IL-13 regulates the immune response to inhaled antigens

The large inhibitory effect of IL-13 blockers on the asthma phenotype prompted us to ask whether IL-13 would play a role in regulating the allergic immune response in addition to its documented effects on structural pulmonary cells. Because IL-13 does not interact with murine T or B cells, but with monocytes, macrophages, and dendritic cells (DCs), we examined the role of IL-13 in the activation of pulmonary macrophages and DCs and in the priming of an immune response to a harmless, inhaled Ag. We found that a majority of cells called "alveolar or interstitial macrophages" express CD11c at high levels (CD11c(high)) and are a mixture of at least two cell types as follows: 1) cells of a mixed phenotype expressing DC and macrophage markers (CD11c, CD205, and F4/80) but little MHC class II (MHC II); and 2) DC-like cells expressing CD11c, CD205, MHC II, and costimulatory molecules. Endogenous IL-13 was necessary to induce and sustain the increase in MHC II and CD40 expression by pulmonary CD11c(high) cells, demonstrated by giving an IL-13 inhibitor as a measure of prevention or reversal to allergen-primed and -challenged mice. Conversely, IL-13 given by inhalation to naive mice increased the expression of MHC II and costimulatory molecules by CD11c(high) cells in an IL-4Ralpha-dependent manner. We found that exogenous IL-13 exaggerated the immune and inflammatory responses to an inhaled, harmless Ag, whereas endogenous IL-13 was necessary for the priming of naive mice with an inhaled, harmless Ag. These data indicate that blockade of IL-13 may have therapeutic potential for controlling the immune response to inhaled Ags.     

Sepsis-induced apoptosis causes progressive profound depletion of B and CD4+ T lymphocytes in humans

Patients with sepsis have impaired host defenses that contribute to the lethality of the disorder. Recent work implicates lymphocyte apoptosis as a potential factor in the immunosuppression of sepsis. If lymphocyte apoptosis is an important mechanism, specific subsets of lymphocytes may be more vulnerable. A prospective study of lymphocyte cell typing and apoptosis was conducted in spleens from 27 patients with sepsis and 25 patients with trauma. Spleens from 16 critically ill nonseptic (3 prospective and 13 retrospective) patients were also evaluated. Immunohistochemical staining showed a caspase-9-mediated profound progressive loss of B and CD4 T helper cells in sepsis. Interestingly, sepsis did not decrease CD8 T or NK cells. Although there was no overall effect on lymphocytes from critically ill nonseptic patients (considered as a group), certain individual patients did exhibit significant loss of B and CD4 T cells. The loss of B and CD4 T cells in sepsis is especially significant because it occurs during life-threatening infection, a state in which massive lymphocyte clonal expansion should exist. Mitochondria-dependent lymphocyte apoptosis may contribute to the immunosuppression in sepsis by decreasing the number of immune effector cells. Similar loss of lymphocytes may be occurring in critically ill patients with other disorders.     

GM-CSF is an essential regulator of T cell activation competence in uterine dendritic cells during early pregnancy in mice

Uterine dendritic cells (DCs) are critical for activating the T cell response mediating maternal immune tolerance of the semiallogeneic fetus. GM-CSF (CSF2), a known regulator of DCs, is synthesized by uterine epithelial cells during induction of tolerance in early pregnancy. To investigate the role of GM-CSF in regulating uterine DCs and macrophages, Csf2-null mutant and wild-type mice were evaluated at estrus, and in the periconceptual and peri-implantation periods. Immunohistochemistry showed no effect of GM-CSF deficiency on numbers of uterine CD11c(+) cells and F4/80(+) macrophages at estrus or on days 0.5 and 3.5 postcoitum, but MHC class II(+) and class A scavenger receptor(+) cells were fewer. Flow cytometry revealed reduced CD80 and CD86 expression by uterine CD11c(+) cells and reduced MHC class II in both CD11c(+) and F4/80(+) cells from GM-CSF-deficient mice. CD80 and CD86 were induced in Csf2(-/-) uterine CD11c(+) cells by culture with GM-CSF. Substantially reduced ability to activate both CD4(+) and CD8(+) T cells in vivo was evident after delivery of OVA Ag by mating with Act-mOVA males or transcervical administration of OVA peptides. This study shows that GM-CSF regulates the efficiency with which uterine DCs and macrophages activate T cells, and it is essential for optimal MHC class II- and class I-mediated indirect presentation of reproductive Ags. Insufficient GM-CSF may impair generation of T cell-mediated immune tolerance at the outset of pregnancy and may contribute to the altered DC profile and dysregulated T cell tolerance evident in infertility, miscarriage, and preeclampsia.     

Immune cell populations in the equine corpus luteum throughout the oestrous cycle and early pregnancy: an immunohistochemical and flow cytometric study

Recent evidence indicates that the cells of the immune system and their large network of secretory products, or cytokines, play an active role in the ovary throughout the oestrous cycle. In the present study, immune cell populations (T and B lymphocytes, macrophages, granulocytes and eosinophils) and expression of major histocompatibility complex (MHC) class II were investigated in corpora lutea from mares in early (days 2-4), mid- (days 7-10) and late (days 12-14) dioestrus, the post-luteolytic phase (days 16-17) and early pregnancy. The number of T lymphocytes within the corpus luteum increased in the late luteal phase. CD4+ cells did not increase until day 16, whereas the number of CD8+ cells increased before functional luteolysis; an apparently selective luteal infiltration of CD8+ cells was observed. MHC class II expression by non-steroidogenic cells was increased in samples from days 16-17, as was the number of infiltrating macrophages. Flow cytometry revealed very low expression of MHC class II by large luteal cells at all stages of the oestrous cycle. In early pregnancy, the number of CD4+ and CD8+ cells and macrophages decreased, as did MHC class II expression, compared with mid-dioestrous samples. B cells were present in very small numbers in all samples examined. Eosinophils were similarly sparsely distributed and numbers decreased further in pregnancy. After exogenous PGF2 alpha administration, populations of CD4+ cells and non-specific esterase staining cells were significantly smaller than after natural luteolysis, whereas eosinophil numbers were increased compared with samples from days 16-17. However, the number of CD8+ and CD5+ cells and MHC class II expression were not significantly different from those observed after natural luteolysis. These findings indicate that populations of immune cells in the equine corpus luteum vary during its lifespan. The selective increase in CD8+ cells before functional luteolysis indicates that they have a physiological role in the regression of the corpus luteum.     

CD8alpha+ and CD11b+ dendritic cell-restricted MHC class II controls Th1 CD4+ T cell immunity

The activation, proliferation, differentiation, and trafficking of CD4 T cells is central to the development of type I immune responses. MHC class II (MHCII)-bearing dendritic cells (DCs) initiate CD4(+) T cell priming, but the relative contributions of other MHCII(+) APCs to the complete Th1 immune response is less clear. To address this question, we examined Th1 immunity in a mouse model in which I-A(beta)(b) expression was targeted specifically to the DCs of I-A(beta)b-/- mice. MHCII expression is reconstituted in CD11b(+) and CD8alpha(+) DCs, but other DC subtypes, macrophages, B cells, and parenchymal cells lack of expression of the I-A(beta)(b) chain. Presentation of both peptide and protein Ags by these DC subsets is sufficient for Th1 differentiation of Ag-specific CD4(+) T cells in vivo. Thus, Ag-specific CD4(+) T cells are primed to produce Th1 cytokines IL-2 and IFN-gamma. Additionally, proliferation, migration out of lymphoid organs, and the number of effector CD4(+) T cells are appropriately regulated. However, class II-negative B cells cannot receive help and Ag-specific IgG is not produced, confirming the critical MHCII requirement at this stage. These findings indicate that DCs are not only key initiators of the primary response, but provide all of the necessary cognate interactions to control CD4(+) T cell fate during the primary immune response.     

B lymphocytes participate in cross-presentation of antigen following gene gun vaccination

Although endocytosed proteins are commonly presented via the class II MHC pathway to stimulate CD4(+) T cells, professional APCs can also cross-present Ags, whereby these exogenous peptides can be complexed with class I MHC for cross-priming of CD8(+) T cells. Whereas the ability of dendritic cells (DCs) to cross-present Ags is well documented, it is not known whether other APCs may also play a role, or what is the relative contribution of cross-priming to the induction of acquired immunity after DNA immunization. In this study, we compared immune responses generated after gene gun vaccination of mice with DNA vaccine plasmids driven by the conventional CMV promoter, the DC-specific CD11c promoter, or the keratinocyte-specific K14 promoter. The CD11c promoter achieved equivalent expression in CD11c(+) DCs in draining lymph nodes over time, as did a conventional CMV-driven plasmid. However, immunization with DC-restricted DNA vaccines failed to generate protective humoral or cellular immunity to model Ags influenza hemagglutinin and OVA, despite the ability of CD11c(+) cells isolated from lymph nodes to stimulate proliferation of Ag-specific T cells directly ex vivo. In contrast, keratinocyte-restricted vaccines elicited comparable T and B cell activity as conventional CMV promoter-driven vaccines, indicating that cross-priming plays a major role in the generation of immune responses after gene gun immunization. Furthermore, parallel studies in B cell-deficient mu-MT mice demonstrated that B lymphocytes, in addition to DCs, mediate cross-priming of Ag-specific T cells. Collectively, these data indicate that broad expression of the immunogen is required for optimal induction of protective acquired immunity.     

Critical role of TRIF and MyD88 in Mycobacterium tuberculosis Hsp70-mediated activation of dendritic cells

As a potent immune regulator, heat shock protein 70 derived from Mycobacterium tuberculosis (Mtb Hsp70) has adjuvant effect and activates immune cells such as macrophages and dendritic cells (DCs). Although Toll-like receptors (TLRs) are known to involve in DCs activation by Mtb Hsp70, there is still a controversy and the underlying mechanism is not well understood. In this study, we examined whether TRIF and MyD88, the core adaptor molecules for TLRs signaling, regulate Mtb Hsp70-induced DCs activation. Although Mtb Hsp70 produced substantial level of cytokines (IL-6, IL-12p40, and TNF-α) in TRIF-deficient DCs in a dose-dependent manner, each level was significantly lower than that in WT cells. The cytokines production was almost abolished in MyD88-deficient DCs. Consistent with cytokine results, Mtb Hsp70-induced activation of NF-κB and MAPKs was also impaired in both TRIF- and MyD88-deficient DCs, as compared with WT cells. Inhibitor assay revealed that NF-κB, ERK, and JNK, but not p38, regulate Mtb Hsp70-induced production of cytokines. In addition, the up-regulation of co-stimulatory molecules and MHC class II was mostly TRIF-dependent in DCs in response Mtb Hsp70, whereas MyD88 was only partially involved. Finally, mixed leukocytes reaction (MLR) assay revealed that both TRIF and MyD88 are critical for DCs ability promoted by Mtb Hsp70 to differentiate naïve T cells into effector T cells of producing IFN-γ. Our findings suggest that both TRIF and MyD88 are essential for the activation and maturation of DCs in response to Mtb Hsp70.     

Brucella abortus lipopolysaccharide in murine peritoneal macrophages acts as a down-regulator of T cell activation

Macrophages play a central role in host immune responses against pathogens by acting as both professional phagocytic cells and as fully competent APCs. We report here that the LPS from the facultative intracellular Gram-negative bacteria Brucella abortus interferes with the MHC class II Ag presentation pathway. LPS inhibits the capacity of macrophages to present hen egg lysozyme (HEL) antigenic peptides to specific CD4(+) T cells but not those of OVA to specific CD8(+) T cells. This defect was neither related to a decrease of MHC class II surface expression nor to a deficient uptake or processing of HEL. In addition, B. abortus LPS did not prevent the formation of SDS-resistant MHC class II complexes induced by HEL peptides. At the cell surface of macrophages, we observed the presence of LPS macrodomains highly enriched in MHC class II molecules, which may be responsible for the significant down-regulation of CD4(+) T cell activation. This phenomenon may account for the avoidance of the immune system by certain bacterial pathogens and may explain the immunosuppression observed in individuals with chronic brucellosis.     

Recruitment of dendritic cells and macrophages during T cell-mediated synovial inflammation

Adoptive transfer of adjuvant-induced arthritis was used in this study to examine local macrophages and dendritic cells (DCs) during T cell-mediated synovial inflammation. We studied the influx of CD11b+CD11c+ putative myeloid DCs and other non-lymphoid CD45+ cells into synovium-rich tissues (SRTs) of the affected hind paws in response to a pulse of autoreactive thoracic duct cells. Cells were prepared from the SRTs using a collagenase perfusion-digestion technique, thus allowing enumeration and phenotypic analysis by flow cytometry. Numbers of CD45+ cells increased during the first 6 days, with increases in CD45+MHC (major histocompatibility complex) II+ monocyte-like cells from as early as day 3 after transfer. In contrast, typical MHC II(-) monocytes, mainly of the CD4(-) subset, did not increase until 12 to 14 days after cell transfer, coinciding with the main influx of polymorphonuclear cells. By day 14, CD45+MHC IIhi cells constituted approximately half of all CD45+ cells in SRT. Most of the MHC IIhi cells expressed CD11c and CD11b and represented putative myeloid DCs, whereas only approximately 20% were CD163+ macrophages. Less than 5% of the MHC IIhi cells in inflamed SRT were CD11b(-), setting a maximum for any influx of plasmacytoid DCs. Of the putative myeloid DCs, a third expressed CD4 and both the CD4+ and the CD4(-) subsets expressed the co-stimulatory molecule CD172a. Early accumulation of MHC IIhiCD11c+ monocyte-like cells during the early phase of T cell-mediated inflammation, relative to typical MHC II(-) blood monocytes, suggests that recruited monocytes differentiate rapidly toward the DC lineage at this stage in the disease process. However, it is possible also that the MHC IIhiCD11c+ cells originate from a specific subset of DC-like circulating mononuclear cells.     

Prion protein expression by mouse dendritic cells is restricted to the nonplasmacytoid subsets and correlates with the maturation state

Expression of the physiological cellular prion protein (PrP(C)) is remarkably regulated during differentiation and activation of cells of the immune system. Among these, dendritic cells (DCs) display particularly high levels of membrane PrP(C), which increase upon maturation, in parallel with that of molecules involved in Ag presentation to T cells. Freshly isolated mouse Langerhans cells, dermal DCs, and DCs from thymus, spleen, and mesenteric lymph nodes expressed low to intermediate levels of PrP(C). Highest levels of both PrP(C) and MHC class II molecules were displayed by lymph node CD8alpha(int) DCs, which represent fully mature cells having migrated from peripheral tissues. Maturation induced by overnight culture resulted in increased levels of surface PrP(C), as did in vivo DC activation by bacterial LPS. Studies on Fms-like tyrosine kinase 3 ligand bone marrow-differentiated B220(-) DCs confirmed that PrP(C) expression followed that of MHC class II and costimulatory molecules, and correlated with IL-12 production in response to TLR-9 engagement by CpG. However, at variance with conventional DCs, B220(+) plasmacytoid DCs isolated from the spleen, or in vitro differentiated, did not significantly express PrP(C), both before and after activation by TLR-9 engagement. PrP knockout mice displayed higher numbers of spleen CD8alpha(+) DCs, but no significant differences in their maturation response to stimulation through TLR-4 and TLR-9 were noticed. Results are discussed in relation to the functional relevance of PrP(C) expression by DCs in the induction of T cell responses, and to the pathophysiology of prion diseases.     

Regulation of MHC class I transport in human dendritic cells and the dendritic-like cell line KG-1

Dendritic cells (DCs) progress through distinct maturational phases; immature DCs capture Ag while mature DCs are optimized for Ag presentation. Proper control of immunity requires regulated compartmentalization of MHC class II molecules. We report that DCs also regulate MHC class I trafficking throughout maturation. Although mature human DCs express high levels of surface MHC class I, immature DCs exhibit lower surface levels while retaining MHC class I-peptide complexes in the Golgi. A cell line, KG-1, behaves similarly. We confirm the similarity of KG-1 to DCs by demonstrating its capacity to present exogenous Ags in an MHC class I-restricted fashion to CD8(+) T cell hybridomas, a phenomenon called cross-presentation. Biochemical characterization of MHC class I trafficking throughout maturation showed that, in early KG-1 dendritic-like cells, surface arrival of MHC class I-peptide complexes is delayed by their retention in the Golgi. In mature dendritic-like cells, these complexes relocate to the surface and their stability increases, concomitant with up-regulation of costimulatory molecules. Maturation induces qualitative changes in the MHC class I-associated peptide repertoire demonstrated by increased thermostability. The differential processing of MHC class I throughout maturation may prevent premature immune activation while promoting T cell responses in lymph nodes to Ags acquired at sites of inflammation.     

Bursopentin (BP5) from chicken bursa of fabricius attenuates the immune function of dendritic cells

Bursopentine (BP5), a novel pentapeptide isolated from chicken bursa of fabricius, has been proved to have immunomodulatory effects on B and T lymphocytes, anti-oxidative stress on macrophages, and antiproliferation on tumor cells. However, the effects of BP5 on the immune function exhibited by dendritic cells (DCs), which are regarded as a major target for immunomodulators, remain unknown. In this study, we examined the effects of BP5 on the activation and maturation of murine bone marrow-derived DCs. Our results showed that BP5 significantly suppressed the secretion of lipopolysaccharide (LPS)-induced pro-inflammatory (TNF-α, IL-1β, IL-6 and IL-12p70) and anti-inflammatory (IL-10) cytokines by DCs, and this impact was not due to its cytotoxicity. Besides, BP5 reversed the morphological changes and attenuated the expression of phenotypic markers (MHC II, CD40, CD80 and CD86 molecules) in LPS-induced DCs. Furthermore, BP5 restored the decreased FITC-dextran uptake in LPS-treated DCs, arrested the LPS-induced migration of DCs and abrogated the promoting ability of LPS-induced DCs for allogeneic T cell proliferation. These findings show a new immunopharmacological capability of BP5 and provide a novel approach in the prevention and therapy of chronic inflammation and autoimmunity via abolishing the immune function of DCs.     

Dynamics of dendritic cell phenotype and interactions with CD4+ T cells in airway inflammation and tolerance

An emerging concept is that different types of dendritic cells (DCs) initiate different immune outcomes, such as tolerance vs inflammation. In this study, we have characterized the DCs from the lung draining lymph nodes of mice immunized for allergic airway inflammation or tolerance and examined their interactions with CD4(+) T cells. The DC population derived from tolerized mice was predominantly CD11c(+), B220(+), Gr-1(+), CD11b(-), and MHC class II(low), which resembled plasmacytoid-type DCs whereas DCs from the inflammatory condition were largely CD11c(+), B220(-), Gr-1(-), CD11b(+), and MHC class II(high) resembling myeloid-type DCs. The DCs from the tolerogenic condition were poor inducers of T cell proliferation. DCs from both conditions induced T cell IL-4 production but the T cells cultured with tolerogenic DCs were unresponsive to IL-4 as indicated by inhibition of STAT6 activation and expression of growth factor-independent 1, which has been recently shown to be important for STAT6-activated Th2 cell expansion. Our data suggest that airway tolerance vs inflammation is determined by the DC phenotype in lung draining lymph nodes.     

Characterization of the systemic loss of dendritic cells in murine lymph nodes during polymicrobial sepsis

Dendritic cells (DCs) play a key role in critical illness and are depleted in spleens from septic patients and mice. To date, few studies have characterized the systemic effect of sepsis on DC populations in lymphoid tissues. We analyzed the phenotype of DCs and Th cells present in the local (mesenteric) and distant (inguinal and popliteal) lymph nodes of mice with induced polymicrobial sepsis (cecal ligation and puncture). Flow cytometry and immunohistochemical staining demonstrated that there was a significant local (mesenteric nodes) and partial systemic (inguinal, but not popliteal nodes) loss of DCs from lymph nodes in septic mice, and that this process was associated with increased apoptosis. This sepsis-induced loss of DCs occurred after CD3(+)CD4(+) T cell activation and loss in the lymph nodes, and the loss of DCs was not preceded by any sustained increase in their maturation status. In addition, there was no preferential loss of either mature/activated (MHCII(high)/CD86(high)) or immature (MHCII(low)/CD86(low)) DCs during sepsis. However, there was a preferential loss of CD8(+) DCs in the local and distant lymph nodes. The loss of DCs in lymphoid tissue, particularly CD8(+) lymphoid-derived DCs, may contribute to the alterations in acquired immune status that frequently accompany sepsis.     

High mobility group box protein 1: an endogenous signal for dendritic cell maturation and Th1 polarization

High mobility group box protein 1 (HMGB1), a DNA binding nuclear and cytosolic protein, is a proinflammatory cytokine released by monocytes and macrophages. This study addressed the hypothesis that HMGB1 is an immunostimulatory signal that induces dendritic cell (DC) maturation. We show that HMGB1, via its B box domain, induced phenotypic maturation of DCs, as evidenced by increased CD83, CD54, CD80, CD40, CD58, and MHC class II expression and decreased CD206 expression. The B box caused increased secretion of the proinflammatory cytokines IL-12, IL-6, IL-1alpha, IL-8, TNF-alpha, and RANTES. B box up-regulated CD83 expression as well as IL-6 secretion via a p38 MAPK-dependent pathway. In the MLR, B box-activated DCs acted as potent stimulators of allogeneic T cells, and the magnitude of the response was equivalent to DCs activated by exposure to LPS, nonmethylated CpG oligonucleotides, or CD40L. Furthermore, B box induced secretion of IL-12 from DCs as well as IL-2 and IFN-gamma secretion from allogeneic T cells, suggesting a Th1 bias. HMGB1 released by necrotic cells may be a signal of tissue or cellular injury that, when sensed by DCs, induces and/or enhances an immune reaction.     

Basic concepts of immune response and defense development

The induction of immune responses requires critical interaction between innate parts of the immune system, which respond rapidly and in a relatively nonspecific manner, and other specific parts, which recognize particular epitopes on an antigen. A critical element in this interaction is the role played by dendritic cells (DCs), which represent "professional antigen-presenting cells." DCs endocytose and process antigen to peptide presented on the cell surface in association with major histocompatibility complex (MHC) molecules. This presentation results in interaction with and stimulation of helper T (Th) lymphocytes, which recognize peptide in association with either MHC class II or cytotoxic T (Tc) lymphocytes, which recognize peptide in association with MHC class I. Stimulation of Th lymphocytes produces the growth and differentiation factors (cytokines) essential for the B lymphocytes that have responded to a more intact form of the antigen and that differentiate into antibody-producing cells. The precise interaction between the cells depends on cognate ligand-receptor recognition between the B and Th lymphocytes. DCs also play a direct role with the stimulation of the B lymphocytes. It appears that DC can deliver antigen to the B lymphocytes in a more intact form than the processed form essential for stimulating T lymphocytes, and can release cytokines that assist the differentiation of the B lymphocytes into antibody-producing cells. This close relationship among the three cell types and the cytokines that are produced ensures the precise control and regulation necessary for immune response development.     

Host dendritic cells alone are sufficient to initiate acute graft-versus-host disease

Alloantigen expression on host APCs is essential to initiate graft-vs-host disease (GVHD); however, critical APC subset remains to be elucidated. We compared the ability of dendritic cells (DCs) and B cells to initiate acute GVHD by an add-back study of MHC class II-expressing APCs (II(+/+)) into MHC class II-deficient (II(-/-)) mice that were resistant to CD4-dependent GVHD. Injection of host-derived, but not donor-derived, II(+/+) DCs or host-derived II(+/+) B cells, was sufficient to break GVHD resistance of II(-/-) mice and induced lethal acute GVHD. By contrast, host-derived II(+/+) B cells, both naive and LPS stimulated, failed to induce activation or tolerance of donor CD4(+) T cells. Similarly, in a model of CD8-dependent GVHD across MHC class I mismatch injection of allogeneic DCs, but not B cells, induced robust proliferation of donor CD8(+) T cells and broke GVHD resistance of chimeric recipients in which APCs were syngeneic to donors. These results demonstrate that host-derived DCs are critical in priming donor CD4(+) and CD8(+) T cells to cause GVHD, and selective targeting of host DCs may be a promising strategy to prevent GVHD.     

Differential MHC class II presentation of a pathogenic autoantigen during health and disease

Glucose-6-phosphate isomerase (GPI) is the target autoantigen recognized by KRN T cells in the K/BxN model of rheumatoid arthritis. T cell reactivity to this ubiquitous Ag results in the recruitment of anti-GPI B cells and subsequent immune complex-mediated arthritis. Because all APCs have the capacity to process and present this autoantigen, it is unclear why systemic autoimmunity with polyclonal B cell activation does not ensue. To this end, we examined how GPI is presented by B cells relative to other immunologically relevant APCs such as dendritic cells (DCs) and macrophages in the steady state, during different phases of arthritis development, and after TLR stimulation. Although all APCs can process and present the GPI:I-A(g7) complex, they do so with different efficiencies. DCs are the most potent at baseline and become progressively more potent with disease development correlating with immune complex uptake. Interestingly, in vivo and in vitro maturation of DCs did not enhance GPI presentation, suggesting that DCs use mechanisms to regulate the presentation of self-peptides. Non-GPI-specific B cells are the weakest APCs (100-fold less potent than DCs) and fail to productively engage KRN T cells at steady state and during arthritis. However, the ability to stimulate KRN T cells is strongly enhanced in B cells after TLR ligation and provides a mechanism whereby polyclonal B cells may be activated in the wake of an acute infection.     

Mast cell-derived exosomes induce phenotypic and functional maturation of dendritic cells and elicit specific immune responses in vivo

Mast cells (MCs) are considered major players in IgE-mediated allergic responses, but have also recently been recognized as active participants in innate as well as specific immune responses. Recent work provided evidence that MCs are able to activate B and T lymphocytes through the release of vesicles called exosomes. Here we demonstrate that exosomes, which are located in the endocytic pathway, harbor exogenous Ags that associate with other molecules endowed with immunomodulatory functions, including 60- and 70-kDa heat shock proteins. Administration to naive mice of Ag-containing exosomes in the absence of conventional adjuvants elicits specific Ab responses across the MHC II haplotype barrier. We demonstrate that MC-exosomes induce immature dendritic cells (DCs) to up-regulate MHC class II, CD80, CD86, and CD40 molecules and to acquire potent Ag-presenting capacity to T cells. Uptake and processing of Ag-associated exosomes by endogenous DCs were also demonstrated. Finally, exosome-associated heat shock proteins are critical for the acquisition by DCs of the Ag-presenting function. This work demonstrates a heretofore unrecognized collaborative interaction between MCs and DCs leading to the elicitation of specific immune responses.     

Major histocompatibility complex class I and II expression on macrophages containing a virulent strain of Brucella abortus measured using green fluorescent protein-expressing brucellae and flow cytometry

Immune responses appropriate for control of an intracellular pathogen are generated in mice infected with Brucella abortus, shown by the ability of T cells to adoptively transfer resistance to naive mice. The infection nevertheless persists for months. It was hypothesized that one factor in maintaining the infection despite the presence of immune T cells was suboptimal expression of major histocompatibility complex (MHC) molecules on macrophages containing brucellae. This would allow B. abortus to elude detection by the host's immune system. To test this, B. abortus organisms expressing green fluorescent protein (GFP-Brucella) were constructed and three-color flow cytometry used to evaluate MHC expression on macrophages following in vitro or in vivo infection. When infected in vitro, the levels of MHC class I and class II expression on J774 macrophages containing GFP-Brucella were the same or higher than on macrophages without GFP-Brucella in the same cultures. Similarly, the MHC expression was higher on GFP(+) peritoneal exudate cells following infection or phagocytosis of heat-killed GFP-Brucella than it was on uninfected peritoneal exudate cells. Following in vivo infection of mice the level of MHC class I and II expression on GFP(+) cells in their spleens (the main site of infection) also tended to be as high as or higher than that on the GFP-negative cells. The only in vivo GFP(+) cells that showed a decreased MHC expression was a population of splenic Mac1(+) cells recovered from interferon-gamma gene-disrupted mice at the time of their death due to an overwhelming number of bacteria per spleen. Overall, it was concluded that decreased MHC expression is not a general principle associated with brucella infection of macrophages and thus not likely to contribute to maintenance of the chronic infection.