Plant-derived recombinant immune complexes as self-adjuvanting TB immunogens for mucosal boosting of BCG.

Progress with protein-based tuberculosis (TB) vaccines has been limited by poor availability of adjuvants suitable for human application. Here, we developed and tested a novel approach to molecular engineering of adjuvanticity that circumvents the need for exogenous adjuvants. Thus, we generated and expressed in transgenic tobacco plants the recombinant immune complexes (RICs) incorporating the early secreted Ag85B and the latency-associated Acr antigen of Mycobacterium tuberculosis, genetically fused as a single polypeptide to the heavy chain of a monoclonal antibody to Acr. The RICs were formed by virtue of the antibody binding to Acr from adjacent molecules, thus allowing self-polymerization of the complexes. TB-RICs were purified from the plant extracts and shown to be biologically active by demonstrating that they could bind to C1q component of the complement and also to the surface of antigen-presenting cells. Mice immunized with BCG and then boosted with two intranasal immunizations with TB-RICs developed antigen-specific serum IgG antibody responses with mean end-point titres of 1 : 8100 (Acr) and 1 : 24 300 (Ag85B) and their splenocytes responded to in vitro stimulation by producing interferon gamma. 25% of CD4+ proliferating cells simultaneously produced IFN-γ, IL-2 and TNF-α, a phenotype that has been linked with protective immune responses in TB. Importantly, mucosal boosting of BCG-immunized mice with TB-RICs led to a reduced M. tuberculosis infection in their lungs from log10 mean = 5.69 ± 0.1 to 5.04 ± 0.2, which was statistically significant. We therefore propose that the plant-expressed TB-RICs represent a novel molecular platform for developing self-adjuvanting mucosal vaccines.

Attenuated expression of A20 markedly increases the efficacy of double-stranded RNA-activated dendritic cells as an anti-cancer vaccine.

A20 is a zinc finger protein with ubiquitin-modifying activity. A20 has been described as negatively regulating signaling induced by the TNF receptor and TLR family in a number of cell types, including mouse bone marrow-derived dendritic cells (DCs). However, the expression and effect of A20 in activated human monocyte-derived DCs have not been previously evaluated. We report that DCs activated with the TLR3 ligand poly(I:C) up-regulate A20. Down-regulating A20 demonstrated its role in the functional activation of DCs. A20 down-regulated DCs showed higher activation of the transcription factors NF-kappaB and activator protein-1, which resulted in increased and sustained production of IL-6, IL-10, and IL-12p70. We additionally silenced the immunosuppressive cytokine IL-10 and demonstrated that IL-10 inhibits T cell proliferation. We further demonstrated that A20 down-regulated DCs skew naive CD4+ T cells toward IFN-gamma producing Th1 cells, a process which is dependent on IL-12p70 and which is unaffected by IL-10. Furthermore, A20 and/or IL-10 down-regulated DCs had an enhanced capacity to prime Melan-A/MART-1 specific CD8+ T cells. Finally, we demonstrated that potent T cell stimulatory DCs are generated by the simultaneous delivery of poly(I:C12U), A20, or A20/IL-10 small interfering RNA and Ag-encoding mRNA, introducing a one step approach to improve DC-based vaccines. Together these findings demonstrate that A20 negatively regulates NF-kappaB and activator protein-1 in DCs and that down-regulation of A20 results in DCs with enhanced T cell stimulatory capacity.

Hydrogen sulfide augments neutrophil migration through enhancement of adhesion molecule expression and prevention of CXCR2 internalization: role of ATP-sensitive potassium channels.

In this study, we have addressed the role of H(2)S in modulating neutrophil migration in either innate (LPS-challenged naive mice) or adaptive (methylated BSA (mBSA)-challenged immunized mice) immune responses. Treatment of mice with H(2)S synthesis inhibitors, dl-propargylglycine (PAG) or beta-cyanoalanine, reduced neutrophil migration induced by LPS or methylated BSA (mBSA) into the peritoneal cavity and by mBSA into the femur/tibial joint of immunized mice. This effect was associated with decreased leukocyte rolling, adhesion, and P-selectin and ICAM-1 expression on endothelium. Predictably, treatment of animals with the H(2)S donors, NaHS or Lawesson's reagent, enhanced these parameters. Moreover, the NaHS enhancement of neutrophil migration was not observed in ICAM-1-deficient mice. Neither PAG nor NaHS treatment changed LPS-induced CD18 expression on neutrophils, nor did the LPS- and mBSA-induced release of neutrophil chemoattractant mediators TNF-alpha, keratinocyte-derived chemokine, and LTB(4). Furthermore, in vitro MIP-2-induced neutrophil chemotaxis was inhibited by PAG and enhanced by NaHS treatments. Accordingly, MIP-2-induced CXCR2 internalization was enhanced by PAG and inhibited by NaHS treatments. Moreover, NaHS prevented MIP-2-induced CXCR2 desensitization. The PAG and NaHS effects correlated, respectively, with the enhancement and inhibition of MIP-2-induced G protein-coupled receptor kinase 2 expression. The effects of NaHS on neutrophil migration both in vivo and in vitro, together with CXCR2 internalization and G protein-coupled receptor kinase 2 expression were prevented by the ATP-sensitive potassium (K(ATP)(+)) channel blocker, glybenclamide. Conversely, diazoxide, a K(ATP)(+) channel opener, increased neutrophil migration in vivo. Together, our data suggest that during the inflammatory response, H(2)S augments neutrophil adhesion and locomotion, by a mechanism dependent on K(ATP)(+) channels.

Regulation of TLR2 expression by prostaglandins in brain glia.

TLR have emerged as important primary sensors for diverse stimuli and are increasingly implicated in various diseases. However, the molecular mechanisms underlying the regulation of the TLR system remain poorly understood. In this study, we report that some PGs may control TLR-mediated inflammatory events through modulation of TLR2 expression in brain immune cells. We first found that 15-deoxy-Delta12,14-PG J(2) (15d-PGJ(2)) markedly altered the expression of TLR2 but not TLR4, TLR1, and TLR9 at the message and protein levels in activated glia. Down-regulation of TLR2 expression and downstream events of TLR2 activation, including phagocytosis by 15d-PGJ(2), were also observed in cells treated with representative TLR2 ligands such as lipoteichoic acid and Pam(3)CSK(4). We further revealed that certain 15d-PGJ(2)-related PGs such as 15d-PGD(2) and PGD(2) also suppressed the ligand-stimulated increase of TLR2 expression, whereas PGE(2) and arachidonic acids did not. Interestingly, TLR2 expression was down-regulated even when such PGs were added at several hours after stimulator treatment. These findings appear to be independent of peroxisome proliferator-activated receptor gamma and D prostanoid receptors (DPs) because potent synthetic peroxisome proliferator-activated receptor gamma agonists, selective DP1 agonist, or DP2 agonist did not mimic the effects of such PGs on TLR2 expression. Taken together, our results suggest that 15d-PGJ(2), 15d-PGD(2), and PGD(2) may play notable roles as modulators of the TLR2-mediated inflammatory events, and provide new insight into the resolution of inflammation in the brain.

Changes in adrenal steroidogenesis in spontaneous bacterial peritonitis and sterile ascites.

Adrenal androgens, particularly dehydroepiandrosterone (DHEA), may have important regulatory effects on the immune system in humans. This study measured the changes in adrenal steroidogenesis in 13 non-infected cirrhosis patients with sterile ascites and 13 patients with spontaneous bacterial peritonitis and the relation with circulating interleukin-6 (IL-6) levels. Comparisons were made with 10 healthy age-matched control subjects. The severity of bacterial peritonitis in liver cirrhosis was significantly associated with enhanced serum IL-6 and cortisol levels, and a decrease in serum DHEA sulfate in relation to serum IL-6 concentrations. Careful, long-term studies on DHEA administered to cirrhosis patients are needed to assess its safety in improving a number of pathological conditions that complicate liver cirrhosis.

LFA-1-mediated T cell costimulation through increased localization of TCR/class II complexes to the central supramolecular activation cluster and exclusion of CD45 from the immunological synapse.

T cell activation is associated with a dramatic reorganization of cell surface proteins and associated signaling components into discrete subdomains within the immunological synapse in T cell:APC conjugates. However, the signals that direct the localization of these proteins and the functional significance of this organization have not been established. In this study, we have used wild-type and LFA-1-deficient, DO11.10 TCR transgenic T cells to examine the role of LFA-1 in the formation of the immunological synapse. We found that coengagement of LFA-1 is not required for the formation of the central supramolecular activation cluster (cSMAC) region, but does increase the accumulation of TCR/class II complexes within the cSMAC. In addition, LFA-1 is required for the recruitment and localization of talin into the peripheral supramolecular activation cluster region and exclusion of CD45 from the synapse. The ability of LFA-1 to increase the amount of TCR engaged during synapse formation and segregate the phosphatase, CD45, from the synapse suggests that LFA-1 might enhance proximal TCR signaling. To test this, we combined flow cytometry-based cell adhesion and calcium-signaling assays and found that coengagement of LFA-1 significantly increased the magnitude of the intracellular calcium response following Ag presentation. These data support the idea that in addition to its important role on regulating T cell:APC adhesion, coengagement of LFA-1 can enhance T cell signaling, and suggest that this may be accomplished in part through the organization of proteins within the immunological synapse.

Mass production of somatic embryos expressing Escherichia coli heat-labile enterotoxin B subunit in Siberian ginseng.

The B subunit of Escherichia coli heat-labile toxin (LTB) is a potent mucosal immunogen and immunoadjuvant for co-administered antigens. In order to produce large scale of LTB for the development of edible vaccine, we used transgenic somatic embryos of Siberian ginseng, which is known as medicinal plant. When transgenic somatic embryos were cultured in 130L air-lift type bioreactor, they were developed to mature somatic embryos through somatic embryogenesis and contained approximately 0.36% LTB of the total soluble protein. Enzyme-linked immunosorbent assay indicated that the somatic embryo-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting the LTB subunits formed active pentamers. Therefore, the use of the bioreactor system for expression of LTB proteins in somatic embryos allows for continuous mass production in a short-term period.

Multiple roles of CLAN (caspase-associated recruitment domain, leucine-rich repeat, and NAIP CIIA HET-E, and TP1-containing protein) in the mammalian innate immune response.

NAIP CIIA HET-E and TP1 (NACHT) family proteins are involved in sensing intracellular pathogens or pathogen-derived molecules, triggering host defense responses resulting in caspase-mediated processing of proinflammatory cytokines and NF-kappaB activation. Caspase-associated recruitment domain, leucine-rich repeat, and NACHT-containing protein (CLAN), also known as ICE protease-activating factor, belongs to a branch of the NACHT family that contains proteins carrying caspase-associated recruitment domains (CARDs) and leucine-rich repeats (LRRs). By using gene transfer and RNA-interference approaches, we demonstrate in this study that CLAN modulates endogenous caspase-1 activation and subsequent IL-1beta secretion from human macrophages after exposure to LPS, peptidoglycan, and pathogenic bacteria. CLAN was also found to mediate a direct antibacterial effect within macrophages after Salmonella infection and to sensitize host cells to Salmonella-induced cell death through a caspase-1-independent mechanism. These results indicate that CLAN contributes to several biological processes central to host defense, suggesting a prominent role for this NACHT family member in innate immunity.

Expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells.

A major function of macrophages is to engulf apoptotic cells to prevent them from releasing noxious materials as they die. Milk fat globule-EGF-factor 8 (MFG-E8) is a glycoprotein secreted by activated macrophages that works as a bridge between apoptotic cells and phagocytes by specifically recognizing phosphatidylserine exposed on apoptotic cells. In this study, we found that developmental endothelial locus-1 (Del-1), originally identified as an embryonic endothelial cell protein that binds alphavbeta3 integrin, is structurally and functionally homologous to MFG-E8. That is, both consist of a signal sequence, two epidermal growth factor domains and two factor VIII-homologous domains (C1 and C2). Del-1 bound to the apoptotic cells by recognizing phosphatidylserine via the factor VIII-homologous domains with an affinity similar to that of MFG-E8. The phagocytic activity of NIH 3T3 cells against apoptotic cells was enhanced by Del-1 through an interaction between the epidermal growth factor domain in Del-1 and alphavbeta3 integrin expressed in the NIH 3T3 cells. Screening of primary macrophages and macrophage cell lines for the expression of MFG-E8 and Del-1 indicated that MFG-E8 and Del-1 are expressed in different sets of macrophages. These results suggest the existence of macrophage subsets that use MFG-E8 or Del-1 differently to engulf apoptotic cells.

KIR2DL4 is an IL-2-regulated NK cell receptor that exhibits limited expression in humans but triggers strong IFN-gamma production.

Killer cell Ig-like receptor (KIR)2DL4 (2DL4, CD158d) was previously described as the only KIR expressed by every human NK cell. It is also structurally atypical among KIRs because it possesses a basic transmembrane residue, which is characteristic of many activating receptors, but also contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). We expressed epitope-tagged 2DL4 in an NK-like cell line to study receptor function. Three distinct 2DL4 cDNA clones were analyzed: one encoding the "conventional" 2DL4 with the cytoplasmic ITIM (2DL4.1) and two encoding different cytoplasmic truncated forms lacking the ITIM (2DL4.2 and 2DL4(*)). Surprisingly, one truncated receptor (2DL4.2), which is the product of a prevalent human 2DL4 allele, was not expressed on the cell surface, indicating that some individuals may lack functional 2DL4 protein expression. Conversely, both 2DL4.1 and 2DL4(*) were expressed on the cell surface and up-regulated by IL-2. Analysis of primary NK cells with anti-2DL4 mAb confirmed the lack of surface expression in a donor with the 2DL4.2 genotype. Donors with the 2DL4.1 genotype occasionally expressed receptor only on CD56(high) NK cells, although their expression was up-regulated by IL-2. Interestingly, Ab engagement of epitope-tagged 2DL4 triggered rapid and robust IFN-gamma production, but weak redirected cytotoxicity in an NK-like cell line, which was the opposite pattern to that observed upon engagement of another NK cell activating receptor, NKp44. Importantly, both 2DL4.1 and 2DL4(*) exhibited similar activation potential, indicating that the ITIM does not influence 2DL4.1 activating function. The unique activation properties of 2DL4 suggest linkage to a distinct signaling pathway.

Immunoregulatory role of ocular macrophages: the macrophages produce RANTES to suppress experimental autoimmune uveitis.

Murine experimental autoimmune uveitis (EAU) is a model of human uveitis. Ocular-infiltrating macrophages play a crucial role in the generation of tissue damage in EAU. In fact, several chemokines are actually produced in the inflamed eye. The aim of this study was to elucidate the role of ocular macrophage-derived chemokines in EAU. C57BL/6 mice were immunized with human interphotoreceptor retinoid binding protein peptide 1-20, and the EAU severity was scored at multiple time points based on microscopic fundus observations (retinal vascular dilatation and exudates) and histological examinations. The peak inflammatory response was observed 1 wk (day 16) after the beginning of macrophage infiltration to the eye (day 9). Ocular-infiltrating cells were enriched or depleted of macrophages by magnetic beads and analyzed by real-time RT-PCR for chemokine mRNA production. We found that only the macrophage-enriched cells from the eye produced RANTES, and thus proposed that macrophage-derived RANTES facilitated the ocular inflammations. In contrast to our postulate, neutralization of RANTES by specific Ab in vivo on days 9 and 13 exacerbated EAU. We also found that the ratio of ocular CD4/CD8 T cells was markedly increased after treatment. As a result, RANTES neutralization might exacerbate EAU by modulating the type of T cell subsets recruited to the eye. In conclusion, our data provide insight into the immunoregulatory role of macrophages and RANTES in the pathogenesis of ocular inflammation. Not all macrophage-derived chemokines cause local inflammation, since RANTES produced by ocular macrophages appears to suppress EAU.

Immunoglobulin activation of T cell chemoattractant expression in fibroblasts from patients with Graves' disease is mediated through the insulin-like growth factor I receptor pathway.

Graves' disease (GD) is associated with T cell infiltration, but the mechanism for lymphocyte trafficking has remained uncertain. We reported previously that fibroblasts from patients with GD express IL-16, a CD4-specific chemoattractant, and RANTES, a C-C chemokine, in response to GD-specific IgG (GD-IgG). We unexpectedly found that these responses result from a functional interaction between GD-IgG and the insulin-like growth factor (IGF)-I receptor (IGF-IR). IGF-I and the IGF-IR-specific IGF-I analog, des(1-3), mimic the effects of GD-IgG. Neither GD-IgG nor IGF-I activates chemoattractant expression in control fibroblasts from donors without GD. Interrupting IGF-IR function with specific receptor-blocking Abs or by transiently transfecting fibroblasts with a dominant negative mutant IGF-IR completely attenuates signaling provoked by GD-IgG. Moreover, GD-IgG displaces specific (125)I-labeled IGF-I binding to fibroblasts and attenuates IGF-IR detection by flow cytometry. These findings identify a novel disease mechanism involving a functional GD-IgG/IGF-IR bridge, which potentially explains T cell infiltration in GD. Interrupting this pathway may constitute a specific therapeutic strategy.

The therapeutic potential of lactoferrin.

Lactoferrin (Lf), a natural defence iron-binding protein, is present in exocrine secretions that are commonly exposed to normal flora: milk, tears, nasal exudate, saliva, bronchial mucus, gastrointestinal fluids, cervicovaginal mucus and seminal fluid. Additionally, Lf is produced in polymorphonuclear leukocytes and is deposited by these circulating cells in septic sites. A principal function of Lf is that of scavenging non-protein-bound iron in body fluids and inflamed areas so as to suppress free radical-mediated damage and decrease accessibility of the metal to invading bacterial, fungal and neoplastic cells. Adequate sources of bovine and recombinant human Lf are now available for development of commercial applications. Among the latter are use of Lf in food preservation, fish farming, infant milk formula and oral hygiene. Other readily accessible body compartments for Lf administration include skin, throat and small intestine. Further research is needed for possible medicinal use in colon and systemic tissues. Although Lf is a natural product and should be highly biocompatible, possible hazards have been documented.

Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) can play a direct role in the inhibitory function of killer cell Ig-like receptors in human NK cells.

The inhibitory forms of killer cell Ig-like receptors (KIR) are MHC class I-binding receptors that are expressed by human NK cells and prevent their attack of normal cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatase, Src homology region 2-containing protein tyrosine phosphatase (SHP)-1, to phosphorylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs). However, the functional significance of parallel recruitment of a SHP-1-related phosphatase, SHP-2, to KIR ITIMs has not been addressed. In the present study, our results with mutant forms of a classical KIR, KIR3DL1, show a direct correlation between SHP-2 recruitment and functional inhibition of target cell conjugation and cytotoxicity. In addition, KIR3DL1 inhibition of target cell cytotoxicity is blocked by overexpression of a dominant-negative form of SHP-2. Finally, KIR3DL1 fused directly with the catalytic domain of SHP-2 inhibits both target cell conjugation and cytotoxicity responses. These results strongly indicate that SHP-2 catalytic activity plays a direct role in inhibitory KIR functions, and SHP-2 inhibits NK cell activation in concert with SHP-1.

Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells.

HIV-1 is an enveloped retrovirus that acquires its outer membrane as the virion exits the cell. Because of the association of apoptosis with the progression of AIDS, HIV-1-infected T cells or macrophages might be expected to express elevated levels of surface phosphatidylserine (PS), a hallmark of programmed cell death. Virions produced by these cells would also be predicted to have PS on the surface of their envelopes. In this study, data are presented that support this hypothesis and suggest that PS is required for macrophage infection. The PS-specific protein annexin V was used to enrich for virus particles and to inhibit HIV-1 replication in primary macrophages, but not T cells. HIV-1 replication was also significantly inhibited with vesicles consisting of PS, but not phosphatidylcholine. PS is specifically required for HIV-1 infection because viruses pseudotyped with vesicular stomatitis virus G and amphotropic murine leukemia virus envelopes were not inhibited by PS vesicles or annexin V. These data indicate that PS is an important cofactor for HIV-1 infection of macrophages.

Gram-negative, but not Gram-positive, bacteria elicit strong PGE2 production in human monocytes.

Gram-positive and Gram-negative bacteria induce different cytokine patterns in human mononuclear cells. We have seen that Gram-positives preferentially induce IL-12 and TNF-alpha, whereas Gram-negatives induce more IL-10, IL-6, and IL-8. In this study, we compared the capacity of these two groups of bacteria to induce PGE2. Monocytes stimulated with Gram-negative bacterial species induced much more PGE2 than did Gram-positive bacteria (5600 +/- 330 vs. 1700 +/- 670 pg/mL, p < 0.001). Blocking of COX-2 by NS398 abolished PGE2 production, but did not alter the cytokine patterns induced by Gram-positive and Gram-negative bacteria. We suggest that Gram-positive and Gram-negative bacteria may stimulate different innate effector functions; Gram-positive bacteria promoting cell-mediated effector functions whereas Gram-negative bacteria inducing mediators inhibiting the same.

DNA-encoded fetal liver tyrosine kinase 3 ligand and granulocyte macrophage-colony-stimulating factor increase dendritic cell recruitment to the inoculation site and enhance antigen-specific CD4+ T cell responses induced by DNA vaccination of outbred animals.

DNA-based immunization is a contemporary strategy for developing vaccines to prevent infectious diseases in animals and humans. Translating the efficacy of DNA immunization demonstrated in murine models to the animal species that represent the actual populations to be protected remains a significant challenge. We tested two hypotheses directed at enhancing DNA vaccine efficacy in outbred animals. The first hypothesis, that DNA-encoding fetal liver tyrosine kinase 3 ligand (Flt3L) and GM-CSF increases dendritic cell (DC) recruitment to the immunization site, was tested by intradermal inoculation of calves with plasmid DNA encoding Flt3L and GM-CSF followed by quantitation of CD1(+) DC. Peak DC recruitment was detected at 10-15 days postinoculation and was significantly greater (p < 0.05) in calves in the treatment group as compared with control calves inoculated identically, but without Flt3L and GM-CSF. The second hypothesis, that DNA encoding Flt3L and GM-CSF enhances immunity to a DNA vector-expressed Ag, was tested by analyzing the CD4(+) T lymphocyte response to Anaplasma marginale major surface protein 1a (MSP1a). Calves immunized with DNA-expressing MSP1a developed strong CD4(+) T cell responses against A. marginale, MSP1a, and specific MHC class II DR-restricted MSP1a epitopes. Administration of DNA-encoding Flt3L and GM-CSF before MSP1a DNA vaccination significantly increased the population of Ag-specific effector/memory cells in PBMC and significantly enhanced MSP1a-specific CD4(+) T cell proliferation and IFN-gamma secretion as compared with MHC class II DR-matched calves vaccinated identically but without Flt3L and GM-CSF. These results support use of these growth factors in DNA vaccination and specifically indicate their applicability for vaccine testing in outbred animals.

1,25(OH)2 vitamin D3-stimulated osteoclast formation in spleen-osteoblast cocultures is mediated in part by enhanced IL-1 alpha and receptor activator of NF-kappa B ligand production in osteoblasts.

We examined the ability of 1,25 (OH)(2) vitamin D(3) (Vit D) to stimulate osteoclast-like cell (OCL) formation in cocultures of spleen cells and primary calvarial osteoblasts from wild-type (WT) and IL-1R type 1-deficient (knockout; KO) mice. Vit D dose dependently increased OCL in cocultures containing WT osteoblasts. In contrast, there was a 90% reduction in OCL numbers in cocultures containing KO osteoblasts. In cocultures with either WT or KO osteoblasts, treatment with Vit D increased receptor activator of NF-kappaB ligand mRNA by 17-, 19-, or 3.5-fold, respectively. Vit D decreased osteoprotegerin mRNA to undetectable in all groups. Intracellular IL-1alpha protein increased after Vit D treatment in cocultures containing WT, but not KO osteoblasts. We also examined direct effects of Vit D, IL-1alpha, and their combination on gene expression in primary osteoblasts. In WT cells, Vit D and IL-1 stimulated receptor activator of NF-kappaB ligand mRNA expression by 3- and 4-fold, respectively, and their combination produced a 7-fold increase. Inhibition of osteoprotegerin mRNA in WT cells was partial with either agent alone and greatest with their combination. In KO cells, only Vit D stimulated a response. IL-1 alone increased IL-1alpha protein expression in WT osteoblasts. However, in combination with Vit D, there was a synergistic response (100-fold increase). In KO cultures, there were no effects of IL-1, Vit D, or their combination on IL-1alpha protein. These results demonstrate interactions between IL-1 and Vit D in primary osteoblasts that appear important in both regulation of IL-1alpha production and the ability of Vit D to support osteoclastogenesis.

Enhanced Th1 response to Staphylococcus aureus infection in human lactoferrin-transgenic mice.

Lactoferrin (Lf) is an iron-binding protein of external secretions and neutrophil secondary granules with antimicrobial and immunomodulatory activities. To further define these properties of Lf, we have investigated the response to Staphylococcus aureus infection in transgenic mice carrying a functional human Lf gene. The transgenic mice cleared bacteria significantly better than congenic littermates, associated with a trend to reduced incidence of arthritis, septicemia, and mortality. We identified two pathways by which S. aureus clearance was enhanced. First, human Lf directly inhibited the growth of S. aureus LS-1 in vitro. Second, S. aureus-infected transgenic mice exhibited enhanced Th1 immune polarization. Thus, spleen cells from infected transgenic mice produced higher levels of TNF-alpha and IFN-gamma and less IL-5 and IL-10 upon stimulation ex vivo with the exotoxin toxic shock syndrome toxin-1 compared with congenic controls. To confirm that these effects of Lf transgene expression could occur in the absence of live bacterial infection, we also showed that Lf-transgenic DBA/1 mice exhibited enhanced severity of collagen-induced arthritis, an established model of Th1-induced articular inflammation. Higher levels of stainable iron in the spleens of transgenic mice correlated with human Lf distribution, but all other parameters of iron metabolism did not differ between transgenic mice and wild-type littermates. These results demonstrate that human Lf can mediate both antimicrobial and immunomodulatory activities with downstream effects on the outcome of immune pathology in infectious and inflammatory disease.

Regulation of NK cell-mediated cytotoxicity by the adaptor protein 3BP2.

Stimulation of lymphocytes through multichain immune recognition receptors activates multiple signaling pathways. Adaptor proteins play an important role in integrating these pathways by their ability to simultaneously bind multiple signaling components. Recently, the 3BP2 adaptor protein has been shown to positively regulate the transcriptional activity of T cells. However, the mechanisms by which signaling components are involved in this regulation remain unclear, as does a potential role for 3BP2 in the regulation of other cellular functions. Here we describe a positive regulatory role for 3BP2 in NK cell-mediated cytotoxicity. We also identify p95(vav) and phospholipase C-gamma isoforms as binding partners of 3BP2. Our results show that tyrosine-183 of 3BP2 is specifically involved in this interaction and that this residue critically influences 3BP2-dependent function. Therefore, 3BP2 regulates NK cell-mediated cytotoxicity by mobilizing key downstream signaling effectors.