Intracellular survival of Mycobacterium tuberculosis is dependent on iron within the mycobacterial phagosome. Yet there is not much information about the concentration or regulation of elements within phagosomes containing intracellular bacteria. Wagner et al. (p. 1491 ) used a hard x-ray microprobe with suboptical spatial resolution to measure concentrations of 10 single elements in vacuoles of live mouse macrophages infected with one of two pathogenic strains or with a nonpathogenic strain of mycobacteria. Iron concentrations increased 1 h after infection with either pathogenic strain and continued to increase to 24 h. In contrast, iron concentrations in macrophages infected with the nonpathogenic strain or with a M. tuberculosis strain lacking bacterial siderophore increased at 1 h after infection but then quickly decreased. Acquisition of radioactive iron (59Fe) by bacteria in the macrophage cytosol was confirmed by electron microscopic examination of macrophages incubated with 59Fe-transferrin for 24 h. Concentrations of other elements within 1 h or 24 h of infection varied among the strains. Addition of TNF-α to macrophages before infection with pathogenic mycobacteria prevented increased iron concentration in the phagosome; addition of the cytokine after infection reversed the increased iron concentration normally seen at 24 h after infection. The data show that phagosomes containing pathogenic mycobacteria have high levels of iron that are retained through the action of a bacterial siderophore.

At least six cytokines with receptors that share the common γ-chain (γc) are implicated in NK cell development and function; the individual role of each cytokine has not been established. Vosshenrich et al. (p. 1213 ) generated a series of Rag−/− mice lacking one to three of the cytokines or lacking γc. Rag−/−γc−/− mice had reduced percentages of immature and mature NK cells in bone marrow but had a higher percentage of committed NK cell precursors compared with Rag−/− mice. Rag−/− mice deficient for IL-2 or IL-7 or multiply deficient for IL-2, IL-4, and IL-7 had normal bone marrow and splenic populations of NK cells at all stages of maturity with normal lytic and in vivo antitumor responses. Lack of IL-4 only resulted in a slight reduction of mature splenic NK cells. In contrast, Rag−/− mice deficient in IL-15 had reduced numbers of immature and mature NK cells in the bone marrow and fewer mature NK cells in the spleen. The few NK cells that remained in Rag−/− IL-15−/− mice exhibited nearly complete phenotypic differentiation but had reduced ability to lyse target cells and produce IFN-γ after IL-12 stimulation compared with controls. The data suggest that γc cytokines are not required for NK commitment of hemopoietic precursors but that generation of mature NK cells in bone marrow and spleen is IL-15 dependent.

T cell lineage commitment occurs at the double-positive (DP) stage when both CD8 and CD4 coreceptors are expressed. A cis-regulatory element, the enhancer E8III, located on mouse genomic DNA between the CD8β and CD8α genes is expressed only in DP thymocytes. Feik et al. (p. 1513 ) mapped E8III enhancer activity to a core fragment of 285 bp using a CD8+ thymus-derived T cell line transiently transfected with luciferase reporter constructs containing deletions of E8III. The 285-bp fragment was inserted in the plasmid next to a human CD2 reporter gene and injected into mouse eggs; several transgenic mouse lines were established. Expression of the reporter gene occurred only in DP T cells. Transgenic mice deleted in E8III and E8II (active in DP and CD8+ T cells) by a targeting vector had normal CD8 T cell development with only small decreases in CD8 DP and single-positive (SP) cells and a slight increase in CD4 SP cells compared with wild-type mice. The CD4 SP thymocytes had maturation markers characteristic of DP thymocytes but failed to up-regulate CD8. CD8α levels on CD8α-positive dendritic cells and intraepithelial lymphocytes were not affected by E8III and E8II deletion. Five regulatory elements (RE) within the 285-bp core fragment were identified by transient transfection assays of deletion mutants cloned in the luciferase reporter plasmid and by EMSA with thymocyte nuclear extracts. One RE contained an E box motif, and a second RE had a c-myb binding site. The definition of elements in E8III responsible for cis-regulation of CD8 gene expression contributes to a better understanding of lineage commitment and CD8 T cell development.

The tick-borne spirochete Borrelia burgdorferi is the causative agent of Lyme disease in humans and induces a similar disease in mice. CD14 is involved in initiating activation of innate immune cells exposed to B. burgdorferi. To examine the role of CD14 in spirochete infections in vivo, Benhnia et al. (p. 1539 ) found that infected CD14−/− mice had thicker tibiotarsal joints, greater joint inflammation, and synovial hyperplasia, and more inflammation of heart tissues than infected wild-type mice even though spirochete burdens were not significantly different. Infected CD14−/− mice had higher serum levels of TNF-α, IFN-γ, MCP-1, and IL-6 than infected wild-type animals whether CD14−/− was on a B. burgdorferi-sensitive or -resistant genetic background. Macrophages from CD14−/− mice had higher mRNA expression levels and production of TNF-α and IFN-γ than wild-type cells after stimulation with live spirochetes; lysed spirochetes induced much lower levels. Microarray analysis of cytokine and receptor gene responses of spirochete-stimulated mutant macrophages showed an up-regulation of molecules involved in recruiting or activating innate immune cells, whereas expression levels of molecules involved in recruiting or activating T cells or down-regulating macrophage effector functions were reduced compared with wild-type cells. TLR2 surface expression was more enhanced and lasted longer on spirochete-stimulated CD14−/− vs control macrophages. The data suggest that the CD14-TLR2 pathway is responsible for controlling inflammation during B. burgdorferi infection in mice.

Skin and the respiratory tract are major areas of exposure to allergens as well as major sites for allergic diseases. However, it is not clear whether the initial site of Ag exposure determines the site of disease. Alvarez et al. (p. 1664 ) sensitized mice via gene gun bombardment of gold particles coated with plasmid-encoding OVA cDNA. OVA-specific IgE and IgG1 were detected in sera 2 wk after a single immunization; 3 weekly immunizations increased the Ig levels further and enhanced production of IL-4, IL-5, IL-10, and IL-13 by OVA-stimulated splenocytes. No response was detected in control mice receiving gold particles alone or empty plasmid vector. Numbers of CD69+CD4+ T cells and CD8+ T cells increased in draining inguinal and axillary lymph nodes in sensitized mice. Dermal-sensitized mice had acute cutaneous responses when challenged intradermally with OVA at a different site 1 wk after sensitization and developed systemic anaphylactic shock after receiving OVA i.p. Challenge with OVA aerosolization had no effect on dermal-sensitized animals but did elicit a robust airway inflammatory response in mice sensitized by OVA aerosolization. Th2 cytokine responses of in vitro OVA-activated lymphocytes were specific to cells taken from lymph nodes draining the site of sensitization. The data demonstrate that the site of initial allergen exposure determines the tissue specificity of the Th2 memory response.

Although viral Ag-specific memory CD8+ T cells are generated and persist long after a viral infection, the site at which the cells are maintained by homeostatic proliferation in the absence of Ag has not been determined. Becker et al. (p. 1269 ) infected C57BL/6 mice with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). LCMV-specific memory cells were detected in all tissues examined. However, the bone marrow had the highest concentration of virus-specific memory cells that stained with propidium iodide and incorporated BrdU by 1 day following labeling; adoptively transferred CFSE-labeled memory cells from spleens of infected mice transferred into naive recipients showed highest levels of division in bone marrow. The second most active site of homeostatic proliferation was spleen. BrdU-labeled cells in bone marrow were greatly reduced by 3 days following injection. Proliferation of virus-specific memory CD8+ T cells injected into either naive or LCMV immune animals was highest in bone marrow after treatment of recipients with poly(I:C); almost no proliferation of injected cells occurred in naiveIL-15−/− recipients. The authors interpret their data to suggest that memory cells migrate to the bone marrow, divide, and then rapidly move to peripheral tissues.

Costimulation of T cells by CD28 involves the activation of several signaling pathways. The E3 ubiquitin ligase Cbl-b negatively regulates several CD28 functions, including Vav1 phosphorylation; however, the effect of Cbl-b on other CD28 functions is unknown. Krawczyk et al. (p. 1472 ) found that mice deficient in Cbl-b alone or in combination with Vav1 or CD28 deficiency developed spontaneous autoimmunity as they aged, whereas cd28−/− mice did not. Anti-viral IgG production after infection with vesicular stomatitis virus (VSV) was comparable in cbl-b−/−cd28−/−, cbl-b−/−vav1−/−, and wild-type mice, but was impaired in cd28−/− and vav1−/− animals. In contrast, VSV-positive splenic germinal center formation, impaired in cd28−/− and vav1−/− mice, was restored to wild-type levels by Cbl-b deficiency only in vav1−/− mice. Spleens of cd28−/− mice contained scattered VSV-specific B cells and lacked follicular dendritic cell clusters. Loss of Cbl-b in the cd28−/− mice restored follicular dendritic cell cluster formation. Decreased IL-4 and IFN-γ production and reduced T cell expression of ICOS in VSV-infected cd28−/− mice were not restored by loss of Cbl-b. The data highlight the different functions of Cbl-b/Vav1-dependent and Cbl-b/Vav1-independent CD28 activation pathways in response to VSV infection.

The BCR consists of surface Ig (sIg), which binds Ag, in association with the Igα/Igβ heterodimer, which transmits an activating signal to downstream signal transducers. Although it is known that capped BCR on activated B cells is internalized, the fate of the individual BCR components is unclear. Kremyanskaya and Monroe (p. 1501 ) stimulated primary BALB/c mouse B cells with anti-mouse Ig Ab or anti-mouse IgG F(ab′)2. A fluorochrome-labeled secondary Ab detected capping by 5 min and a 99% loss of sIg by 60 min by flow cytometry. Comparable results were obtained using Fab specific for L or H chains. However, staining of stimulated cells with FITC-conjugated Igβ, or BCR cross-linking by anti-Ig Ab, followed by staining with a fluorescent-labeled secondary Ab before stimulation, demonstrated retention of Igβ on the surface by immunofluorescence microscopy. Stimulated B cells coexpressing mouse and human Igβ chains and B cells expressing an Ag-specific BCR transgene internalized only Igκ at high levels whereas significant levels of Igβ were detected on the cell surface. Mice lacking surface expression of MHC class II also retained Igβ and internalized sIg. Presence of an inhibitor of membrane trafficking during anti-Ig stimulation reduced Igβ surface expression by 30%. The results show that the stimulated BCR disassociates to permit internalization of sIg with its bound ligand and retention of the signaling complex at the cell surface.

Viral and bacterial dsRNA interact with TLR3 on the surface of cells of the innate immune system to induce IFN-β via activation of IFN regulatory factor-3 (IRF-3) and NF-κB signaling pathways. A negative regulator of NF-κB signaling, A20, has been identified, but its role in regulating IRF-3 signaling has not been established. Saitoh et al. (p. 1507 ) used the IFN-stimulation response element (ISRE) and the IFN-β promoter cloned in separate luciferase reporter plasmids to measure induction of IRF-3 in human embryonic kidney cells in response to stimulation by poly(I:C) or Newcastle Disease Virus (NDV). Stimulated luciferase activity by either element was dependent on IRF-3 and a NF-κB-activating kinase (NAK/TBK1) and was inhibited in a dose-dependent manner by a cotransfected plasmid expressing A20. IRF-3 dimerization that normally occurred after stimulation was prevented by the presence of A20. A20 was coimmunoprecipitated with NAK/TBK1 or a second IRF-3 kinase. Both kinases associated with A20 mutants deleted at the C or N terminus. IRF-3 was phosphorylated by NAK/TBK1 only in cells expressing the N terminus of A20 but not in cells expressing the full-length A20 or its C terminus. Full-length A20 or its C terminus suppressed ISRE-dependent transcription induced after TLR3 stimulation by poly(I:C) or NDV infection, whereas expression of a small inhibitory A20 RNA construct enhanced the ISRE-dependent transcription. The results show that A20 negatively regulates IRF-3 signaling in cells stimulated by TLR3 interaction with dsRNA or NDV.

Mast cell degranulation is precipitated by IgE interaction with its Fc receptor. The receptor for CD200, a member of the Ig supergene family, negatively regulates myeloid functions, but its effect on mast cells has not been determined. Cherwinski et al. (p. 1348 ) detected surface expression of CD200R on mouse and human mast cells. A fusion molecule mCD200-Ig, consisting of the extracellular domain of CD200 fused to the FcR binding domain of mouse IgG, or a rat anti-mCD200R Ab inhibited TNP-induced degranulation and cytokine secretion of TNP-specific IgE-coated transfected mouse mast cells overexpressing CD200R. Cross-linking the anti-mCD200R Abs or coligating the mCD200R and FcεRI enhanced the inhibitory responses. Eliminating FcR binding capability or murinizing the rat anti-mCD200R mAb with a mouse IgG1 isotype did not decrease the inhibitory effect. In contrast, inhibition of human mast cell degranulation occurred by an anti-hCD200R Ab alone and was enhanced by cross-linking the Ab or coligating the anti-hCD200R Ab to FcεRI. Expression of CD200R in frozen mouse and human skin was determined by immunofluorescent microscopy to be predominantly on skin mast cells and infiltrating leukocytes, whereas CD200 was expressed on dermal fibroblasts and other skin cells but not on mast cells. Mice injected i.v. with anti-mCD200R mAb 24 h prior to intradermal treatment with DNP-specific mouse IgE had reduced IgE-dependent passive cutaneous anaphylactic reactions compared with an isotype mAb control. The data show that CD200 engagement of its receptor inhibits mast cell degranulation without coligation to the activating FcεRI.

Summaries written by Dorothy L. Buchhagen, Ph.D.