The Journal of Immunology, 2007, 178: 4003-4004.
Copyright © 2007 by The American Association of Immunologists, Inc.
IN THIS ISSUE
Controlling Actin Polymerization
Although B cell lymphoma/leukemia-10 (Bcl10) protein, important in the adaptive immune response, is recruited to the TCR/CD3 complex after TCR triggering and is phosphorylated, the functional importance of the phosphorylation is not known. Rueda et al. (p. 4373
) determined by serine to alanine point mutations within the C terminus of Bcl10 that serine 138 was required for formation of Bcl10 phosphoprotein isoforms and for IL-2 production in Bcl10-transduced human T cells treated with PMA/ionomycin or superantigen, respectively. In contrast, serine 138 phosphorylation was not necessary for expression of a NF-
B luciferase reporter construct in the activated T cells. Levels of F-actin increased in primary Bcl10+/+, but not Bcl10–/–, T cells stimulated with anti-CD3 Ab in vitro. Human T cells in which Bcl10 protein expression was silenced by a transfected short hairpin RNA did not have increased F-actin levels, had almost no spreading on anti-CD3 Ab-coated coverslips, and had impaired conjugate formation with superantigen-loaded human B cells. Fc
R-triggered Bcl10-silenced human monocytes had reduced phagocytosis of IgG-SRBCs compared with control cells in which Bcl10 expression was not silenced. F-actin cups were shown by three-dimensional reconstructions of wide-field microscopic images to be smaller in Bcl10-silenced monocytes. Actin formation and spreading were restored in Bcl10-silenced cells by transfection with wild-type Bcl10 but not with the serine 138/alanine mutant. The results detail a mechanistic link between phosphorylation at serine 138 of Bcl10 and regulation of actin polymerization during lymphocyte and monocyte activation that is independent of the NF-B pathway.
Annexin-1 Message: Eat Me
Phagocytosis of apoptotic leukocytes is important in resolving inflammation. To identify the prophagocytic ("eat me") signal required for cell clearance, Scannell et al. (p. 4595
) found that human macrophages and leukemic monocytes incubated with a supernatant of apoptotic, but not necrotic, human polymorphonuclear neutrophils (PMNs), mesangial cells, or T cells had increased phagocytosis of apoptotic PMNs compared with controls incubated with supernatants from viable cells. The phagocytic effect could be diluted with fresh medium, induced localized polymerization of actin filaments and release of TGF-
1, and was sensitive to proteinase K, to a caspase inhibitor, to an antagonist of the binding of lipoxin (an anti-inflammatory eicosanoid) to its receptor, or to an increase in intracellular cAMP. Annexin-1 was identified as a potential prophagocytic factor by immunodepletion and nanoelectrospray liquid chromatography mass spectrometry analysis of 73 proteins present in the supernatant of apoptotic PMNs, human mesangial cells, and human T cells. Both full-length annexin-1 and a small serine protease cleavage product stimulated phagocytosis and were inhibited by an anti-annexin-1 Ab. The authors suggest that annexin-1 released by apoptotic cells and its processed fragment are responsible for the prophagocytic activity that results in macrophage clearance of apoptotic cells from inflammatory sites.
SLP-ed Neutrophils
The Koretzky laboratory has shown that the adaptor molecule, Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), is required for integrin-dependent ROI production and other responses by neutrophils defending the host against invading microorganisms. But an in vivo determination of SLP-76 function is hampered by severe lethality and abnormalities of Slp-76–/– mice. To determine SLP-76 involvement in neutrophil function in vivo, Clemens et al. (p. 4606
) in the Koretzky group developed a mouse model in which the Slp-76 gene is deleted only in cells of myeloid lineage. Neutrophils lacking SLP-76 did not adhere to or spread on a polymer of an amino acid trimer that stimulates integrins and reduces phosphorylation of several key signaling mediators; the cells also had less ROI production or granulation when plated onto integrin ligand-coated dishes in the presence of TNF-
. Signaling events from FcR activation were partially intact in the Slp-76–/– neutrophils. Although neutrophil migration into air pouches infected with Staphylococcus aureus and bacterial killing were normal in mutant and wild-type animals, mice with Slp-76–/– neutrophils had a milder localized Shwartzman reaction with less redness, swelling, and vascular injury in LPS-injected ears. Similar reductions in hemorrhage were not seen in either of two strains of mast cell-deficient mice after LPS injection. The data from this novel mouse strain demonstrate that myeloid lineage-specific loss of SLP-76 expression lessens tissue inflammation by decreasing neutrophil release of regions of interest and other inflammatory mediators but has no impact on neutrophil migration or ability to clear infection with S. aureus.
Encounter/Docking of Anti-HIV-1 mAbs
Two human mAbs that neutralize HIV-1 by reacting with viral envelope glycoprotein 41 have been shown by Haynes and collaborators to bind the anionic phospholipid, cardiolipin, and other human autoantigens. To compare binding kinetics of the two mAbs with their specific epitopes vs cardiolipin, Alam et al. (p. 4424
) in the Hayneslaboratory used surface plasmon resonance assays to measure affinity and specificity of the mAbs or their Fabs for surface-bound molecules. Both mAbs and their Fabs specifically bound cardiolipin; the Fabs bound more weakly to their own epitopes and to cardiolipin than the intact mAbs. One of two anti-cardiolipin autoantigens from patients with Anti-Phospholipid Syndrome bound as strongly to cardiolipin as one anti-HIV-1 mAb; the second anti-cardiolipin autoantibody and the other anti-HIV-1 mAb bound weakly. In contrast to the anti-HIV-1 mAb binding to cardiolipin, the anti-cardiolipin mAbs did not cross-react with any HIV-1 envelope proteins with one exception. Sequence analyses of the four mAbs revealed common CDR3 features, including a highly hydrophobic domain, several key arginine residues, and an IgG3 isotype. Binding of the anti-HIV-1 mAbs or Fabs to their respective peptide-liposome conjugates was biphasic with the first step (encounter) rate limiting and the second step (docking) less stable at higher temperatures. The authors propose a two-step model in which encounter of mAb with the target is followed by a docking step requiring a conformational change that is constrained by the presence of lipid.
Specifically Recognizing S. aureus
Recognition of invading pathogens triggers a variety of inflammatory responses that must be modulated to prevent damage to the host. However, the mechanisms by which different pathogens elicit specific responses are unclear. Nakayama et al. (p. 4250
) identified receptors that specifically recognize Staphylococcus aureus by transfecting NIH3T3 cells that do not bind the bacteria with a cDNA expression library from bone marrow-derived C57BL/6 macrophages that do. Paired Ig-like receptor (PIR)-B was identified by sequence analysis of the inserts from three transfected clones that bound S. aureus; anti-PIR mAb prevented the binding. Cells transfected with PIR-A1 but not other PIR-As, all of which have greater than 92% aa identity with PIR-B, also bound the bacteria; polyanionic reagents blocked the binding. Mutation of charged amino acids at three positions in the conserved Ig-like domain D2 reduced binding. Interaction of S. aureus with Pirb–/– macrophages resulted in enhanced IL-6 and reduced IL-10 production compared with wild-type controls. Exogenous IL-10 suppressed IL-6 production from Pirb–/– macrophages exposed to S. aureus, but anti-IL-10 Ab enhanced IL-6 production from bacteria-stimulated wild-type macrophages. PIR-B recognized only Helicobacter pylori and Escherichia coli in addition to S. aureus. Two human orthologs of PIRs bound S. aureus and one of them also bound E. coli. The data demonstrate specific recognition of S. aureus by PIR-B and PIR-A1 on mouse macrophages and by two related human Ig-like transcript receptors.
Zebrafish Fight Virus Infections
Kim and colleagues described the first fish type I IFN and demonstrated its antiviral effects. However, questions about its activation were raised because the zebrafish gene structure is so distinct from that of mammalian and avian type I IFNs. In a continuation of their studies, Sullivan et al. (p. 4517
) in the Kim laboratory cloned a full-length Toll/IL-1R domain-containing adaptor molecule 1 (TICAM1) from zebrafish genomic DNA. By sequence analyses, the zebrafish adaptor had 26 and 46% aa identity with human and mouse TICAM1s, respectively (zebrafish lack a TICAM2 ortholog). Comparative genomic analyses with TICAMs from other species indicate that two TICAM gene duplications ultimately produced TICAM1 and TICAM2, with the TICAM2 gene being lost after a divergence 
450 million years ago. Coimmunoprecipitation experiments of cloned molecules overexpressed in human and zebrafish cells indicated that zebrafish TICAM1 associated with three of four binding partners, including TLR3, comparable to those found in mammalian cells. Overexpressed full-length zebrafish TICAM1 activated a reporter construct with a NF-B promoter or a zebrafish IFN promoter in human and zebrafish cells; activation was greater with a N-terminal deletion mutant. NF-B and IFN activation diminished with constructs mutated in a specific C-terminal domain, but only NF-B activation was reduced by deletion of the Toll/IL-1R domain or the C terminus. The authors conclude that zebrafish type I IFN expression is induced by TICAM1 during TLR signaling and that the TICAM1 N terminus might inhibit NF-B and IFN activation.
PlGF and Uterine NK Cells
Low concentrations of blood and urinary placenta growth factor (PlGF) in humans are associated with onset of hypertensive pre-eclampsia. Maternal uterine NK cells expressing PlGF promote early vascular changes in the pregnant endometrium, but mice lacking PlGF reproduce normally. Tayade et al. (p. 4267
) found increased endometrial expression of PlGF mRNA at gestational days 10–16 in wild-type pregnant mice compared with virgin controls. Gestational day 10 implantation sites from wild-type mice had strong immunohistochemical staining for PlGF mRNA in stroma around spiral arteries, in smooth muscle cells, and in uterine NK cells that was lacking in alymphoid controls; laser capture-microdissected uterine NK cells from pregnant wild-type uteri showed a lack of association of PlGF mRNA expression with uterine NK cell maturation. Implantation sites from PlGF–/– mice were grossly larger due to more endometrial (maternal) subregions and significant enlargement of the mesometrial lymphoid aggregate of pregnancy (MLAp) containing immature proliferative uterine NK cells surrounding the uterine artery; PlGF–/– placentae were smaller. MLAp uterine NK cells in the mutant mice were more numerous and smaller and had scant cytoplasm, fewer granules, and a high frequency of binucleation compared with those from wild-type mice. Ratios of spiral artery to lumen area were higher, and the smooth muscle coat around spiral arteries was thicker in PlGF–/– mice. The authors conclude that PlGF is important for uterine NK cell proliferation and/or differentiation within the MLAp and for timely spiral arterial modification.
Summaries written by Dorothy L. Buchhagen, Ph.D.
Related articles in The JI:
- Paired Ig-Like Receptors Bind to Bacteria and Shape TLR-Mediated Cytokine Production
- Masafumi Nakayama, David M. Underhill, Timothy W. Petersen, Bin Li, Toshio Kitamura, Toshiyuki Takai, and Alan Aderem
The JI 2007 178: 4250-4259.
[Abstract]
[Full Text]
- Genetic Deletion of Placenta Growth Factor in Mice Alters Uterine NK Cells
- Chandrakant Tayade, David Hilchie, Hong He, Yuan Fang, Lieve Moons, Peter Carmeliet, Robert A. Foster, and B. Anne Croy
The JI 2007 178: 4267-4275.
[Abstract]
[Full Text]
- Bcl10 Controls TCR- and FcR-Induced Actin Polymerization
- Daniel Rueda, Olivier Gaide, Liza Ho, Elodie Lewkowicz, Florence Niedergang, Stephan Hailfinger, Fabien Rebeaud, Montserrat Guzzardi, Béatrice Conne, Marcus Thelen, Jérôme Delon, Uta Ferch, Tak W. Mak, Jürgen Ruland, Jürg Schwaller, and Margot Thome
The JI 2007 178: 4373-4384.
[Abstract]
[Full Text]
- The Role of Antibody Polyspecificity and Lipid Reactivity in Binding of Broadly Neutralizing Anti-HIV-1 Envelope Human Monoclonal Antibodies 2F5 and 4E10 to Glycoprotein 41 Membrane Proximal Envelope Epitopes
- S. Munir Alam, Mildred McAdams, David Boren, Michael Rak, Richard M. Scearce, Feng Gao, Zenaido T. Camacho, Daniel Gewirth, Garnett Kelsoe, Pojen Chen, and Barton F. Haynes
The JI 2007 178: 4424-4435.
[Abstract]
[Full Text]
- Evidence for Evolving Toll-IL-1 Receptor-Containing Adaptor Molecule Function in Vertebrates
- Con Sullivan, John H. Postlethwait, Christopher R. Lage, Paul J. Millard, and Carol H. Kim
The JI 2007 178: 4517-4527.
[Abstract]
[Full Text]
- Annexin-1 and Peptide Derivatives Are Released by Apoptotic Cells and Stimulate Phagocytosis of Apoptotic Neutrophils by Macrophages
- Michael Scannell, Michelle B. Flanagan, Andreas deStefani, Kieran J. Wynne, Gerard Cagney, Catherine Godson, and Paola Maderna
The JI 2007 178: 4595-4605.
[Abstract]
[Full Text]
- Loss of SLP-76 Expression within Myeloid Cells Confers Resistance to Neutrophil-Mediated Tissue Damage while Maintaining Effective Bacterial Killing
- Regina A. Clemens, Laurie E. Lenox, Taku Kambayashi, Natalie Bezman, Jonathan S. Maltzman, Kim E. Nichols, and Gary A. Koretzky
The JI 2007 178: 4606-4614.
[Abstract]
[Full Text]
