro to determine whether they could maintain HIV infectivity. FDC-trapped virus remained infectious in vivo at all time points examined over a 9-mo period. Remarkably, as few as 100 FDCs were sufficient to transmit infection throughout the 9-mo period. Human FDCs maintained HIV infectivity for at least 25 days in vitro, whereas virus without FDCs lost infectivity after only a few days. These data indicate that HIV retained on FDCs can be long lived even in the absence of viral replication and suggest that FDCs stabilize and protect HIV, thus providing a long-term reservoir of infectious virus. These trapped stores of HIV may be replenished with replicating virus that persists even under highly active antiretroviral therapy and would likely be capable of causing infection on cessation of drug therapy.     

Isolation and long-term cultivation of human tonsil follicular dendritic cells

Highly purified follicular dendritic cells (FDC) were isolated from human tonsils and cultivated for up to 150 days. The cell separation method employed produced pure aggregates (FDC-clusters) composed of FDC and germinal center lymphoid cells, useful for the analysis of the relationship between these two cell types and of the behavior of FDC in culture. During the first few days of culture, lymphoid cells located between FDC extensions survived better than those which were free or partly covered by FDC. After 6 days, the lymphoid population degenerated and only the FDC survived. The unique antigenic pattern of FDC (positive for HLA-DR. DRC-1, CD14b, CD21, CD23, CD35) disappeared within a few days of culture. Recombinant interferon-gamma exerted a positive effect either on retaining HLA-DR expression or on the reexpression of these antigens by FDC. HLA-ABC antigens were traced until the 10th day and desmosomal junctions until the 14th day. Subsequently, FDC presented peculiar features, including oval and rhomboid shapes, one to ten nuclei, fine amoeboid extensions, stress fibers and a radical dense zone in their cytoplasm. FDC possessed actin, tubulin and vimentin, but neither desmin nor cytokeratin. After 40 days of culture, FDC enlarged and were covered with abundant membrane extensions. Even when kept as long as 150 days in vitro. FDC did not proliferate in any of the culture conditions employed.     

PTEN expression in ovine granulosa cells increases during terminal follicular growth

In the present paper, we have studied the expression of the Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) and its putative biological role in the sheep ovary. We found by Northern-blot, immunohistochemistry and immunoblot that PTEN is highly expressed in granulosa cells from large differentiated follicles (LF) in comparison with small proliferating follicles (SF) (P < 0.001), with no clear effect of follicle quality. Moreover, the PTEN lipid phosphatase activity is also higher in LF than in SF (P < 0.01). In contrast, levels of the phosphorylated form of AKT (pAKT) are lower in LF than in SF (P < 0.0001). IGF-I and insulin but not FSH, LH or forskolin are able to stimulate the expression of PTEN mRNA (P < 0.001) and protein by ovine granulosa cells after 48 h of culture in vitro. An IGF-1 time course analysis showed that expression of PTEN protein appeared after 12h of culture, concomitant with the fall of the pAKT levels, which peaked after 6h of stimulation with IGF-I. Moreover, transfection experiments showed that overexpression of PTEN in ovine granulosa cells induced a decrease and an increase in E2F and p27 promoter activity, respectively (P < 0.05). Overall, our present data show for the first time that the expression of PTEN increases during terminal follicular growth. This increase, that might be induced by IGF-I but not FSH, would participate in the proliferation/differentiation transition of ovine granulosa cells in differentiating follicles.     

Subpopulations of physiological and ovarian follicular fluid peptide induced apoptotic cells

Ovarian follicular fluid peptide (OFFP) purified from sheep ovaries enhances apoptotic changes in ovarian granulosa cells of mice. To get an insight into the cell subpopulations responding to OFFP, the heterogeneity of granulosa cells was resolved. Subpopulations of granulosa cells were obtained from ovaries of immature mice treated with PMSG alone and autopsied 48 hr (control) and 72 hr after injection (atretic) and from animals injected OFFP 24 hr after PMSG injection and autopsied 24 hr later (OFFP treated) by separation on discontinuous Percoll gradient. Four fractions were collected and studied for their relative distributions and percent apoptotic cells measured by acridine orange staining. FSH binding to granulosa cell (sedimenting as a major) fraction was studied by radio receptor assay. There is a difference in densities in subpopulations of apoptotic cells induced by OFFP and those generated during the physiological process of atresia. This difference may be a reflection of different granulosa cell subpopulations involved in peptide response or differences in phases as the cells transit from normal to apoptotic phenotype. FSH binding to granulosa cells from OFFP treated animals was significantly less than those from control and atretic group.     

Foxp3+ follicular regulatory T cells control the germinal center response

Follicular helper (T(FH)) cells provide crucial signals to germinal center B cells undergoing somatic hypermutation and selection that results in affinity maturation. Tight control of T(FH) numbers maintains self tolerance. We describe a population of Foxp3(+)Blimp-1(+)CD4(+) T cells constituting 10-25% of the CXCR5(high)PD-1(high)CD4(+) T cells found in the germinal center after immunization with protein antigens. These follicular regulatory T (T(FR)) cells share phenotypic characteristics with T(FH) and conventional Foxp3(+) regulatory T (T(reg)) cells yet are distinct from both. Similar to T(FH) cells, T(FR) cell development depends on Bcl-6, SLAM-associated protein (SAP), CD28 and B cells; however, T(FR) cells originate from thymic-derived Foxp3(+) precursors, not naive or T(FH) cells. T(FR) cells are suppressive in vitro and limit T(FH) cell and germinal center B cell numbers in vivo. In the absence of T(FR) cells, an outgrowth of non-antigen-specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, the T(FH) differentiation pathway is co-opted by T(reg) cells to control the germinal center response.     

Reassessing the function of immune-complex retention by follicular dendritic cells

The close association of follicular dendritic cells (FDCs) and germinal-centre B cells has fostered the idea that B-cell recognition of retained antigen that is presented on the surface of FDCs is important for affinity maturation and memory B-cell development. We argue that the retention of immune complexes is not required for germinal-centre development, affinity maturation and memory B-cell maintenance. Instead, it is probable that FDCs support B-cell proliferation and differentiation in a non-specific manner. Other potential roles of immune complexes retained by FDCs are discussed.     

Gonadotropic regulation of prostaglandin production by ovarian follicular cells of the pig

Prepubertal gilts were treated with 750 IU pregnant mare's serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa cells (GC) and theca interna cells (TC) from follicles of gilts 72 h (GC-72 and TC-72, respectively) and 108 h (GC-108 and TC-108 h, respectively) after PMSG treatment were cultured for 0, 12, 24, and 36 h in medium with or without luteinizing hormone (LH), dibutyryl cyclic adenosine 3',5'-monophosphate [Bu)2cAMP), calcium ionophore (A23187), and/or arachidonic acid (AA), and the production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. TC-72 was the principal source of PGs 72 h after PMSG. At 108 h, the production of PGE and PGF by GC was increased 10- and 30-fold, respectively, whereas corresponding increases by TC were 2-fold. LH and A23187 significantly stimulated PGE and PGF production by both GC-72 and TC-72, but only thecal PG production was stimulated by (Bu)2cAMP. LH had minimal or no effect on PG production by GC-108 and TC-108, but A23187 (GC-108, TC-108) and (Bu)2cAMP (TC-108) were stimulatory. Basal PG production by GC-72, GC-108, and TC-108 was stimulated by AA. However, production by GC and TC cultured in medium containing AA and LH, A23187, or (Bu)2cAMP was not different from that produced by AA alone. These findings suggested that GC and TC can synthesize PGs in vitro, but AA availability is rate-limiting in GC. After exposure to hCG in vivo, the capacity of both cell types to produce PGs is increased but is limited by AA availability.(ABSTRACT TRUNCATED AT 250 WORDS)