Breaking and repairing DNA
Activation-induced cytidine deaminase (AID) is involved in class switch recombination. However, it is not known whether AID acts alone or in concert with other proteins to induce DNA double-strand breaks. Wu et al. (p. 934 ) transfected human cells, including B cells, with a vector expressing a tagged AID protein. Proteins that bound AID were isolated by anti-tag mAb immunoprecipitation. The largest coprecipitated protein was DNA-protein kinase cs (DNA-PKcs). AID/DNA-PKcs coprecipitates were obtained from nuclear fractions only, although AID was found predominantly in the cytoplasm. Using a series of AID deletion mutants, the authors determined that both the AID C terminus and the deamination activity were required for binding with DNA-PKcs. DNA was a cofactor in the binding reaction. Lower survival rates and greater accumulation of a specific histone that bound to DNA double-strand breaks were seen in cells transiently transfected with a C-terminal deletion mutant of AID that could not bind DNA-PKcs than in cells transfected with wild-type AID or empty vector. Similarly, LPS-activated mouse B cells transiently transfected with the same deletion mutant had lower survival rates than controls. Transfected wild-type AID induced a high level of double-strand breaks in cells defective for DNA-PKcs. Mutating the AID nuclear export signal or adding nuclear localization signals did not disrupt the cytoplasmic association of AID with β-tubulin. The authors conclude that the AID C terminus both induces DNA double-strand breaks and recruits DNA-PKcs to resolve them.
Anaphylatoxin receptors and allergic asthma
Recent data from several laboratories suggest that the anaphylatoxins (ATs), C3a and C5a, and their receptors play a role in the development of allergic asthma. However, neither the contribution of C3aR or C5aR nor its activity during the sensitization or effector phase has been determined. Baelder et al. (p. 783 ) found that mAb blocking of C5aR improved midexpiratory flow in a mouse model of airway hyperresponsiveness (AHR) induced by Aspergillus fumigatus, whereas pharmacological blocking of C3aR had no impact on AHR during allergen challenge. Animals sensitized with A. fumigatus had higher total leukocyte numbers and higher levels of IL-5 and IL-6 in bronchoalveolar lavage after allergen challenge compared with nonsensitized controls. Blocking both AT receptors decreased IL-4 levels and the total numbers of eosinophils, lymphocytes, and neutrophils after allergen challenge; C5aR blockade alone reduced the numbers of eosinophils and lymphocytes and was more effective than C3aR blockade in reducing IL-6 concentration. Allergen-treated mice had elevated serum IgE levels that were not altered by AT blockade. The data suggest that C5aR plays a significant role in AHR during the effector phase and that C3a and C5a have different roles in recruiting infiltrating cells after challenge in a sensitized host.
Mimotope vaccination against melanoma
Inducing Ab responses against tumor-specific Ags is one approach in developing a vaccine against malignant melanoma. Wagner et al. (p. 976 ) identified two mimotopes, i.e., conformational epitopes, of the human high m.w.-melanoma-associated Ag (HMW-MAA) by screening a random phage peptide library with a mAb specific for HMW-MAA. Synthetic peptides containing the phage displayed peptide plus an additional 5-aa phage coat protein linker inhibited binding of the mAb to HMW-MAA in ELISA. A rabbit polyclonal Ab raised against one of the peptides conjugated to tetanus toxoid reacted with HMW-MAA and with an anti-Id mAb to the anti-HMW-MAA mAb. Purified anti-peptide Abs inhibited proliferation by 62% and induced 26% lysis of an HMW-MAApositive melanoma cell line. The results indicate that a vaccine based on a melanoma protein mimotope induces a humoral immune response against the native protein and inhibits growth of the tumor cells in vitro.
A BAFFling discovery
The finding that mature B cells reside in the joint tissue of patients with rheumatoid arthritis (RA) raises the question as to what factors are involved in the survival of the cells. Ohata et al. (p. 864 ) detected by immunohistochemical staining the expression of B cell-activating factor of the TNF family (BAFF) in fibroblast-like synoviocytes (FLS) in joint tissue from RA patients but not in joint tissue from patients with osteoarthritis (OA) or from normal controls. Both BAFF mRNA and protein were expressed in serially passaged FLS from RA joints; FLS from OA joints expressed lower levels of BAFF mRNA, whereas FLS from normal joints had no expression. FLS from healthy donors treated in vitro with IFN-γ or TNF-α exhibited increased levels of BAFF mRNA and protein. Pretreatment of FLS with very small concentrations of IFN-γ resulted in higher expression levels of BAFF mRNA after TNF-α treatment compared with TNF-α treatment alone. Isolated mature B cells from healthy donors had greater viability when cultured with healthy FLS compared with B cells cultured alone, but cell viability was highest when the B cells were cultured with FLS pretreated with IFN-γ or TNF-α. Addition of a soluble antagonist of BAFF interaction with its receptor significantly reduced survival of B cells cocultured with FLS pretreated with either cytokine. The study shows that synovial cells of mesenchymal origin exposed to TNF-α or IFN-γ express BAFF and promote the survival of B cells.
Genes controlling autoimmunity
Treatment of chronic inflammatory diseases relies on identifying the involved genes and understanding their interactions. Jagodic et al. (p. 918 ) studied experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) in tenth generation (F10) mice derived from EAE-susceptible DA rats carrying a chromosome 4 EAE-resistant region obtained by backcrossing to MHC-identical PVG rats. Induced EAE incidence in the F10 mice was 29%. High-resolution mapping of quantitative trait loci (QTL) on chromosome 4 identified three loci involved in MOG-induced EAE. Eae20 correlated with weight loss, maximum EAE score, and incidence and onset of disease at one specific chromosome 4 locus; Eae21 at a locus 3.1 Mb away correlated with weight loss, maximum EAE score, cumulative EAE score, and duration of EAE. Linkage analyses revealed epistatic interactions between Eae20 and a third QTL, Eae22, 10 Mb telomeric to Eae20. Highest cumulative EAE scores occurred in rats that were PVG homozygous at Eae20 and DA homozygous at Eae22. Paradoxically, DA rats congenic for a 1.44-Mb PVG region within the Eae20 QTL were more resistant than DA rats to MOG-induced EAE and mortality. The study uses F10 mice to define three loci within a 16.8-Mb region of rat chromosome 4 that regulate incidence and severity of MOG-induced EAE and offers a method for analysis of other multigenic chronic autoimmune diseases.
p53 increases CTL antitumor killing
Chouaib and coworkers have demonstrated that introduction of wild-type tumor suppressor p53 (wtp53) into tumor cells induces FasR expression and enhances CTL-mediated killing. Yet, the molecular basis of the wtp53 effect is unknown. In work from the same laboratory, Thiery et al. (p. 871 ) transfected Fas cDNA into a human nonsmall cell lung carcinoma (NSCLC) cell line lacking FasR and carrying a mutated p53. The cells became more susceptible to killing by autologous CTL or anti-Fas mAb only after cotransfection with wtp53. No changes in expression of apoptosis-associated genes, other than decreased expression of one inhibitor of apoptosis, were detected in wtp53-infected cells by immunoblot analysis; p21 expression was induced. Cleavages of caspase 8 and caspase 3 were increased in wtp53-transfected cells after incubation with autologous CTLs or anti-Fas mAb, and both cleavages were blocked by an anti-Fas mAb. Mitochondrial transmembrane potential was shown by cytofluorometric analysis to be disrupted in the cells containing wtp53 after their exposure to the CTLs or anti-Fas mAb, and a protein linking caspase 8 to the mitochondrial pathway was cleaved and translocated from the cytosol to mitochondria. Cytochrome c was released from mitochondria to the cytoplasm and nuclear fragmentation occurred. None of these changes were detected in tumor cells that were not transfected with wtp53. The data suggest that wtp53 sensitizes tumor cells to CTL-mediated lysis at the mitochondrial level.
Understanding CD40/CD154 interactions
Ligation of CD40 on monocytes and macrophages is a precipitating event in inflammatory diseases. Yet the details of the signaling pathways activated and the molecules involved are only partially understood. Mukundan et al. (p. 1081 ) found that a specific Src kinase inhibitor eliminated production of TNF-α, IL-1β, and IL-6, and prevented Src and ERK1/2 phosphorylation in primary human monocytes stimulated with soluble recombinant CD154. The involvement of TNFR-associated factors (TRAFs) in CD40-mediated phosphorylations was established by transfecting a CD40-deficient murine macrophage cell line with wild-type CD40 or with CD40 mutants lacking TRAF binding sites. CD40-deficient cells transfected with wild-type CD40 produced IL-6 and TNF-α after CD40 ligation, whereas cells transfected with CD40 lacking a TRAF6 binding site did not; cells transfected with CD40 lacking a TRAF2/3/5 binding site had reduced production. Cytokine production also was inhibited in stimulated primary human monocytes preincubated with a TRAF6 binding peptide and in stimulated mouse macrophages expressing a TRAF6 mutant able to bind CD40 but lacking catalytic activity. Luciferase assays showed that TRAF6-CD40 interactions were required for NF-κB activation and IκBα phosphorylation. The data indicate that CD40 interacts with TRAF6 to activate signaling pathways and to produce inflammatory cytokines in CD154-activated monocytes and macrophages.
Summaries written by Dorothy L. Buchhagen, Ph.D.