Human angiotensin II type 1 receptor isoforms encoded by messenger RNA splice variants are functionally distinct.

Human tissues that express the angiotensin II (Ang II) type 1 receptor (hAT(1)R) can synthesize four distinct alternatively spliced hAT(1)R mRNA transcripts. In this study, we show that the relative abundance of these mRNA transcripts varies widely in human tissues, suggesting that each splice variant is functionally distinct. Here we demonstrate, for the first time, that the hAT(1)R-B mRNA splice variant encodes a novel long hAT(1)R isoform in vivo that has significantly diminished affinity for Ang II (i.e. >3-fold) when compared with the short hAT(1)R isoform (encoded by hAT(1)R-A mRNA splice variant). This reduced agonist affinity caused a significant shift to the right in the dose-response curve for Ang II-induced inositol trisphosphate production and Ca(2+) mobilization of the long hAT(1)R when compared with that of the short hAT(1)R. The functional differences between these isoforms allows Ang II responsiveness to be fine-tuned by regulating the relative abundance of the long and short hAT(1)R isoform expressed in a given human tissue.

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.

Susceptibility of S49 lymphoma cell membranes to hydrolysis by secretory phospholipase A(2) during early phase of apoptosis.

During cell death, plasma membranes of cells become vulnerable to attack by extracellular secretory phospholipase A(2). The purpose of this study was to identify the timing of this phenomenon relative to other events that occur during the process of cell death. Death was induced in S49 murine lymphoma cells by treatment with dexamethasone, dibutyryl cAMP, ionomycin, thapsigargin, or heat shock (1 h at 43 degrees C). The appearance of membrane susceptibility to secretory phospholipase A(2) was compared to the following apoptotic events: loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, early DNA damage assessed by the comet assay, and changes in cell size and internal complexity assessed by flow cytometry. Each inducer of death was distinct in the time course of events produced. Although dead cells were susceptible to the action of phospholipase A(2), live cells (impermeable to propidium iodide) also became vulnerable to the enzyme during characteristic time courses after exposure to each inducer. In fact, susceptibility to sPLA(2) was observed in each case prior to or concurrent with the earliest of the markers of apoptosis. These results demonstrate that the onset of susceptibility to sPLA(2) is an early event in apoptosis suggesting that changes in membrane structure may be relevant to initial aspects of the apoptotic process.

Follicular dendritic cells protect malignant B cells from apoptosis induced by anti-Fas and antineoplastic agents.

The observation that follicular dendritic cells (FDC) reduce apoptosis in B cells prompted the hypothesis that FDC might enhance tumor cell survival by protecting malignant B cells from apoptotic death. To test this notion, apoptosis was induced in B cell lymphomas by anti-Fas or various antineoplastic agents in the presence and absence of FDC. Apoptosis was detected and quantified by TUNEL analysis. Induction of apoptosis with anti-Fas, etoposide, cyclophosphamide, and busulfan was markedly antagonized by FDC at FDC to B cell ratios of >/=1:16. For example, treatment with 10 ng/ml anti-Fas caused 60-90% of A20 cells to undergo apoptosis in 6 h, whereas addition of FDC reduced apoptosis to background levels (3-15%). Similarly, treatment with busulfan induced apoptosis in 55-80% of A20 cells, whereas addition of FDC reduced B cell death to

Follicular dendritic cells mediated maintenance of primary lymphocyte cultures for long-term analysis of a functional in vitro immune system.

Primary lymphocyte cultures are important for analysis of cellular and molecular events occurring during immune responses. However, the lymphoid cells (especially B cells) typically only survive for a few days in vitro which limits studies. Establishment of long-term primary lymphocyte cultures where a functioning humoral immune responses can be maintained and regulated is still a challenge. Follicular dendritic cells (FDC) are immune accessory cells that reside in the follicles of secondary lymphoid organs and are known to protect lymphocytes from apoptosis. We hypothesized that addition of FDC to primary lymphocyte cultures may help maintain humoral immune responses in vitro as they do in vivo. To test the hypothesis, freshly isolated lymphocytes were cultured with or without FDC. The B cells in cultures were labeled using B220 and apoptotic cells were labeled using the TUNEL assay. Antibody production was monitored in supernatant fluids using ELISA. The results showed that FDC reduced apoptosis and helped sustain primary lymphocyte cultures and antibody production was maintained throughout the entire period (e.g., 8 weeks). This FDC dependent system should be useful for analysis of cellular and molecular events over extended periods in vitro.

Tingible body macrophages in regulation of germinal center reactions.

Tingible body macrophages (TBM), long thought simply as scavengers of apoptotic lymphocytes, are located in the unique microenvironment of germinal centers in close proximity to antigen-retaining follicular dendritic cells (FDC). Observations that TBM endocytose FDC-iccosomal (immune-complex coated bodies) antigen suggested that TBM might present this antigen and help regulate the germinal center reaction. To test for antigen presentation, the ovalbumin (OVA)-specific T(H) hybridoma, 3DO-54.8, which produces IL-2 on receiving effective presentation of OVA, were used as responders to OVA-bearing TBM. Results showed that OVA-bearing TBM failed to induce IL-2 production. Furthermore, addition of TBM to IL-2-inducing positive controls (B cells) not only failed to augment IL-2 production, but rather TBM significantly (55-90%) reduced B-cell induction of IL-2. We found that TBM were rich in prostaglandin by comparison with other nongerminal center lymph node macrophages and that addition of indomethacin to the cultures reversed the inhibitory effect of TBM. Depletion of TBM from enriched preparations, prior to addition to positive control cultures, also abrogated the inhibitory effect on IL-2 production. These data support the concept that TBM, within the unique microenvironment of germinal centers, may be specialized to downregulate the germinal center reaction.

Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG responses.

The addition of Ags to mononuclear leukocyte cultures typically elicits modest Ab responses, implying that cosignals beyond those provided by T cells and macrophages may be needed. Recently, we reported that Ab responses could be dramatically enhanced (10-1000-fold) by the addition of follicular dendritic cells (FDC), suggesting that FDC may provide an important costimulatory signal. This result prompted a study of molecules involved in FDC-mediated enhancement of Ab responses stimulated by specific Ag with memory T and B cells or nonspecifically by the addition of LPS. In this study, we report evidence supporting the concept that FDC bear a ligand that engages complement receptor II (CR2 or CD21) on B cells and provides a critical cosignal for both Ag-specific and polyclonal responses. A blockade of the CR2 ligand on FDC by the use of soluble CR2 or a blockade of CR2 on B cells by use of CR2 knockout mice (or B cells with CR2 blocked) reduced Ab responses from the microg/ml to the ng/ml range (10-1000-fold reductions). FDC from C3 knockout mice, which cannot generate the CR2-binding fragments (iC3b, C3d, and C3dg), were unable to provide costimulatory activity, suggesting the CR2 ligand on FDC consists of C3 fragments. FDC trap complement-activating Ag-Ab complexes, and it appears that FDC present B cells with both specific Ag to engage B cell receptors and a CR2 ligand to engage B cell-CR2. In short, optimal induction of specific Ab responses appears to require the combination of specific Ag and costimulatory molecules from both T cells and FDC.

Follicular dendritic cells and presentation of antigen and costimulatory signals to B cells.

This review focuses on how immunogens trapped by FDC in the form of Ag-Ab complexes productively signal B cells. In vitro. Ag-Ab complexes are poorly immunogenic but in vivo immune complexes elicit potent recall responses. FDC trap Ag-Ab complexes and make immune complex coated bodies or "iccosomes". B cells endocytose iccosomes, the Ag is processed, and T-cell help is elicited. In vitro, addition of FDC bearing appropriate Ag-Ab complex to memory T and B cells provoke potent recall responses (IgG and IgE). FDC also provide nonspecific costimulatory signals which augment B-cell proliferation and Ab production. B cell-FDC contact is important and interference with ICAM-1-LFA-1 interactions reduces FDC-mediated costimulation. Preliminary data suggest that a costimulatory signal may be delivered via CR2L on FDC binding CR2 on B cells. FDC can also stimulate B cells to become chemotactically active and can protect lymphocytes from apoptosis. FDC also appear to be rich in thiol groups and may replace reducing compounds such as 2 mercaptoethanol in cultures. In short, FDC-Ag specifically signals B cells through BCR, and FDC provide B cells with iccosomal-Ag necessary for processing to elicit T-cell help. In addition, FDC provide nonspecific signals that are important to promote B-cell proliferation, maintain viability, and induce chemotactic responsiveness.

Follicular dendritic cells (FDC) in retroviral infection: host/pathogen perspectives.

Follicular dendritic cells (FDC) are found in the follicles of virtually all secondary lymphoid tissues. In health, these cells trap and retain antigens (Ag) in the form of immune complexes and preserve them for months in their native conformation. FDC thus serve as a long-term repository of extracellular Ag important for induction and maintenance of memory responses. In retroviral infection, FDC trap and retain large numbers of retroviral particles with profound effects on FDC. FDC-trapped retrovirus induces follicular hyperplasia, and conventional Ag trapped prior to infection are lost and new Ag cannot be trapped. Concomitantly, antibody-forming cells (AFC) specific for Ag lost from FDC decrease followed by loss of specific serum antibody (Ab). Eventually, FDC die and follicular lysis occurs. From the pathogen perspective, binding to FDC is remarkably beneficial, bringing together virus and activated target cells that are highly susceptible to infection. Furthermore, FDC permit HIV to infect surrounding cells even in the presence of a vast excess of neutralizing Ab. Preliminary data suggest that FDC maintain virus infectivity-even when the virus cannot replicate. Thus retrovirus infection monopolizes FDC networks, thereby transforming the FDC Ag repository into a highly infectious retroviral reservoir.

Follicular dendritic cell-derived antigen and accessory activity in initiation of memory IgG responses in vitro.

Follicular dendritic cells (FDC) release Ag in developing germinal centers in the form of immune complex-coated bodies (iccosomes). Iccosomes are endocytosed by B cells, and the B cells process and present the FDC-derived Ag to T cells. By 3 days after Ag challenge, Ab-forming cells producing IgG specific for the iccosomal Ag emerge from the developing germinal centers and home to the bone marrow, where most Ab in a secondary response are produced. In addition to providing Ag, FDC exhibit potent accessory activities that promote B cell proliferation. These observations prompted the hypothesis that both FDC-derived Ag and FDC-derived Ag-independent secondary signal(s) are essential for optimal secondary Ab responses. To test this hypothesis, methods were developed to separate Ag-bearing iccosomes from intact FDC, and we then examined the ability of isolated iccosomes to elicit secondary Ab responses in vitro in the presence and absence of intact FDC. In the absence of FDC, iccosomes bearing the appropriate Ag elicited only minimal levels of specific IgG. Proliferation studies revealed that iccosomes lacked the FDC accessory activity necessary to augment B cell proliferation. When a source of FDC lacking the relevant Ag but exhibiting accessory activity was added to the iccosomal/Ag/lymphocyte mixture, dramatic increases in IgG specific for the iccosomal Ag were obtained (increases were from low ng/ml to microg/ml levels of specific IgG). The results suggest the concept that Ag on FDC or on iccosomes signals B cells through B cell Ag receptor, the iccosome provides these B cells with Ag necessary to process and elicit T cell help, and a secondary signal(s) necessary to optimize the memory response is delivered to B cells by FDC in an Ag-nonspecific fashion.

Alternations in splenocyte and thymocyte subpopulations in B6C3F1 mice exposed to cocaine plus diazinon.

Our laboratory has proposed a working model which asserts that cocaine's effects on immunity are mediated by reactive metabolites generated by the cytochrome P-450 system. This metabolic pathway is normally a minor one in humans, but takes on significance when metabolism of cocaine by the P-450 system is increased, as may occur with excessive alcohol consumption (enzyme induction) or after exposure to organophosphate pesticides (esterase inhibition). Results from our laboratory demonstrate that cocaine exerts its most dramatic effects on immunocompetence when administered to mice that have been pretreated with diazinon, an organophosphate esterase inhibitor. Most notably, we observed decreases in both the splenic T-dependent antibody response to sheep erythrocytes and the splenic T-independent antibody response to DNP-ficoll and a dramatic thymic atrophy in mice exposed to cocaine + diazinon, which were not seen in mice exposed to cocaine alone. The primary objective of the present investigation was to determine whether the exposure conditions used to produce the changes noted above are also capable of causing changes in lymphocyte cell types by use of flow cytometric analysis. Administration of cocaine after pretreatment with diazinon only modestly affected splenic lymphocyte subsets, which caused a slight decrease in the number of B cells. No effect was observed in the macrophage, T-helper or T-suppressor subpopulations in the spleen. These results suggest that changes in splenocyte subpopulations induced by cocaine + diazinon cannot account for the suppression of the antibody response. In contrast, all T-cell subsets in the thymus were decreased significantly, with immature double-positive thymocytes suffering the greatest loss in cell number. These results indicate that T cells, especially immature thymocytes located in the thymus, are sensitive to effects associated with the combined treatment of cocaine + diazinon.

Follicular dendritic cells and human immunodeficiency virus infectivity.

Large amounts of human immunodeficiency virus (HIV) localize on follicular dendritic cells (FDC) in the follicles of secondary lymphoid tissues following viral infection. During clinical latency, active viral infection occurs primarily at these sites. As HIV on FDC is in the form of immune complexes, some of which may be formed with neutralizing antibody, we investigated whether HIV on FDC is infectious. We report here that HIV on FDC is highly infectious. Furthermore, FDC can convert neutralized HIV into an infectious form even in the presence of a vast excess of neutralizing antibody. Thus FDC may provide a mechanism whereby HIV infection can continue in the presence of neutralizing antibody.

Loss of follicular dendritic cells in murine-acquired immunodeficiency syndrome.

BACKGROUND: The disease caused by HIV-1 leads to the destruction of follicular dendritic cells (FDC) and the follicular architecture in secondary lymphoid tissues. The murine acquired immunodeficiency syndrome (MAIDS, caused by LP-BM5) serves as an animal model for study of mechanisms involved in development of retrovirus-induced immunodeficiencies. The present study was undertaken to determine whether LP-BM5 infection leads to the destruction of murine FDC and the normal follicular architecture in secondary lymphoid tissues. EXPERIMENTAL DESIGN: Mice were infected with LP-BM5, and the follicular architecture and FDC networks were assessed. The pathologic changes observed were correlated with FDC function. RESULTS: Three weeks after infection, FDC networks were present, and they often appeared hyperplastic. However, by 1 month after infection, distorted lymphoid follicles were apparent, and the intensity of FDC labeling began to decline. FDC disappeared first in the spleen, and in hyperimmunized mice, FDC in draining lymph nodes disappeared before FDC in nondraining lymph nodes. By 4 months, the normal follicular localization of B cells was missing, and FDC were not detectable in most tissues. As the FDC and the normal lymphoid architecture degenerated, extrafollicular foci of immunoblasts and plasma cells appeared in areas typically reserved for T cells, and the Thy 1.2+ T cells were dispersed. Of interest, the total number of Ig-producing cells increased as the disease progressed; in contrast, the number of anti-human serum albumin-producing cells in mice immunized with human serum albumin before infection decreased. CONCLUSIONS: These data indicate that, like HIV-1 infection, LP-BM5 infection leads to the loss of FDC and the normal follicular architecture. However, morphologic changes were not observed until after FDC had lost their normal ability to trap and retain Ag. These data indicate that retroviral infections may cause FDC dysfunctions long before FDC are destroyed.

Follicular dendritic cells and the maintenance of IgE responses.

Antigen (Ag) is retained for long periods of time in secondary lymphoid tissues in the form of immune complexes on follicular dendritic cells (FDC). Ag retained on FDC is thought to play a role in maintaining antibody (Ab) responses in vivo. A model for study of Ab production induced by retained Ag in vitro is the spontaneous Ab response. In this response, specific Ab production is induced spontaneously (no exogenous Ag needed) in cultures derived from secondary lymphoid tissues containing persisting Ag. Specific IgG is spontaneously induced and we reasoned that FDC may also play a role in the maintenance of specific IgE responses. To test this hypothesis, we monitored spontaneous anti-ovalbmin (OVA) IgE production in cultures of lymph node (LN) fragments from OVA-immunized mice. In addition, highly enriched preparations of OVA bearing FDC were added to OVA-specific memory cells in an attempt to stimulate OVA-specific IgE production. Months after secondary immunization, anti-OVA IgE responses were spontaneously induced when fragments from draining LN were placed into culture. Furthermore, FDC bearing OVA from draining LN induced anti-OVA IgE production when incubated with spleen cells from OVA-immune mice whereas identical cultures with FDC bearing environmental Ag from non-draining LN of the OVA immune animals did not. The anti-OVA IgE responses were elicited only in cultures containing OVA-immune memory cells indicating that specific memory cells were critical for these anti-OVA IgE responses. Removal of FDC from cultures with an FDC-specific mAb dramatically decreased anti-OVA IgE production. These studies demonstrate that FDC can induce specific memory T and B cells to produce IgE and help support the concept that FDC-associated antigen may be involved in the long-term maintenance of specific IgE responses.

Follicular dendritic cells and B cell chemotaxis.

B cells isolated from germinal centers (GC) of immune mice 2-5 days after antigen (Ag) challenge migrate in response to chemotactic signals, whereas GC B cells isolated at other times and resting B cells do not. Since B cells are in direct contact with follicular dendritic cells (FDC) in GC we reasoned that FDC might play a role in enabling B cells to become chemotactically active. Resting B cells were co-cultured with FDC either with or without anti-mu-dextran (anti-mu-dex) as an Ag surrogate and/or recombinant interleukin (rIL)-4 as a T cell surrogate. After 3 days, the B cells were isolated and their migration to chemotactic factors contained in zymosan-activated serum assessed in microchemotaxis chambers. B cells incubated alone or with anti-mu-dex or rIL-4 showed minimal migration, which could be increased if both anti-mu-dex or rIL-4 were present. However, maximal migration was obtained when B cells were cultured with FDC, and this was not increased by addition of anti-mu-dex and/or rIL-4, indicating that the FDC signal was a primary signal and did not require pre-activation of the B cells. Checkerboard analysis using variation in concentration and location of the chemoattractant in chemotaxis chambers indicated that both chemotaxis and chemokinesis occurred. B cell migration began within 6 h of culture, peaked by 48 h and decreased thereafter. Removal of FDC or interference with FDC-B cell contact ablated or significantly decreased induction of B cell migration. Furthermore, induction did not require functional T cells. These data indicate that FDC can induce resting B cells to become responsive to chemotactic signals.

Expression of the intercellular adhesion molecule-1 on high endothelial venules and on non-lymphoid antigen handling cells: interdigitating cells, antigen transporting cells and follicular dendritic cells.

Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the development of germinal center reactions in vitro, and the present study was undertaken to determine the distribution of ICAM-1 in active germinal centers in vivo and in murine secondary lymphoid tissues in general. Anti-ICAM-1-specific monoclonal antibodies were used in conjunction with immunohistochemistry at both the light and ultrastructural levels of resolution. Examination of cryostat sections of lymph nodes, spleens, and Peyer's patches revealed that anti-ICAM-1 distinctly labeled cells in the light zones of germinal centers, a few cells in the T cell zones (e.g. paracortex of lymph nodes), cells in the sinus floor of the subcapsular sinuses of lymph nodes, and high endothelial venules (HEV). Ultrastructural studies revealed that the cells labeling with anti-ICAM-1 in germinal centers were follicular dendritic cells (FDC) which appeared to have more ICAM-1 than any other cell type. The surfaces of well-developed, intricate, convoluted FDC processes were intensely labeled even under conditions where B cells appeared negative. Interdigitating cells (IDC) were also labeled as were certain endothelial cells in the HEV. The cells in the subcapsular sinus floor labeling with anti-ICAM-1 were the "antigen transporting cells" (ATC) that carry antigen-antibody complexes into lymph node follicles. We suspect ATC are FDC precursors which mature into FDC in the follicles. Interestingly, FDC, IDC, and ATC are 3 important accessory cells known to handle antigens in specific compartments of lymphoid tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Follicular dendritic cell function and murine AIDS.

Infection of mice with LP-BM5 elicits an immunodeficiency state referred to as murine acquired immune deficiency syndrome (MAIDS). Shortly after infection, retrovirus particles become associated with follicular dendritic cells (FDC) and this study was undertaken to determine whether retroviruses alter FDC functions. The FDC functions examined included the ability to: (1) retain antigen (Ag) trapped prior to infection; (2) trap new Ag after infection; (3) maintain specific IgG responses; and (4) provide co-stimulatory signals to B cells. Mice were infected with LP-BM5 and the ability of their FDC to trap and retain 125I-Ag (HSA) was assessed. Serum anti-HSA levels were monitored and FDC co-stimulatory activity was indicated by increased B-cell proliferation. HSA trapped on FDC prior to infection began to disappear by 3 weeks and was practically gone by 6 weeks. Serum anti-HSA titres were maintained normally for about 3 weeks after infection and then declined precipitously. The ability of FDC to trap new Ag began to disappear around the second and third week of infection and was markedly depressed by the fourth week. However, FDC recovered from infected mice retained their ability to co-stimulate anti-mu- and interleukin-4 (IL-4)-activated B cells throughout a 5-week period. In short, the ability of FDC to trap and retain specific Ag and maintain specific antibody levels was markedly depressed after retrovirus infection. However, FDC from infected mice continued to provide co-stimulatory signals and these signals may contribute to the lymphadenopathy and splenomegaly characteristic of MAIDS.