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.

Anti-cardiolipin and increased serum adhesion molecule levels in patients with aggressive periodontitis.

BACKGROUND: We observed that a significant proportion of patients with periodontitis have elevated serum levels of beta2-glycoprotein-I-dependent anti-cardiolipin (anti-CL). These prothrombotic autoantibodies, commonly found to be elevated in patients with systemic lupus erythematosus and the antiphospholipid syndrome, are associated with adverse pregnancy outcomes, such as fetal involution, prematurity, and low birth weight, and with cardiovascular sequelae, such as atherosclerosis, stroke, and myocardial infarction. Anti-CL is known to promote vascular inflammation and thrombosis. METHODS: We measured serum levels of markers of vascular inflammation, including soluble intercellular adhesion molecule (sICAM)-1, soluble vascular cell adhesion molecule (sVCAM)-1, and sE-selectin, in 190 subjects with generalized aggressive or chronic periodontitis and in 90 periodontally healthy subjects. RESULTS: sVCAM-1 and sE-selectin levels were significantly higher in patients with elevated anti-CL (>15 U/ml). This relationship also was observed in the never-smoker subset of subjects, even after correction for demographic and periodontal variables. Within the diagnostic categories, sICAM-1, sVCAM-1, and sE-selectin were significantly higher in generalized aggressive periodontitis patients who had elevated anti-CL compared to those with normal anti-CL. Statistical correction for demographic and periodontal variables indicated that elevated anti-CL remained significantly associated with increased sVCAM-1 and sE-selectin in generalized aggressive periodontitis patients. CONCLUSIONS: Systemic markers of vascular inflammation in patients with aggressive periodontitis are associated with elevated levels of anti-CL. We hypothesize that a subset of periodontitis patients with elevated antiphospholipid antibodies could represent a subgroup at increased risk for obstetrical and cardiovascular sequelae.

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.

Prostaglandin E2-mediated regulation of immunoglobulin G2 via interferon gamma.

BACKGROUND: Patients with localized aggressive periodontitis (LAgP) produce elevated levels of IgG2 both in vivo and in vitro. Responses to the periodontitis-associated pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are dominated by IgG2, and these IgG2 responses are associated with reduced extent and severity of disease. Little is known about regulation of the IgG2 subclass, although prostaglandin E2 (PGE2) (a mediator known to polarize responses toward Th-2) and interferon (IFN)-gamma (a Th-1 mediator) both promote IgG2 production. This unusual relationship prompted the hypothesis that, in certain circumstances, PGE2 enhances rather than inhibits IFN-gamma production. METHODS: To test this hypothesis, indomethacin (IND)-treated peripheral blood mononuclear cell (PBL) cultures from healthy volunteers were stimulated with pokeweed mitogen (PWM), and the cultures were manipulated by adding PGE2, rIFN-gamma, rIL-Ialpha, rIL-1beta, rIL-6, or rIL-12. Production of IgG1, IgG2, IFN-gamma, and PGE2 was monitored by enzyme-linked immunosorbent assay. RESULTS: Indomethacin treatment inhibited IgG1 and IgG2 production, and PGE2 restored both immunoglobulins in PWM-stimulated cultures. Remarkably, addition of IFN-gamma also restored IND-suppressed IgG2 but not IgG1. In contrast, addition of rIL (interleukin)-1alpha, rIL-1beta, rIL-6, or rIL-12 did not restore IgG2 responses. Furthermore, IND suppressed IFN-gamma production and PGE2 increased IFN-gamma levels. Kinetic studies indicate that PGE2 production occurred on the first day of culture, followed by IFN-gamma two days later. CONCLUSIONS: These findings support the concept that in certain circumstances, PGE2 production can lead to increased IFN-gamma and that this IFN-gamma selectively promotes IgG2 responses. These data suggest that the elevated PGE2 observed in LAgP patients may contribute to increased IFN-gamma production and help explain elevated IgG2 responses.

Locally produced anti-phosphorylcholine and anti-oxidized low-density lipoprotein antibodies in gingival crevicular fluid from aggressive periodontitis patients.

BACKGROUND: Sera from patients with periodontal attachment loss contain higher concentrations of IgG anti-phosphorylcholine (anti-PC) than sera from healthy subjects. Furthermore, a large proportion of plaque bacteria bear PC-containing surface antigens, implicating the oral flora as a source of immunogen for anti-PC. Additionally, anti-PC is cross-reactive with a variety of oral bacterial antigens and human antigens such as oxidized low-density lipoprotein (oxLDL). We hypothesized that, if the oral flora is a source of PC antigens, then we should be able to detect local anti-PC and anti-oxLDL production in gingival crevicular fluid (GCF). METHODS: To test this, we collected 66 GCF samples from 15 patients with aggressive periodontitis and examined both the GCF samples and serum samples for their content of IgG anti-PC, IgG anti-LDL, and IgG anti-oxLDL by enzyme-linked immunosorbent assay. We also determined levels of anti-tetanus toxoid (anti-TT) as a non-oral antigen control. Serum and GCF concentrations of serum albumin (HSA) were also determined for use as a dilution marker. A conservative GCF:serum antibody ratio of greater than 1.5 was considered to be evidence of local antibody production. RESULTS: For the non-oral antigen TT, only one out of 62 samples contained locally produced antibody. Eight out of 64 samples (7 from a single subject) demonstrated local production of anti-LDL. In contrast, 28 out of 66 samples demonstrated local production of anti-PC, and 47 out of 66 samples contained locally produced anti-oxLDL. It was observed that A. actinomycetemcomitans strains containing or devoid of PC could absorb anti-oxLDL from human sera. Although there was a correlation between the ratios of anti-PC and anti-oxLDL (Spearman's rho = 0.35, P = 0.0037), local production of both antibodies was found in only 17 out of 65 samples, indicating that these antibodies are not always reflective of reactivity to the same antigens. CONCLUSION: The local production of anti-PC and anti-oxLDL further implicates the oral flora as a source of antigen that may mediate immune reactions of relevance to cardiovascular and other systemic diseases.

Comparison of type 1 and type 2 cytokine production by mononuclear cells cultured with streptococcus mutans and selected other caries bacteria.

A feature of pulpal immune responses is the predominance of type 1 cytokine mRNA under shallow caries and a mixed (type 1/type 2) profile under deep caries. These results prompted an examination of the cytokine profiles induced by bacteria in shallow caries (Streptococcus mutans and Actinomyces viscosus) and deep caries (Lactobacillus casei, Pseudoramibacter alactolyticus, and Prevotella intermedia). All isolates induced interferon-gamma and interleukin-10, whereas interleukin-4 and interleukin-2 titers were low to undetectable. S. mutans was the most potent and persistent interferon-gamma inducer. Differences in interleukin-10 were apparent at low doses but were less dramatic, with L. casei the dominant producer. S. mutans induced substantially more interferon-gamma than interleukin-10 over all doses and time points, suggesting strong type 1 polarization. P. alactolyticus induced significantly more interleukin-10 than interferon-gamma at higher concentrations, suggesting polarization toward type 2. A similar amount of interferon-gamma and interleukin-10 induced by L. casei, A. viscosus, and P. intermedia reflected a mixed profile. A better understanding of pulpal immune response to caries bacteria may enable us to develop an immune system-based pulp therapy in the future.

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.

ADAM10 is essential for Notch2-dependent marginal zone B cell development and CD23 cleavage in vivo.

The proteolytic activity of a disintegrin and metalloproteinase 10 (ADAM10) regulates cell-fate decisions in Drosophila and mouse embryos. However, in utero lethality of ADAM10(-/-) mice has prevented examination of ADAM10 cleavage events in lymphocytes. To investigate their role in B cell development, we generated B cell-specific ADAM10 knockout mice. Intriguingly, deletion of ADAM10 prevented development of the entire marginal zone B cell (MZB) lineage. Additionally, cleavage of the low affinity IgE receptor, CD23, was profoundly impaired, but subsequent experiments demonstrated that ADAM10 regulates CD23 cleavage and MZB development by independent mechanisms. Development of MZBs is dependent on Notch2 signaling, which requires proteolysis of the Notch2 receptor by a previously unidentified proteinase. Further experiments revealed that Notch2 signaling is severely impaired in ADAM10-null B cells. Thus, ADAM10 critically regulates MZB development by initiating Notch2 signaling. This study identifies ADAM10 as the in vivo CD23 sheddase and an important regulator of B cell development. Moreover, it has important implications for the treatment of numerous CD23- and Notch-mediated pathologies, ranging from allergy to cancer.

Mature dendritic cells in inflamed human pulps beneath deep caries.

OBJECTIVE: Dendritic cells (DCs) in pulps have been identified with markers for immature DCs and the relationship of DC maturity to pulpal health has not been carefully examined. We sought to test the hypothesis that the frequency of CD83+ mature DCs would correlate with caries invasion. STUDY DESIGN: Pulps were collected from extracted teeth exhibiting (I) no caries (n = 9), (II) shallow dentinal caries (n = 5), and (III) deep caries (n = 9). Immature DCs (CD209+), mature DCs (CD83+), and monocytes/macrophages (CD14+) in three groups were enumerated immunohistochemically. RESULTS: Mature DCs were frequently found beneath deep caries (21.3 mature DCs/3 grids versus <1 in groups I and II) with increasing numbers of CD209+ DCs and CD14+ cells. Co-localization of CD4+ T cells with mature DCs and macrophages was observed in deep caries. CONCLUSION: Mature DCs were frequently found only beneath deep caries and these DCs were co-localized with CD4+ T cells suggesting antigen presentation.

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.

Dexamethasone inhibits maturation and alters function of monocyte-derived dendritic cells from cord blood.

Critically ill infants are treated with dexamethasone (Dx) and other glucocorticoids to reduce inflammation and to promote lung and cardiac function. The neonatal immune system is immature, so neonatal dendritic cells (DCs) might be especially sensitive to glucocorticoid-mediated immunosuppression. To test this, we compared Dx treatment of monocyte-derived DCs from cord (CB) and adult blood (AB). Dx decreased CD1a levels on both AB and CB DCs. CB-treated cells also exhibited decreased expression of CD83 and increased expression of CD14, alterations not observed in AB DCs. Characteristic immature endocytic activity was sustained and enhanced in Dx-treated CB DCs, whereas AB DCs matured normally. Maintenance of endocytosis corresponded with CD14 expression. Dx markedly increased CB DC IL-10, a T cell helper 2 (Th2)-preferential cytokine, while reducing IL-12, a counterbalancing Th1 cytokine. AB DCs were also affected, but increases in IL-10 and decreases in IL-12 were more modest. Dx treatment also inhibited DC-induced T cell proliferation, but CB DCs were inhibited more. In short, neonatal DCs seemed to be especially sensitive to the immunosuppressive effects of Dx as indicated by altered phenotype, endocytic function, ability to stimulate T cells, and cytokine shift favoring Th2. These alterations in DC function are consistent with an increased risk for certain infections and atopic diseases.

Endocarditis-associated oral streptococci promote rapid differentiation of monocytes into mature dendritic cells.

Endocarditis is frequently attributable to oral streptococci, but mechanisms of pathogenesis are not well understood, although monocytes appear to be important. High titers of interleukin-12 (IL-12) are produced by peripheral blood mononuclear cells (PBMC) after engaging Streptococcus mutans, but monocytes in developing endocardial vegetations tend to disappear rather than become macrophages. These data prompted the hypothesis that streptococcus-infected monocytes differentiate into short-lived IL-12-producing dendritic cells (DCs) rather than macrophages. PBMC from healthy subjects were stimulated with six isolates of oral streptococci, three nonstreptococcal oral bacteria, or IL-4 plus granulocyte-macrophage colony-stimulating factor, and the appearance of cells with markers typical of mature DCs (CD83(+), CD86(+), CD11c(+), and CD14(-)) was monitored. Supernatant fluids from the PBMC cultures were harvested and IL-12 p70 levels were determined. S. mutans-stimulated monocytes were analyzed for their ability to elicit allogeneic mixed-lymphocyte reactions. All streptococci examined, except one strain of Streptococcus oralis (35037), rapidly induced up-regulation of CD83 and CD86 and a loss of CD14 in the CD11c(+) monocyte population within 20 h. Induction of IL-12 was CD14 dependent and correlated with streptococcal isolates that promoted the DC phenotype. Major histocompatibility complex (MHC) class II expression was up-regulated by S. mutans, and these cells were short-lived and elicited potent allogeneic mixed-lymphocyte reactions typical of DCs. In summary, monocytes stimulated with endocarditis-associated oral streptococci rapidly exhibited the DC phenotype and functions. These data suggest that the initiation of bacterial endocarditis by oral streptococci may involve monocyte-to-DC differentiation, and this may help explain the low levels of macrophages in the site.

Altered lipoprotein subclass distribution and PAF-AH activity in subjects with generalized aggressive periodontitis.

In this study, we examined whether the documented increase of plasma triglycerides in patients with generalized aggressive periodontitis (GAgP) is associated with changes in lipoprotein subclass distribution and/or LDL-associated platelet-activating factor acetylhydrolase (PAF-AH) activity. Lipoprotein subclasses were analyzed in whole plasma samples using nuclear magnetic resonance methods. Compared with subjects without periodontitis (NP subjects; n = 12), GAgP subjects (n = 12) had higher plasma levels of large, medium, and small VLDL (35.0 +/- 6.7 vs. 63.1 +/- 9.6 nmol/l; P = 0.025), higher levels of intermediate density lipoprotein (24.8 +/- 11.6 vs. 87.2 +/- 16.6 nmol/l; P = 0.006), lower levels of large LDL (448.3 +/- 48.5 vs. 315.8 +/- 59.4 nmol/l; P = 0.098), and higher levels of small LDL (488.2 +/- 104.2 vs. 946.7 +/- 151.6 nmol/l; P = 0.021). The average size of LDL from NP and GAgP subjects was 21.4 +/- 0.2 and 20.6 +/- 0.3 nm, respectively (P = 0.031). Compared with NP subjects, GAgP subjects had a greater number of circulating LDL particles (961.3 +/- 105.3 vs. 1,349.0 +/- 133.2 nmol/l; P = 0.032). Differences in the plasma levels of large, medium, and small HDL were not statistically significant. NP and GAgP subjects had similar plasma levels of total LDL-associated PAF-AH activity; however, LDL of GAgP subjects contained less PAF-AH activity per microgram of LDL protein (1,458.0 +/- 171.0 and 865.2 +/- 134 pmol/min/microg; P = 0.014). These results indicate that, in general, GAgP subjects have a more atherogenic lipoprotein profile and lower LDL-associated PAF-AH activity than NP subjects. These differences may help explain the increased risk of GAgP subjects for cardiovascular disease.