Activation of B cells by antigens on follicular dendritic cells.

A need for antigen-processing and presentation to B cells is not widely appreciated. However, cross-linking of multiple B cell receptors (BCRs) by T-independent antigens delivers a potent signal that induces antibody responses. Such BCR cross-linking also occurs in germinal centers where follicular dendritic cells (FDCs) present multimerized antigens as periodically arranged antigen-antibody complexes (ICs). Unlike T cells that recognize antigens as peptide-MHC complexes, optimal B cell-responses are induced by multimerized FDC-ICs that simultaneously engage multiple BCRs. FDC-FcgammaRIIB mediates IC-periodicity and FDC-BAFF, FDC-IL-6 and FDC-C4bBP are co-stimulators. Remarkably, specific antibody responses can be induced by FDC-ICs in the absence of T cells, opening up the exciting possibility that people with T cell insufficiencies may be immunized with T-dependent vaccines via FDC-ICs.

T-independent antibody responses to T-dependent antigens: a novel follicular dendritic cell-dependent activity.

Follicular dendritic cells (FDCs) periodically arrange membrane-bound immune complexes (ICs) of T-dependent Ags 200-500A apart, and in addition to Ag, they provide B cells with costimulatory signals. This prompted the hypothesis that Ag in FDC-ICs can simultaneously cross-link multiple BCRs and induce T cell-independent (TI) B cell activation. TI responses are characterized by rapid IgM production. OVA-IC-bearing FDCs induced OVA-specific IgM in anti-Thy-1-pretreated nude mice and by purified murine and human B cells in vitro within just 48 h. Moreover, nude mice immunized with OVA-ICs exhibited well-developed GL-7(+) germinal centers with IC-retaining FDC-reticula and Blimp-1(+) plasmablasts within 48 h. In contrast, FDCs with unbound-OVA, which would have free access to BCRs, induced no germinal centers, plasmablasts, or IgM. Engagement of BCRs with rat-anti-mouse IgD (clone 11-26) does not activate B cells even when cross-linked. However, B cells were activated when anti-IgD-ICs, formed with Fc-specific rabbit anti-rat IgG, were loaded on FDCs. B cell activation was indicated by high phosphotyrosine levels in caps and patches, expression of GL-7 and Blimp-1, and B cell proliferation within 48 h after stimulation with IC-bearing FDCs. Moreover, anti-IgD-IC-loaded FDCs induced strong polyclonal IgM responses within 48 h. Blockade of FDC-FcgammaRIIB inhibited the ability of FDC-ICs to induce T-independent IgM responses. Similarly, neutralizing FDC-C4BP or -BAFF, to minimize these FDC-costimulatory signals, also inhibited this FDC-dependent IgM response. This is the first report of FDC-dependent but TI responses to T cell-dependent Ags.

IL-6 produced by immune complex-activated follicular dendritic cells promotes germinal center reactions, IgG responses and somatic hypermutation.

Reports that follicular dendritic cells (FDCs) produce IL-6 prompted the hypotheses that immune complexes (ICs) induce FDCs to produce IL-6 and that FDC-IL-6 promotes germinal center (GC) reactions, somatic hypermutation (SHM) and IgG production. FDCs were activated in vitro by addition of ICs and FDC-IL-6 production was determined. Wild-type (WT) and IL-6 knockout (KO) mice, as well as chimeras with WT and IL-6 KO cells, were immunized with (4-hydroxy-3-nitrophenyl)-acetyl (NP)-chicken gamma globulin (CGG) and used to study anti-(4-hydroxy-3-iodo-5-nitrophenyl) acetyl (NIP) responses, GC formation and SHM in the VH186.2 gene segment in Ig-gamma. FDC-IL-6 increased when FDCs encountered ICs. At low immunogen dose, 1 microg NP-CGG per mouse, the IgG anti-NIP response in IL-6 KO mice was low and immunohistochemistry revealed a reduction in both the number and size of GCs. The physiological relevance of FDC-IL-6 was apparent in the chimeric mice where total splenocytes from WT mice were unable to provide the IL-6 needed for normal IgG and GC responses in IL-6 KO animals with IL-6-defective FDCs. Moreover, the rate of mutation decreased from 18 to 8.9 mutations per 1000 bases (P < 0.001) in WT versus IL-6 KO mice. Addition of anti-IL-6 to GC reactions in vitro reduced antibody levels and SHM from 3.5 to 0.65 mutations per 1000 bases (P < 0.02). Thus, the absence of FDC-IL-6 correlated with a reduction in SHM that coincided with the reduction in GCs and specific anti-NIP. This is the first study to document that ICs induce FDC-IL-6 and that FDC-derived IL-6 is physiologically relevant in generating optimal GC reactions, SHM and IgG levels.

Fibroblastic reticular cells of the peripheral lymphoid organs: unique features of a ubiquitous cell type.

The highly ordered structure in peripheral lymphoid tissues is maintained by continuous interactions between their hemopoietic and stromal components. The main reticular cell type, fibroblastic reticular cells (FRCs) emerged as a considerably heterogeneous group of the stroma. These cells have diverse roles beyond architectural scaffolding. Their functions include the formation of nests for recirculating lymphocytes with subset-preference and a dynamic filtration system for facilitating encounter between antigen, antigen-presenting cells and antigen-receptor bearing cells. FRCs are influenced by lymphocyte-derived morphogenic signals and factors necessary for lymphoid tissue formation and lymphocyte homeostasis. Moreover, FRCs may also interact with other stromal elements during both lymphoid organ development and immune responses. FRCs are profoundly affected by pathogens, which may limit the lymphoid cells' capacity to establish efficient protection. This review focuses on the ontogenic, phenotypic and functional complexities of FRCs and their role in the stromal rearrangement of lymphoid tissues.

Isolation of functionally active murine follicular dendritic cells.

Biochemical, genetic, and immunological studies of follicular dendritic cells (FDCs) have been hampered by difficulty in obtaining adequate numbers of purified cells in a functional state. To address this obstacle, we enriched FDCs by irradiating mice to destroy most lymphocytes, excised the lymph nodes, and gently digested the nodes with an enzyme cocktail to form single cell suspensions. The FDCs in suspension were selected using the specific mAb FDC-M1 with magnetic cell separation technology. We were able to get nearly a million viable lymph node FDCs per mouse at about 90% purity. When examined under light and transmission electron microscopy, the cytological features were characteristic of FDCs. Furthermore, the cells were able to trap and retain immune complexes and were positive for important phenotypic markers including FDC-M1, CD21/35, CD32, CD40, and CD54. Moreover, the purified FDCs exhibited classical FDC accessory activities including: the ability to co-stimulate B cell proliferation, augment antibody responses induced by mitogens or antigens, maintain B cell viability for weeks, and protect B lymphocytes from anti-FAS induced apoptosis. In short, this combination of methods made it possible to obtain a substantial number of highly enriched functional murine FDCs.

Follicular dendritic cells in aging, a "bottle-neck" in the humoral immune response.

Senescence leads to the appearance of atrophic follicular dendritic cells (FDCs) that trap and retain little immune complexes (IC), generate few memory B cells, and induce a reduced number of germinal centers (GC). Deficiencies in antibody responses to T cell dependent exogenous antigens such as pneumonia and influenza vaccines may reflect intrinsic FDC defects or altered FDC-B cell interactions. We recently studied antigen handling capacity and co-stimulatory activity of old FDCs and determined age-related changes in the expression or function of FcgammaRII or CR1 and 2 on FDCs. Here, we present an overview of FDC function in recall responses with known deficiencies in FDCs and GC development. Then, we review our recent work on aged FDCs and discuss age-related changes in molecular interactions between FDCs and B cells. We also discuss the causes underlying the impaired humoral immune response with respect to age-related molecular changes in FDC and B cell interactions. In vitro evidence suggests that FcgammaRII on aged FDCs is regulated abnormally and this in turn might cause the development of a defective FDC-network (reticulum) that retains few ICs, promotes ITIM signaling, prevents B cell proliferation and GC formation, and antibody production.

Immunoarchitecture of distinct reticular fibroblastic domains in the white pulp of mouse spleen.

The development of peripheral lymphoid tissues requires a series of cognate interactions between hemopoietic and stromal cell populations, including reticular fibroblasts, which form the mesenchymal scaffolding of distinct tissue compartments. Here we describe the formation of different fibroblastic domains in the mouse spleen white pulp by using two new rat monoclonal antibodies (MAbs). In the white pulp, MAb IBL-10 labels both T- and B-cell zone reticular elements at various intensities. The IBL-10hi subset was found primarily at the edge between the peripheral part of the PALS and follicles, and the IBL-10lo compartment was distributed evenly within the white pulp. The IBL-10hi subset appeared during the first 2 postnatal weeks and was absent in SCID mice. The white pulp fibroblast subset identified with MAb IBL-11 had a different tissue distribution and kinetics of ontogeny, with an appearance overwhelmingly restricted to the PALS and a narrow rim at the edge of the follicular border area toward the marginal zone. The appearance of IBL-11-positive reticular cells was delayed compared with that of the IBL-10lo-positive subset. The formation was independent of the influence of antigen receptor-bearing lymphocytes, as evidenced by the presence of IBL-11-positive fibroblasts in SCID mice. By transferring various lymphocyte subsets into SCID mice, partial compartmentalization of the white pulp fibroblasts could be induced, indicating that these mesenchymal fibroblast precursors retain their ability to differentiate upon encountering mature T- or B-cells.

Multi-therapeutic potential of autoantibodies induced by immune complexes trapped on follicular dendritic cells.

Induction of autoantibodies (autoAbs) targeting disease drivers / mediators is emerging as a potential immunotherapeutic strategy. Auto-immune complex (IC)-retaining follicular dendritic cells (FDCs) critically regulate pathogenic autoAb production in autoreactive germinal centers (GCs); however, their ability to induce potentially therapeutic autoAbs has not been explored. We hypothesized that deliberate display of clinically targeted antigens (Ags) in the form of ICs on FDC membranes induces target-specific autoreactive GCs and autoAbs that may be exploited therapeutically. To test our hypothesis, three therapeutically relevant Ags: TNF-α, HER2/neu and IgE, were investigated. Our results indicated that TNF-α-, HER2/neu- and IgE-specific autoAbs associated with strong GC reactions were induced by TNF-α-, HER2/neu- and IgE-IC retention on FDCs. Moreover, the induced anti-TNF-α autoAbs neutralized mouse and human TNF-α with half maximal Inhibitory Concentration (IC50) of 7.1 and 1.6 nM respectively. In addition, we demonstrated that FDC-induced Ab production could be non-specifically inhibited by the IgG-specific Endo-S that accessed the light zones of GCs and interfered with FDC-IC retention. In conclusion, the ability of FDCs to productively present autoAgs raises the potential for a novel immunotherapeutic platform targeting mediators of autoimmune disorders, allergic diseases, and Ab responsive cancers.

Ultrastructural study of highly enriched follicular dendritic cells reveals their morphology and the periodicity of immune complex binding.

Follicular dendritic cells (FDCs) are immune accessory cells found in the follicles of secondary lymphoid organs where they promote B cell maturation in germinal centers (GCs) that develop following antigen exposure. Recently, we published a method for isolating functional murine FDCs in high purity. We reasoned that disruption of FDC reticula in vivo would alter FDC morphology. The present study was undertaken to determine the morphological features of isolated FDCs. FDC-M1 and immune complex (IC) labeling were used to identify FDCs in isolated preparations. Results at the light-microscopic level revealed that isolated FDCs trapped ICs, expressed FDC-M1 and cadherins, but generally appeared non-dendritic. However, at the ultrastructural level, the majority of FDCs exhibited dendrites and typical euchromatic nuclei that appeared as single, bilobed, or double nuclei. Based on morphology, four varieties of FDCs were distinguishable, possibly indicative of differences in maturity. Remarkably, ICs trapped by FDCs showed a distinctive periodic arrangement consistent with that known to induce immune responses by thymus independent-2 (TI-2) antigens that engage and cross-link multiple B cell receptors. The ability of FDCs to trap ICs and then display these T-cell-dependent antigens with repeating periodicity suggests that multiple B cell receptors are cross-linked by antigen on FDCs, thus promoting B cell stimulation and proliferation. Rapid proliferation is characteristic of the GC reaction, and the arrangement of T-dependent antigens in this periodic fashion may help to explain the profuse B cell proliferation in the GC microenvironment.

Immune complex-bearing follicular dendritic cells deliver a late antigenic signal that promotes somatic hypermutation.

We reasoned that immune complex (IC)-bearing follicular dendritic cells (FDCs) promote somatic hypermutation (SHM). This hypothesis was tested in murine germinal center reactions induced in vitro by coculturing 6-day (4-hydroxy-3-nitrophenyl) acetyl-primed but unmutated lambda+ B cells, chicken gamma-globulin (CGG) memory T cells, FDCs, and ICs (anti-CGG plus NP-CGG). Mutations in primed lambda+ B cells were obtained only when both FDCs and immunogen were present. FDCs alone promoted B cell survival and Ab production but there were no mutations without more immunogen. Moreover, the mutation rate was enhanced when FDCs were activated. Trapped ICs ranged from 200 to 500 A apart on FDC membranes and this correlated with the periodicity known to optimally signal BCRs. FDCs are unique in their ability to retain ICs for months and a second signal mediated by FDC-ICs appeared to be needed a week or more after immunization by immunogen persisting on FDCs. However, the time needed to detect extensive SHM could be reduced to 7 days if ICs were injected together with memory T cells in vivo. In marked contrast, no mutations were apparent after 7 days in vivo if ICs were replaced by free Ag that would not load on FDCs until Ab was produced. The data suggest that specific Ab production leads to the following events: Ab encounters Ag and ICs are formed, ICs are trapped by FDCs, B cells are stimulated by periodically arranged Ag in ICs on FDCs, and this late antigenic signal promotes SHM.

TLR4 on follicular dendritic cells: an activation pathway that promotes accessory activity.

Microbial molecular patterns engage TLRs and activate dendritic cells and other accessory cells. Follicular dendritic cells (FDCs) exist in resting and activated states, but are activated in germinal centers, where they provide accessory function. We reasoned that FDCs might express TLRs and that engagement might activate FDCs by up-regulating molecules important for accessory activity. To test this hypothesis, TLR4 expression on FDCs was studied in situ with immunohistochemistry, followed by flow cytometry and RT-PCR analysis. TLR4 was expressed on FDC reticula in situ, and flow cytometry indicated that TLR4 was expressed on surface membranes and TLR4 message was readily apparent in FDCs by RT-PCR. Injecting mice or treating purified FDCs with LPS up-regulated molecules important for accessory activity including, FDC-Fc gammaRIIB, FDC-ICAM-1, and FDC-VCAM-1. Treatment of purified FDCs with LPS also induced intracellular phospho-IkappaB-alpha, indicating NF-kappaB activation, and that correlated with increased Fc gammaRIIB, ICAM-1, and VCAM-1. FDCs in C3H/HeJ mice were not activated with LPS even when mice were reconstituted with C3H/HeN leukocytes, suggesting that engagement of FDC-TLR4 is necessary for activation. Moreover, activated FDCs exhibited increased accessory activity in anti-OVA recall responses in vitro, and the FDC number could be reduced 4-fold if they were activated. In short, we report expression of TLR4 on FDCs for the first time and that engagement of FDC-TLR4 activated NF-kappaB, up-regulated expression of molecules important in FDC accessory function, including Fc gammaRIIB, ICAM-1, and VCAM-1, as well as FDC accessory activity in promoting recall IgG responses.

Follicular dendritic cell (FDC)-FcgammaRIIB engagement via immune complexes induces the activated FDC phenotype associated with secondary follicle development.

Follicular dendritic cell (FDC)-FcgammaRIIB levels are up-regulated 1-3 days after challenge of actively immunized mice with Ag. This kinetics suggested that memory cells are not driving this response, prompting the hypothesis that immune complex (IC)-FDC interactions lead to FDC activation. To test this, mice passively immunized with anti-OVA Ab were OVA challenged to produce IC. After 3 days, levels of IC, FcgammaRIIB, ICAM-1, and VCAM-1 on FDC were analyzed. FDC were also stimulated with IC in vitro, and mRNA for FcgammaRIIB, ICAM-1, and VCAM-1 was quantified by quantitative RT-PCR. IC labeling in passively immunized WT and FcgammaRIIB-/- mice revealed five to six FDC-reticula per LN midsagittal section. In WT mice, these IC-bearing FDC-reticula corresponded with FDC-reticula labeling for FcgammaRIIB, ICAM-1, and VCAM-1. Increases in these molecules on IC-stimulated FDC were confirmed by flow cytometry. In marked contrast, in FcgammaRIIB-/- mice, no increased VCAM-1 or ICAM-1 was seen on IC-bearing FDC-reticula or on purified FDC. Addition of IC in vitro resulted in dramatic increases in mRNA for FcgammaRIIB, ICAM-1 and VCAM-1 in WT FDC, but not in FDC from FcgammaRIIB-/- mice, 2.4G2-pretreated WT FDC, B cells, or macrophages. Thus, although FDC-FcgammaRIIB was not essential for IC trapping, engagement of FDC-FcgammaRIIB with IC initiated an FDC activation pathway.

Differential T cell-mediated regulation of CD23 (Fc epsilonRII) in B cells and follicular dendritic cells.

Differences in murine follicular dendritic cells (FDC)-CD23 expression under Th1 vs Th2 conditions prompted the hypothesis that T cells help regulate the phenotype of FDCs. FDCs express CD40, suggesting that T cell-CD40L and lymphokines may be involved in regulating FDC-CD23. To test this, highly enriched FDCs were incubated with CD40L trimer or anti-CD40 to mimic T cell signaling in the presence of IFN-gamma or IL-4. Surface expression of CD23 was determined by flow cytometry, whereas mRNA levels of CD23 and its isoforms CD23a and CD23b were independently measured by quantitative PCR. When FDCs were incubated with either CD40L trimer or agonistic anti-CD40 Ab, the expression of FDC-CD23 was increased both at the mRNA and protein levels. Moreover, engagement of FDC-CD40 enhanced mRNA levels for both CD23a and CD23b isoforms. In addition, IFN-gamma substantially enhanced CD23a and CD23b mRNA levels in CD40-stimulated FDCs. Curiously, IL-4 could also up-regulate FDC-CD23a but not -CD23b. Anti-IFN-gamma dramatically inhibited FDC-CD23 in mice immunized with CFA, whereas anti-IL-4 had only a modest inhibitory effect. In contrast with FDCs, IFN-gamma inhibited surface expression of murine B cell-CD23 as well as mRNA for B cell CD23a and -CD23b, whereas IL-4 dramatically enhanced message for both isoforms as well as protein expression. In short, CD23 was regulated very differently in FDCs and B cells. Previous studies suggest that high levels of FDC-CD23 inhibit IgE production, and this IFN-gamma and CD40L-mediated up-regulation of FDC-CD23 may explain, at least in part, why Th1 responses are associated with low IgE responses in vivo.

The influence of immune complex-bearing follicular dendritic cells on the IgM response, Ig class switching, and production of high affinity IgG.

It is believed that Ag in immune complexes (ICs) on follicular dendritic cells (FDCs) selects high affinity B cells and promotes affinity maturation. However, selection has been documented in the absence of readily detectable ICs on FDCs, suggesting that FDC-ICs may not be important. These results prompted experiments to test the hypothesis that IC-bearing murine FDCs can promote high affinity IgG responses by selecting B cells after stimulating naive IgM(+) cells to mature and class switch. Coculturing naive lambda(+) B cells, FDCs, (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin (CGG) + anti-CGG ICs, and CGG-primed T cells resulted in FDC-lymphocyte clusters and production of anti-4-hydroxy-5-iodo-3-nitrophenyl acetyl. Class switching was indicated by a shift from IgM to IgG, and affinity maturation was indicated by a change from mostly low affinity IgM and IgG in the first week to virtually all high affinity IgG anti-4-hydroxy-5-iodo-3-nitrophenyl acetyl in the second week. Class switching and affinity maturation were easily detectable in the presence of FDCs bearing appropriate ICs, but not in the absence of FDCs. Free Ag plus FDCs resulted in low affinity IgG, but affinity maturation was only apparent when FDCs bore ICs. Class switching is activation-induced cytidine deaminase (AID) dependent, and blocking FDC-CD21 ligand-B cell CD21 interactions inhibited FDC-IC-mediated enhancement of AID production and the IgG response. In short, these data support the concept that ICs on FDCs can promote AID production, class switching, and maturation of naive IgM(+) B cells, and further suggest that the IC-bearing FDCs help select high affinity B cells that produce high affinity IgG.

Role of CD44 and its v7 isoform in staphylococcal enterotoxin B-induced toxic shock: CD44 deficiency on hepatic mononuclear cells leads to reduced activation-induced apoptosis that results in increased liver damage.

Exposure to bacterial superantigens such as staphylococcal enterotoxin B (SEB) leads to the induction of toxic shock syndrome which results in multiorgan failure, including liver damage. In the present study, we investigated the role of CD44 in SEB-induced liver injury. Injection of SEB into d-galactosamine-sensitized CD44 wild-type (WT) mice led to a significant increase in CD44 expression on liver T cells, NK cells, and NKT cells. Administration of SEB to CD44 knockout (KO) mice caused significantly enhanced liver damage which correlated with elevated numbers of T cells, NK cells, NKT cells, and macrophages in the liver and increased production of tumor necrosis factor alpha and gamma interferon compared to CD44 WT mice. Furthermore, liver mononuclear cells from CD44 KO mice were resistant to SEB-induced apoptosis, and cDNA microarray analysis revealed that SEB activation of such cells led to the induction of several antiapoptotic genes and repression of proapoptotic genes. Examination of CD44 isoforms revealed that SEB exposure altered CD44 variant 7 (v7) isoform expression. Interestingly, mice bearing a specific deletion of the CD44v7 exon exhibited increased susceptibility to SEB-induced hepatitis. Finally, treatment of CD44 WT mice with anti-CD44 monoclonal antibodies reduced expression of CD44 in liver mononuclear cells and caused increased susceptibility to SEB-induced liver injury. Together, these data demonstrate that the expression of CD44 and/or CD44v7 on SEB-activated liver mononuclear cells facilitates their rapid apoptosis, thereby preventing severe liver injury in wild-type mice, and suggest that CD44 plays an important role in the regulation and elimination of immune cells in the liver.

Simultaneous blockade of Fc gamma receptors and indirect labeling of mouse lymphocytes by the selective detection of allotype-restricted epitopes on the kappa chain of rat monoclonal antibodies.

BACKGROUND: Incubation of mouse hemopoietic cells with rat monoclonal antibodies (mAbs) of the IgG class sometimes results in Fc-region mediated binding of immunoglobulins by Fc-receptors. This unwanted binding can be prevented by preincubation of target cells with the rat anti-mouse anti-CD16/32 (2.4G2) mAb. METHODS: To avoid the cross-reactivity of fluorochrome-conjugated secondary anti-rat antibodies with the Fc-receptor blocking 2.4G2, direct fluorochrome-conjugated immunoglobulins need to be used. However, we report that a mouse mAb (MRC OX12) with a strong rat Igk(a) allotype preference can be used for flow cytometric measurements in conjunction with unlabeled rat mAbs with the simultaneous blockade of Fc gamma receptors by the 2.4G2 mAb. Results and Discussion This lack of reactivity of OX12 against the 2.4G2 mAb is remarkable, as it could efficiently detect another Sprague-Dawley-derived rat mAb. This staining procedure (unlabeled rat mAb of the appropriate strain detected by OX12 mAb in the presence of 2.4G2 IgG) is an attractive alternative to using direct antibody conjugates, while satisfying the need for an effective Fc gamma-receptor blockade.

Altered regulation of Fc gamma RII on aged follicular dendritic cells correlates with immunoreceptor tyrosine-based inhibition motif signaling in B cells and reduced germinal center formation.

Aging is associated with reduced trapping of Ag in the form of in immune complexes (ICs) by follicular dendritic cells (FDCs). We postulated that this defect was due to altered regulation of IC trapping receptors. The level of FDC-M1, complement receptors 1 and 2, FcgammaRII, and FDC-M2 on FDCs was immunohistochemically quantitated in draining lymph nodes of actively immunized mice for 10 days after Ag challenge. Initially, FDC FcgammaRII levels were similar but by day 3 a drastic reduction in FDC-FcgammaRII expression was apparent in old mice. FDC-M2 labeling, reflecting IC trapping, was also reduced and correlated with a dramatic reduction in germinal center (GC) B cells as indicated by reduced GC size and number. Nevertheless, labeling of FDC reticula with FDC-M1 and anti-complement receptors 1 and 2 was preserved, indicating that FDCs were present. FDCs in active GCs normally express high levels of FcRs that are thought to bind Fc portions of Abs in ICs and minimize their binding to FcRs on B cells. Thus, cross-linking of B cell receptor and FcR via IC is minimized, thereby reducing signaling via the immunoreceptor tyrosine-based inhibition motif. Old FDCs taken at day 3, when they lack FcgammaRII, were incapable of preventing immunoreceptor tyrosine-based inhibition motif signaling in wild-type B cells but old FDCs stimulated B cells from FcgammaRIIB(-/-) mice to produce near normal levels of specific Ab. The present data support the concept that FcR are regulated abnormally on old FDCs. This abnormality correlates with a reduced IC retention and with a reduced capacity of FDCs to present ICs in a way that will activate GC B cells.

FcgammaRII expression on follicular dendritic cells and immunoreceptor tyrosine-based inhibition motif signaling in B cells.

Immune complexes (IC) initiate immunoreceptor tyrosine-based inhibition motif (ITIM) signaling and inhibit B cell activation by coligating B cell receptor for antigen (BCR) and FcgammaRII. Nevertheless, IC on follicular dendritic cells (FDC) stimulate rapid germinal center (GC) B cell proliferation suggesting that interactions between IC and FDC render IC capable of B cell activation. To understand this, we studied the kinetics of FDC FcgammaRII and complement receptors 1 and 2 (CR1&2) expressions during the GC reaction and determined whether FDC FcgammaRII could bind Fc in IC and block ITIM signaling. Mice were immunized with sheep red blood cells (SRBC), and CR1&2 and FcgammaRII levels in FDC reticula were monitored. The role of FDC FcgammaRII was studied using anti-BCR-stimulated A20 cells. Levels of FDC FcgammaRII in spleens of SRBC-injected mice increased within 24 h and were dramatically increased (approximately 50-fold) on days 3 and 5. In contrast, CR1&2 levels increased less than twofold. Addition of normal FDC, but not FDC lacking FcgammaRII, reduced and reversed anti-BCR-induced SH2 domain-containing inositol phosphatase (SHIP)-1 phosphorylation in A20 cells. FDC were able to induce normal recall responses even after overnight incubation of the lymphocytes with IC to stimulate ITIM signaling. Engagement of Ig Fc with numerous FcgammaRII on FDC appears to minimize IC-induced ITIM signaling. Thus, rapid up-regulation of FDC FcgammaRII may explain why poorly immunogenic IC are rendered highly immunogenic when presented by FDC in GC.

Appearance and phenotype of murine follicular dendritic cells expressing VCAM-1.

The architecture of lymphoid follicles is determined by a series of interactions between lymphoid and follicular stromal cells. A cardinal population in the non-lymphoid compartment is the follicular dendritic cell (FDC), whose communication with resting and activated B cells involves various adhesive interactions. The FDC phenotype variably includes the display of vascular cell adhesion molecule (VCAM-1). In this report we investigated the appearance and follicular tissue distribution of VCAM-1 in murine peripheral lymphoid tissues, and compared VCAM-1 with other FDC markers using immunohistochemistry. Correlating the appearance of VCAM-1 with other murine FDC-associated markers (CR1.2 [complement receptor 1.2 or CD35/21] and FDC-M1) revealed that the display of VCAM-1 is restricted to a subset of CR1.2-positive FDCs. We found that the expression of VCAM-1 antigen in the spleen or peripheral lymph nodes on FDCs requires antigenic stimulus, and that it coincides with germinal center formation. The VCAM-1 expression is associated with the appearance of mucosal addressin cell adhesion molecule (MAdCAM-1), with some slight differences in occurrence. The appearance of VCAM-1 and MAdCAM-1 antigens on FDCs may serve as indicators of FDC activation.

Age-related depression of FDC accessory functions and CD21 ligand-mediated repair of co-stimulation.

Morphological and kinetic studies of immune complex (IC) trapping by follicular dendritic cells (FDC) show marked age-related deficits. We postulated that a reduction in trapped IC, which generate CD21 ligands (L) on FDC, would lead to inadequate FDC-Ag-B cell interactions resulting in depressed Ab responses. To determine whether the age-related defect was the result of the aging of FDC or changes in the in vivo microenvironment of FDC (i.e. aging B and T cells), FDC-B cell-T cell-Ag interactions were studied in in vitro germinal centers where various combinations of old and young cells could be compared. Since we reasoned that reduced IC on FDC would generate less CD21L needed to stimulate the B cell co-receptor via CD21, we also examined the role of complement (C'). The hypothesis that aging reduces the accessory activity of FDC was tested with increasing numbers of FDC from young (12 weeks) or old (20 months) mice in the presence of young (12 weeks) B and T lymphocytes. The Ag-specific stimulatory activity of FDC was studied using the OVA-specific Ab response which was reduced by 40-50% in the presence of old FDC. Antigen-independent FDC-mediated co-stimulation was studied by using LPS to stimulate B-lymphocytes to produce immunoglobulin (Ig). In the presence of old FDC, co-stimulation was decreased by 70-80% in the LPS system. Incubation of aged FDC with IC and C' to provide FDC with CD21L restored co-stimulatory activity to near normal levels. In marked contrast, no defects in old B and T cells were apparent. The data suggest that the Ag handling capacity and co-stimulatory activity of old FDC become defective with aging and this appears to be a consequence of reduced trapping and presentation FDC-Ag and CD21L to B cells.