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Interactions in the CD19 signaling complex
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2 (PLC2), Grb2, the p85 subunit of PI3K, and Lyn kinase in bacteria as GST fusion proteins. The expressed proteins were incubated with either a phosphorylated or an unphosphorylated CD19 cytoplasmic domain. Each of the SH2 domains of p85, PLC2, Grb2, and Vav, coupled to Sepharose beads, removed phosphorylated CD19 protein from reaction mixtures; p85 and Vav constructs also weakly interacted with unphosphorylated CD19. The SH2 domain of Lyn bound strongest to phosphorylated CD19, whereas the Lyn kinase domain bound phosphorylated and unphosphorylated CD19 equally well. By surface plasmon resonance, both Lyn domains interacted strongly with a CD19 peptide containing Y513 but the Lyn kinase domain also interacted strongly with a CD19 peptide containing Y482. The p85 and Lyn SH2 interactions with CD19 were of high affinity. PLC2 or Vav constructs competed for binding to a CD19 peptide containing Y391 but did not interfere with binding of Grb2 to a CD19 peptide containing Y330. The authors conclude that the interactions between CD19 and the five downstream effectors are direct, are strongest with phosphorylated Y residues but also occur with unphosphorylated Y residues, and lead to formation of a multicomponent complex. Regulating TTP expression
The zinc finger protein tristetraprolin (TTP) is up-regulated rapidly in macrophages in response to LPS stimulation. TTP binds to an AU-rich element (ARE) in the 3'-untranslated region (UTR) of TNF-
mRNA to increase the rate of message turnover in unstimulated cells. Yet factors regulating TTP expression and function were unknown. Brooks et al. (p. 7263
) used specific inhibitors of signal transduction pathways in a LPS-stimulated human myelomonocytic cell line to show strong involvement of the p38 and JNK (JNK) pathways and moderate involvement of the ERK (extracellular-regulated kinase) pathway on TTP protein expression. LPS also increased both TTP and TNF- mRNA stability over that seen in unstimulated cells, but only TTP message stability required ERK signal transduction. The p38 pathway was required for stability of TNF- mRNA, but not TTP mRNA. The JNK pathway had no effect on stability of either message but was required for LPS induction of TTP message. TTP was shown to interact directly with both its own and TNF- mRNA at three AREs located within the 3'-UTR. The results indicate that the LPS-mediated increases in TTP protein level and mRNA stability require ERK activity and that TTP binds AREs within its own 3'-UTR, leading to autoregulation.
Adenosine and asthma
The detection of increased levels of adenosine in bronchoalveolar lavage fluid from asthmatics suggests that this molecule may be involved in pulmonary inflammation. In addition, mast cells have been implicated in isotype switching of B cells to produce IgE. The relationship among these observations was explored by Ryzhov et al. (p. 7726 ). They detected increased IL-4, IL-13, and other cytokine mRNA and protein in a human mast cell line, HMC-1, following incubation of the cells with the adenosine analog NECA (5'-N-ethylcarboxamidoadenosine) and adenosine deaminase. Incubation of the cells with adenosine and an inhibitor of adenosine deaminase also increased IL-13 secretion. A selective A2B adenosine receptor antagonist blocked NECA induction of IL-4 and IL-13. Agonists for other adenosine receptors had no effect. B cells cocultured for 12 days with NECA-stimulated HMC-1, that constitutively express CD40L, produced IgE as did B cells stimulated with anti-CD40 and IL-4. However, B cells cocultured with nonstimulated HMC-1, or cultured alone with or without NECA, did not produce IgE. The authors propose that high local concentrations of adenosine in human lung activate an inflammatory loop whereby mast cells secrete IL-4 and IL-13 that act on B cells to induce synthesis of IgE that, in turn, promotes mast cell degranulation and further IL-4 and IL-13 production.
Rejecting heart allografts
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A MHC class I cargo receptor
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2-microglobulin (2m) heterodimers ended when HC was glycosylated in the Golgi. Only the HC/2m, but not the free HC, bound to Bap31 in sequential IP; Bap31 also was found associated with tapasin, the transporter associated with Ag processing (TAP2), and calnexin, but not with calreticulin. However, the peptide loading complex formed properly in cells transfected with Bap29/31 small interfering RNA (Bap29 is closely homologous to Bap31). Cells, each lacking one component of the peptide loading complex, were used for sequential IP. In these experiments, Bap31 remained associated with tapasin in the absence of 2m and remained associated with HC in the absence of TAP2 or calreticulin. However, the binding of TAP2 to Bap31 did not occur in the absence of tapasin. Cells deficient in Bap29/31 had a delay in maturation of HC from the ER to the Golgi and a reduction in the number of HC/2m dimers at ER exit sites. The results suggest that Bap31, and perhaps Bap29, is a cargo receptor that interacts directly with MHC class I, and possibly tapasin, in the peptide loading complex. Generating CD8+ T central memory cells in vitro
Naive CD8+ T cells differentiate into effector CTL after Ag encounter. Although induction and persistence of central memory cells are critical to effective vaccines, the conditions under which they develop are poorly defined. Carrio et al. (p. 7315 ) exposed naive OVA-specific MHC class I-restricted CD8+ T cells from OT-I TCR transgenic mice to an activating OVA peptide in vitro. Ag exposure for 3 days in the presence of IL-2 generated effector CTL. Cells cultured without Ag and IL-2 but with either IL-7 or IL-15 for an additional 2 days developed a central memory cell phenotype; cells cultured without Ag but with IL-2 for the additional 2 days retained the CTL phenotype. Extending the initial exposure to Ag and IL-2 to 5–8 days led to a loss of the IL-7 and IL-15 effect. Cells primed with Ag in the presence of IL-7 or IL-15 developed into CTL only; removal of Ag at day 3 and continued growth in IL-7 or IL-15 were required for central memory cell development. OT-I T cells primed in vitro for 3 days were detected up to 4 wk after adoptive transfer into syngeneic mice and had the phenotype of central memory cells. Longer in vitro priming resulted in fewer memory cells in vivo. Most in vivo phenotypic changes occurred within the time periods seen during in vitro culturing with IL-7 or IL-15. The authors conclude that most CD8+ T cells can develop into central memory cells by incubation with Ag and IL-2 for up to 3 days in vitro followed by exposure to IL-7 or IL-15 in vitro or by adoptive transfer into normal mice after removal of Ag and IL-2.
Stages in rheumatoid arthritis induction
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RIII, their order of participation in the disease process is unknown. Wipke et al. (p. 7694
) used rodent-scale positron emission tomography (microPET) and classical biodistribution studies in the K/BxN model of arthritis to measure localization of -labeled anti-glucose-6-phosphate isomerase Ab in limb joints. C5-deficient mice and wild-type controls, but not FcR-deficient mice, mast cell-deficient mice, or neutrophil-depleted mice, accumulated glucose-6-phosphate isomerase Ab in their joints after transfer of serum from mice injected with a glucose-6-phosphate isomerase peptide. No joint localization was seen in BALB/c mice injected with anti-type II collagen mAb or a control Ab unless irrelevant immune complexes were coinjected. The time span for joint accumulation of injected Ab was rapid and comparable to that seen in mice injected with anti-glucose-6-phosphate isomerase Ab. Mice that received anti-type II collagen mAb with LPS or only anti-glucose-6-phosphate isomerase Ab developed joint inflammation. The authors propose a four-stage model of RA induction in which the first three stages are reversible. Autoantibodies gain entry (stage 1) into joints with the assistance of FcR, mast cells, neutrophils, and immune complexes, and bind (stage 2) to target autoantigens. A strong inflammatory response (stage 3) occurs within 24–48 h via activation of polymorphonuclear leukocytes, NK cells, mast cells, and macrophages. Continued influx of autoantibodies leads to chronic RA (stage 4) with joint pathology. Summaries written by Dorothy L. Buchhagen, Ph.D.
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