IKKε and rheumatoid arthritis
Although the NF-κB pathway is a promising target for treating rheumatoid arthritis (RA), there are concerns that normal immune responses and cell survival might be negatively impacted. Sweeney et al. (p. 6424 ) looked at an alternative target involved in NF-κB signaling, namely the IκB kinase-related kinase, IKKε. IKKε protein was detected in synovial lysates of human RA and osteoarthritis (OA) samples using a polyclonal anti-IKKε Ab, and the protein was localized to synovial intimal lining of RA and OA tissue by immunohistochemistry. IKKε, constitutively expressed in RA and OA fibroblast-like synoviocytes (FLS), had increased IκB phosphorylating activity in an in vitro kinase assay following stimulation of FLS with TNF-α or LPS. Additionally, c-Jun was phosphorylated by IKKε immunoprecipitated from the stimulated FLS, and the phosphorylation was blocked by an anti-IKKε Ab immunoprecipitate from LPS-stimulated FLS transfected with an adenoviral construct expressing a dominant-negative IKKε. A JNK inhibitor blocked IKKε-mediated c-Jun phosphorylation, and no anti-JNK2 mAb reactivity was detected on immunoblots. Induction of mRNA for metalloproteinases MMP3 and MMP13 was seen in wild-type mouse FLS, but not in IKKε−/− FLS, stimulated with TNF-α or LPS. The authors propose that IKKε, which appears to regulate MMP expression, might be an attractive target to reduce synovial inflammation, extracellular matrix destruction, and other aspects of RA pathology.
Response of intestinal mucosa to rotaviruses
Rotavirus, the dsRNA agent of severe gastroenteritis, infects epithelial cells lining the villi of the small intestine. Yet the specific molecules involved in, and responding to, virus-epithelial cell interactions are not known. Vijay-Kumar et al. (p. 6322 ) found that poly(I:C), a synthetic analog of dsRNA, induced IL-8 secretion from human intestinal epithelial cells in vitro more slowly than did flagellin. The two inducers stimulated different patterns of epithelial gene expression as measured by cDNA microarray analysis. Slow increases in expression of mRNA and protein for lipocalin-2/neutrophil gelatinase-associated lipocalin (NGAL) and matrix metalloproteinase-7 and a modest increase in STAT-1 phosphorylation also were induced by poly(I:C). Secretion of IL-8 and NGAL was greater in response to basolateral application of poly(I:C) compared with apical application. An inhibitor of endocytosis blocked poly(I:C)-induced, but not flagellin-induced, expression of IL-8 and NGAL. Loss of poly(I:C) from culture supernatants confirmed its cellular uptake; the inhibitor of endocytosis prevented its loss. The dsRNA-dependent protein kinase R (PKR) was up-regulated at 48 h in response to poly(I:C). Two inhibitors of PKR prevented IL-8 secretion and NGAL protein expression induced by the poly(I:C) or by a laboratory-adapted strain of rotavirus. The authors suggest that intestinal epithelial cells respond to endocytosed rotaviral dsRNA via intracellular PKR.
Regulating IFN-γ-induced transcription
Kalvakolanu and colleagues have shown that IFN-γ-induced gene expression involves a novel cis-acting enhancer element, IFN-γ-activated transcriptional element (GATE), that binds two transactivators, C/EBP-β and GATE binding factor 1 (GBF1). In contrast to C/EBP-β, GBF1 binds DNA poorly. In their report on p. 6203 , Meng et al. found that IFN-γ treatment of mouse embryo fibroblasts (MEFs), transfected with a GBF1 expression vector, stimulated transcription of a cotransfected IFN-responsive luciferase reporter plasmid that contained GATE. Less stimulation was seen with a mutant lacking the GBF1 C terminus, and no stimulation was seen with a mutant lacking the N terminus. IFN-γ-treated C/EBP-β−/− MEFs cotransfected with C/EBP-β and the reporter plasmid strongly induced luciferase activity; adding GBF1 in the transfection greatly increased the response. Minimal luciferase activity was induced by GBF1 alone. Physical association of C/EBP-β and GBF1 was demonstrated by their coprecipitation by anti-C/EBP-β Ab or anti-GBF1 Ab from IFN-γ-treated cotransfected MEFs and untransfected wild-type cells and from a mixture of the two in vitro-translated proteins. Chromatin immunoprecipitation assays demonstrated that both proteins interacted at the IRF9 promoter. C/EBP-β and GBF1 interactions did not occur in cells given an inhibitor of ERK1/2 activation, and a requirement for phosphorylation by ERK1/2 at a specific amino acid residue was determined using C/EBP-β mutants. The minimal C/EBP-β-interacting domain was mapped to aa 174-211 in GBF1 using deletion mutants. The authors show that C/EBP-β and GBF1 interact to activate GATE-driven transcription in response to IFN-γ treatment of cells and define the minimal requirements for the interaction.
Extracorporeal photophoresis (ECP), involving treatment of the patient or isolated T cells with the photosensitizer 8-methoxypsoralen (8-MOP) and UVA radiation, is effective against a variety of T cell-mediated pathologies. However, the mode of action of ECP has not been determined. Maeda et al. (p. 5968 ) developed an experimental in vivo model of ECP in which spleen and lymph node cells from 2,4-dinitrofluorobenzene (DNFB)-sensitized mice were incubated ex vivo with 8-MOP followed by exposure to UVA before injection into naive mice. Ear swelling in response to subsequent DNFB sensitization and challenge was reduced greatly in these recipients compared with controls that received cells treated with only 8-MOP or UVA, or untreated cells. Injection of 8-MOP/UVA-treated cells from naive donors did not reduce ear swelling after DNFB sensitization and challenge. Removal of CD11c+ cells from the 8-MOP/UVA-treated cell population abrogated the reduction of ear swelling in the recipients. Adoptive transfer of splenocytes and lymph node cells from DNFB-sensitized recipients of 8-MOP/UVA-treated cells suppressed a DNFB response, but not an oxazolone-mediated response, in naive recipients. Ear swelling was not suppressed in DNFB-challenged naive mice receiving 8-MOP/UVA-treated cells depleted of CD4+ or CD25+ T cells from DNFB-sensitized donors. Boosting of primary recipients with DNFB was required for transfer of the same level of suppression to secondary recipients. The experiments indicate that immunomodulation occurs only with cells treated with both 8-MOP and UVA, is Ag specific, requires CD11c+ cells, and is mediated by CD4+CD25+ T cells.
TGF-β1 regulation of IFN-γ
Mice lacking TGF-β1 develop rapidly fatal autoimmune diseases. Although IFN-γ-producing CD4+ T cells are directly involved, the mechanism by which TGF-β1 regulates IFN-γ production is unknown. Lin et al. (p. 5950 ) found that liver CD4+ T cells from TGF-β1−/− BALB/c mice expressed more IFN-γ mRNA than those from wild-type controls; mRNAs for transcription factors T-bet and STAT4 also were elevated in the TGF-β1-deficient cells. IFN-γ mRNA was elevated slightly in naive wild-type CD4+ T cells stimulated ex vivo in the absence of APC or cytokines, and addition of IL-12 dramatically increased the level at 48 and 72 h. IL-12 plus TGF-β1 inhibited IFN-γ mRNA expression during priming and IFN-γ production during recall. The results were confirmed by intracellular cytokine analyses and shown by CFSE staining to be independent of cell division. Expression of T-bet and STAT4 mRNA and protein in wild-type liver CD4+ T cells was suppressed by priming with IL-12 plus TGF-β1. In the presence of high levels of retrovirally expressed T-bet, IL-12 plus TGF-β1 suppressed IFN-γ expression during priming, but not during recall stimulation; inhibition occurred at both priming and recall in cells infected with the control vector. In contrast, in the presence of retrovirally expressed STAT4, IL-12 plus TGF-β1 treatment did not inhibit IFN-γ expression at priming, but did inhibit recall expression. TGF-β1 was able to inhibit T-bet expression during priming and IFN-γ expression at recall stimulation only during the first 45 h of priming. The authors suggest that TGF-β1 suppresses IFN-γ expression through two pathways involving inhibition of STAT4 during the priming phase and inhibition of T-bet during the effector phase.
Increasing CTL avidity
Agoal of vaccines against infectious diseases and cancer is to generate Ag-specific CTL with high avidity. To date, no systematic evaluation of strategies to accomplish that goal has been undertaken. Hodge et al. (p. 5994 ) compared the abilities of a foreign Ag, β-gal, or a “self” Ag, carcinoembryonic Ag (CEA), cloned in a vaccinia virus vector to induce CTL in mice. Quantitation of Ag-specific CTL was by tetramer binding; tetramer dissociation and quantitative lyticactivity measured avidity. Mice were vaccinated with a vector containing either β-gal or CEA and three costimulatory molecules, B7-1, cell adhesion molecule-1, and LFA-3 (TRICOM). Controls were injected with a β-gal or CEA peptide, with vector containing one peptide alone or with vector containing one peptide and B7-1. Splenic CTL from mice receiving either Ag/TRICOM vaccine exhibited a modest increase in tetramer binding cells but a large increase in tetramer dissociation time and lysis of target cells compared with controls. In all three assays, responses were greater against β-gal than CEA. Administration of anti-CTLA-4 mAb or GM-CSF in the initial injection with either Ag/TRICOM vaccine resulted in more tetramer-positive CTL and a dramatic increase in tetramer dissociation time and lysis. All three measurements increased dramatically when both anti-CTLA-4 mAb and GM-CSF were administered with either Ag/TRICOM. In vivo, 40% of CEA transgenic mice vaccinated with CTLA-4 mAb, GM-CSF, and CEA/TRICOM survived challenge with CEA-expressing mouse tumor cells through 22 wk; no evidence of autoimmunity was detected in survivors. Only 10% of mice receiving CEA/TRICOM with GM-CSF and no untreated mice or mice receiving only anti-CTLA-4 mAb survived past 6 wk. The authors demonstrate that the additive effect of multiple modalities is effective in increasing avidity of Ag-specific CTL.
Interallelic class switch recombination
Germline transcription precedes class switch recombination (CSR) of IgH chains in activated B cells. Although interchromosomal recombination has been documented for rabbit Cα genes, the frequency of interallelic CSR in mouse B cells is unknown. Reynaud et al. (p. 6176 ) established a homozygous mouse line lacking IgH gene expression by inserting an out-of-frame VκJκ exon into the IgH locus. The mice lacked mature B cells in spleen, bone marrow, and Peyer’s patches, and had no serum Ig. Heterozygous mice expressed both wild-type and larger mutant VDJ-Cμ transcripts, as determined by RT-PCR, but only wild-type IgM was detected on B cells. However, the C region from the mutant gene was expressed in 7% of 120 individual VDJ-Cα cDNA clones obtained from LPS-stimulated splenocytes from two heterozygous mice; ∼18% of VDJ-Cγ3 transcripts contained the C region of the mutant gene as determined by restriction enzyme digestion at single-nucleotide polymorphisms. DNA sequencing confirmed the allotype assignments. Heterozygous mice expressed a low level of mutant allotype serum IgA, and some mutant IgA-producing plasmacytes (∼6% of cells) were detected in their Peyer’s patches using confocal microscopy. The authors interpret their data to indicate that interallelic CSR occurs in murine B cells, resulting in the production of IgH chains expressing part of the mutated allele.
Summaries written by Dorothy L. Buchhagen, Ph.D.