Retinoic acid-related orphan receptor γ (RORγ) regulates thymocyte survival through control of Bcl-xL expression. The molecular mechanism by which RORγt, a truncated isoform of RORγ expressed only in thymocytes, regulates thymocyte survival is unknown. Xie et al. (p. 3800 ) found a conserved activation function-2 (AF-2) domain within the twelfth α helix of RORγt by sequence analysis. Abs against two steroid receptor coactivators (SRCs) coimmunoprecipitated RORγt with the corresponding SRC transiently coexpressed in a mouse T cell line. Mutation of an amino acid residue in the AF-2 domain of RORγt eliminated its binding with either SRC. Mutations of two or three SRC LXXLL motifs abolished SRC interactions with the AF-2 domain of RORγt. Wild-type RORγt, but not the AF-2 mutant, cooperated with either wild-type SRC to enhance transcription from a reporter plasmid containing RORγt binding sites. However, wild-type RORγt or the AF-2 mutant inhibited overexpression of a NFAT reporter plasmid even in the presence of a SRC. SRC-specific Abs coimmunoprecipitated RORγt with the appropriate SRC from thymocytes of RORγ−/− mice transgenic for wild-type or mutated RORγt attached to a GFP coding region under control of a CD4 promoter. Expression of the wild-type RORγt transgene in RORγ−/− thymocytes restored apoptosis, Bcl-xL, CD4+ T cells, TCRlow T cells, and thymic cellularity to levels seen in wild-type mice. The data suggest that RORγt regulates thymocyte survival through interaction with SRCs.

Both IL-13 and IL-4 bind the type II IL-4R to activate STAT6 and produce asthma-like symptoms in mice. Previous studies from the Erle laboratory showed that mucus hyperplasia occurred in mice with both STAT6 expression and IL-13 overexpression restricted to Clara cells found only in lung and trachea. However, their results did not definitively implicate IL-4R in the effect. Kuperman et al. (p. 3746 ) from the same laboratory developed a transgenic mouse with selective disruption of Il4ra gene expression in Clara cells. Wild-type and Il4ra gene-disrupted mice sensitized and challenged intranasally with OVA had comparable serum IgE levels, equivalent increases in numbers of macrophages, neutrophils, eosinophils, lymphocytes, and IL-4 and IL-13 concentrations in bronchoalveolar lavage fluid and similar decreases in airway reactivity. In contrast, allergen-induced mucus production was reduced in mice with IL-4ra gene disruption compared with controls as measured by computer-assisted stereology. Mutant mice also had decreased lung mRNA expression for genes associated with asthma pathogenesis, including two mucin genes, chitinase and calcium-activated chloride channel 3, compared with wild-type mice. The data support the interpretation that IL-13 and/or IL-4 act directly on Clara cells to induce mucus metaplasia in experimental allergic asthma in mice.

Trafficking of stem cells and progenitor cells to and from the bone marrow in response to CXCL12 is enhanced by the anaphylatoxin C3a. Yet, the mechanism of C3a interaction with CXCL12 is not known. Honczarenko et al. (p. 3698 ) found that C3a, C3a-desArg (the cleaved form of C3a), and C4a, but not C5a, increased the chemotactic response of human bone marrow CD34+ progenitor cells and all B cell subsets to CXCL12 and CCL19; none of the anaphylatoxins were chemotactic alone. None of those cells bound anti-C3aR mAb, and an inhibitor of C3a binding to C3aR did not affect the C3a-enhanced chemotactic response to CXCL12. C3a also influenced CXCL12-induced migration of a human pro-B cell line that expressed CXCR4, the receptor for CXCL12, but lacked surface C3aR and of C3aR−/− mouse bone marrow progenitor B and T cells. 125I-CXCL12 binding to CXCR4+C3aR−/− human cells was enhanced by preincubation of the cells with C3a. Specific binding of 125I-CXCL12 to plate-adhered C3a was competed by nonradiolabeled CXCL12. Primary human bone marrow stromal cells, but not established human B cell lines, generated physiological amounts of C3a, C4a, and CXCL12 when cultured for 24 h in serum-free conditions. The data suggest that C3a directly interacts with CXCL12 to increase binding of the chemokine to CXCR4 and to induce the migration of both human and mouse hemopoietic progenitor cells.

Overexpression of matrix metalloproteinase 9 (MMP-9) in the joint capsule results in cartilage degradation characteristic of osteoarthritis (OA). However, the mechanism by which MMP-9 overexpression is induced is not known. Ray et al. (p. 4039 ) found high levels of MMP-9 activity and protein in synovial fluids and high levels of MMP-9 mRNA in knee cartilage of dogs with OA compared with normal controls. Reporter gene constructs containing progressively deleted MMP-9 promoter DNAs were transfected into chondrocyte or synovial cells, and four regulatory element binding sites were mapped by the response of the transfected cells to IL-1β or TNF-α stimulation. Single site mutant reporter constructs of each element further revealed that MMP-9 stimulation in response to IL-1β or TNF-α was regulated by NF-κB, AP-1, and GT-box elements. Proteins from OA cartilage or stimulated synovial cell nuclear extracts formed complexes with a nucleotide encompassing the GT-box element. Binding was competed by oligonucleotides containing serum amyloid A-activating factor (SAF-1) and specific promoter 1-binding sequences; anti-SAF-1 Ab supershifted the protein-DNA complexes in gels. SAF-1 overexpression in synoviocytes and chondrocytes stimulated high levels of MMP-9 production. Coexpression of SAF-1 and AP-1 family members c-Jun plus c-Fos synergistically induced MMP-9, whereas mutation of either AP-1 family member inhibited the stimulatory effect. Immunohistochemical analysis showed elevated levels of MMP-9, c-Jun, and c-Fos in OA cartilage. The authors conclude that cooperative SAF-1/AP-1 binding at the GT-box element within the promoter of MMP-9 regulates its expression in canine OA.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) are involved in a variety of cell-mediated immune responses, including intracellular signaling. However, participation of the uPA/uPAR system in immune complex (IC)-dependent diseases is unknown. Shushakova et al. (p. 4060 ) found increased levels of uPA and uPAR mRNAs and proteins in mouse alveolar macrophages stimulated with heat aggregated mouse IgG1 IC and in lung homogenates, alveolar macrophages, and bronchoalveolar lavage (BAL) fluids from mice undergoing an IC-induced reverse passive pulmonary Arthus reaction. IC-challenged wild-type mice had high lung levels of polymorphonuclear cells and hemorrhage compared with controls deficient in uPA or uPAR. BAL fluids from both IC-treated deficient strains of mice had lower TNF-α and MIP-2 levels and reduced chemotactic activity for wild-type polymorphonuclear cells compared with wild-type mice. Release of TNF-α and MIP-2 from alveolar macrophages, stimulated by interaction of recombinant C5a with C5aR, was enhanced by uPA. Anti-uPAR Ab prevented the enhanced chemokine production and attenuated the C5a-dependent increase of MIP-2 release. The C5a-induced MIP-2 response was abrogated in uPAR−/− mice and reduced in uPA−/− mice but was restored in uPA−/− mice by uPA. C5aR and uPAR were coprecipitated by anti-uPAR or anti-C5a Abs from lysates of alveolar macrophages stimulated with C5a. Induction of FcγRIII and FcRγ and suppression FcγRII occurred in alveolar macrophages from wild-type mice but not in those from uPAR−/− mice in IC-induced alveolitis. The authors demonstrate that uPA/uPAR mediates C5a/C5aR signaling on alveolar macrophages in IC-induced inflammation in mice.

Activation of protein kinase B (PKB) by lipid messengers downstream of TCR stimulation increases T cell viability. However, the mechanism by which PKB prevents Fas-mediated apoptosis has not been deciphered. Jones et al. (p. 3790 ) found that activated T cells expressing a PKB transgene, but not wild-type cells, remained viable when deprived of IL-2. Cycloheximide rendered the transgenic cells susceptible to Fas-mediated apoptosis but had no effect on survival in the absence of IL-2. An ELISA-based assay detected higher levels of nuclear NF-κB DNA-binding complexes in the transgenic cells vs wild-type controls following CD3/CD28 stimulation. Increased nuclear translocation of NF-κB-binding complexes in the transgenic T cells was reduced when the cells also constitutively expressed a NF-κB repressor that prevented IκB degradation. The double-transgenic cells were protected against apoptosis due to IL-2 deficiency but were sensitive to Fas-mediated apoptosis in vitro and superantigen-induced Fas-dependent apoptosis in vivo. CD3/CD28-activated nf-κb1−/− T cells had reduced viability in the absence of IL-2; transgenic expression of PKB rescued the mutant cells. PKB expression did not rescue Fas-induced apoptosis of nf-κb1−/− T cells. T cells carrying the NF-κB repressor, with or without PKB, had enhanced caspase-8 activity following Fas activation compared with controls. Caspase-8 activation was enhanced significantly in nf-κb1−/− and PKB/nf-κb1−/− T cells compared with nf-κb1+/− cells, in the absence or the presence of PKB. Mice expressing PKB plus the NF-κB repressor transgene lacked the lymphoid hyperplasia and increased numbers of activated T cells seen in young mice expressing PKB. The authors show that NF-κB mediates PKB inhibition of Fas-induced T cell death.

NKT cells are positively selected in the thymus by interaction of the semi-invariant NKT cell TCR with glycolipids presented on CD1d on surrounding thymocytes. However, it is not known if CD1d also plays a role in transition of the cells to a mature NK1.1+ phenotype. McNab et al. (p. 3762 ) injected CD4+ NK1.1 T cells into thymi of wild-type or CD1d−/− mice. Seven days after transfer, more than 70% of donor NKT cells had become NK1.1+ in wild-type recipients compared with less than 25% in CD1d−/− recipients. More than 70% of recent thymic emigrant NKT cells from FITC-injected wild-type thymus grafts became NK1.1+ six weeks after engraftment in congenic wild-type mice; up-regulation of NK1.1 did not occur in CD1d−/− mice. There was no difference in proportions of donor T and NKT cells, activation status, or proliferation of donor NKT cells in spleen, liver, and blood of wild-type or CD1d−/− recipients six weeks after transfer of CFSE-labeled wild-type thymocytes. The data suggest that ongoing contact of NKT cells with CD1d is required for their maturation in the thymus or periphery but not for maintenance of an activated phenotype.

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