Rania El Sayed

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.

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.

Follicular dendritic cell (FDC)‐FcγRIIB engagement via immune complexes induces the activated FDC phenotype associated with secondary follicle development

Follicular dendritic cell (FDC)‐FcγRIIB 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, FcγRIIB, ICAM‐1, and VCAM‐1 on FDC were analyzed. FDC were also stimulated with IC in vitro, and mRNA for FcγRIIB, ICAM‐1, and VCAM‐1 was quantified by quantitative RT‐PCR. IC labeling in passively immunized WT and FcγRIIB–/– 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 FcγRIIB, ICAM‐1, and VCAM‐1. Increases in these molecules on IC‐stimulated FDC were confirmed by flow cytometry. In marked contrast, in FcγRIIB–/– 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 FcγRIIB, ICAM‐1 and VCAM‐1 in WT FDC, but not in FDC from FcγRIIB–/– mice, 2.4G2‐pretreated WT FDC, B cells, or macrophages. Thus, although FDC‐FcγRIIB was not essential for IC trapping, engagement of FDC‐FcγRIIB with IC initiated an FDC activation pathway.

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 200–500Å 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-FcγRIIB 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.

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-FcγRIIB, FDC-ICAM-1, and FDC-VCAM-1. Treatment of purified FDCs with LPS also induced intracellular phospho-IκB-α, indicating NF-κB activation, and that correlated with increased FcγRIIB, 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-κB, up-regulated expression of molecules important in FDC accessory function, including FcγRIIB, ICAM-1, and VCAM-1, as well as FDC accessory activity in promoting recall IgG responses.

Regulation of auto-antibody production by persisting auto-immune complexes on follicular dendritic cells

Backgroundand objectives Follicular dendritic cells (FDCs) are characteristic components of organised tertiary lymphoid structures (TLS) containing germinal centres (GCs) in chronically inflamed tissues. However, the role of FDCs in initiation, maintenance and regulation of autoreactive GCs and auto-antibody (Ab) production has not been analysed. The authors hypothesised that auto-immune complex (IC) retention on FDCs critically regulates auto-Ab production and that FDC-IC unloading would significantly inhibit auto-Ab levels. Materials and methods To test our hypothesis, purified human FDCs, B cells, T cells, complement, fibrinogen (Fib) and anti-Fib from high anti-citrullinated peptide protein Ab (ACPA) sera were used to set up controlled in vitro autoreactive GC reactions. The reactions were differentially reconstituted by the cellular and molecular components of GCs. The immunoglobulin G (IgG) specific Endoglycosidase S (EndoS) was used to inhibit Fib-IC retention on FDCs and Fib-specific auto-Ab production. Moreover, the ability of IC retention on FDCs to break B cell tolerance and induce auto-Ab production in tolerant animal models was verified. Results Our in vitro studies indicated that: (1) In the presence of Fib-IC loaded FDCs alone, significant amounts of Fib-specific IgM are produced; (2) In the presence of FDCs alone or T cells alone, Fib-specific IgG is insignificant; (3) The co-stimulatory effect of both FDCs and T cells is required for induction of significantly high levels of Fib-specific IgG; (4) EndoS significantly inhibited Fib-specific IgG production in FDC-T cell-assisted cultures via mechanisms involving interference with FDC-IC retention, inhibition of complement activation, and, possibly, IgG B cell receptor deglycosylation. In vivo, FDC-ICs induced autoreactive GCs and auto-Ab production in well-characterised animal models tolerant to hen egg lysozyme (HEL) as well as targets of therapeutic importance including TNF-α, IgE, and the extracellular domain two of the neu antigen (ECDII). However, safe termination of these induced auto-Ab responses was challenged by the non specific activity of EndoS as indicated by its inhibitory effect on protective immune responses to the foreign antigen ovalbumin (OVA). To avoid the non-selective activity of EndoS, the authors designed bait and pray ‘Split-EndoS conditional re-functionalisation’ system that selectively deglycosylates target Abs (pray) engaging their antigens (baits) on the EndoS molecules. The system was successfully tested as indicated by the ability of biotin-baited EndoS to selectively deglycosylate anti-biotin IgG. Conclusion This is the first report analysing the critical role of FDC-ICs in auto-Ab production in in vitro autoreactive germinal centre reactions, with potential therapeutic targeting of clinically important antigens and safe termination using EndoS.

Age-Associated Decline in Peripheral Lymphoid Organ Functions

Immunosenescence occurs after maturation and is expressed as a series of failing immunologic functions. Aging is best studied in the peripheral organs of the immune system such as lymph nodes, spleen, and isolated lymphoid tissues. The functional immunological building blocks of peripheral lymphoid organs are the lymphoid follicles. Aging defects occur in the components of lymphoid follicles, such as the T cells, B cells, and accessory cells, e.g., the dendritic cells. Among the dendritic cells, the most influential on the aging humoral immune response is the follicular dendritic cell (FDC). In aged mice, the Ab response is depressed because of the reduced capacity of FDCs to trap ICs needed for stimulation of B cells. This path is interrupted by the 70% age-related reduction of FcγRII on FDCs and the 88% age-related reduction in IC trapping. Thus, we conclude that these reductions account, in large part, for the declining Ab response in aged animals.

Breakage of B cell tolerance by antigens on follicular dendritic cells

Background and objectives Autoimmune disorders frequently display autoreactive germinal centres (GCs) with immune complex (IC)-bearing follicular dendritic cells (FDCs) suggesting that FDCs are involved in the pathogenesis of autoimmune diseases. The authors recently described induction of T cell-independent (TI) antibody (Ab) responses to T dependent (TD) antigens (Ags) by presenting the TD Ags on FDCs. Rather than presenting peptide fragments, FDCs multimerize monomeric Ags by trapping them in ICs via FDC-FcγRIIB and then present them polyvalently with a characteristic periodic spacing between epitopes of 200–500 Å. This allows extensive B cell receptor (BCR) cross-linking that induces TI B cell activation, GC formation, and Ag-specific Ig secretion in less than 48 h in the presence of FDC-derived co-stimulatory signals. These correlations prompted the hypothesis that multimerized self/neo-Ags on FDCs would break B cell tolerance; induce autoreactive GC formation, and auto-Ab responses. Materials and methods ICs of autologous tumour necrosis factor α or IgE, were injected into BALB/c mice. In addition, soluble hen egg lysozyme transgenic (sHEL-Tg) and rat neu-Tg FVBN202 mice were challenged with HEL or the subdomain II of the extracellular domain of rat neu (ECDII), respectively, in the form of ICs. These auto/neo Ags were selected because Abs reactive with these Ags are used clinically or, in the case of sHEL-Tg mice, the profound tolerance to sHEL has been well verified. Results Remarkably, in all cases, the ICs were trapped by FDCs; and autoreactive GCs, plasmablast differentiation and strong auto-Ab responses were induced within 48 h. In marked contrast, free Ag that would have unfettered access to BCRs did not load on FDCs and did not induce detectable auto-Abs. Conclusions This is the first report of deliberately inducing auto-Abs by self/neo-Ags on FDCs. The data provide a mechanism by which FDCs may contribute to autoimmunity, and suggest a novel approach for targeting mediators of chronic inflammation, hypersensitivity and cancer. Therapeutic induction of auto-Abs by FDC-ICs may provide self-regulated high affinity Abs efficient in long-term disease control.