Rcan1 negatively regulates FcεRI-mediated signaling and mast cell function

Aggregation of the high affinity IgE receptor (FcεRI) activates a cascade of signaling events leading to mast cell activation. Subsequently, inhibitory signals are engaged for turning off activating signals. We identified that regulator of calcineurin (Rcan) 1 serves as a negative regulator for turning off FcεRI-mediated mast cell activation. FcεRI-induced Rcan1 expression was identified by suppression subtractive hybridization and verified by real-time quantitative polymerase chain reaction and Western blotting. Deficiency of Rcan1 led to increased calcineurin activity, increased nuclear factor of activated T cells and nuclear factor κB activation, increased cytokine production, and enhanced immunoglobulin E–mediated late-phase cutaneous reactions. Forced expression of Rcan1 in wild-type or Rcan1-deficient mast cells reduced FcεRI-mediated cytokine production. Rcan1 deficiency also led to increased FcεRI-mediated mast cell degranulation and enhanced passive cutaneous anaphylaxis. Analysis of the Rcan1 promoter identified a functional Egr1 binding site. Biochemical and genetic evidence suggested that Egr1 controls Rcan1 expression. Our results identified Rcan1 as a novel inhibitory signal in FcεRI-induced mast cell activation and established a new link of Egr1 and Rcan1 in FcεRI signaling.     

The glycosyl phosphatidylinositol-linked FcγRIII(PMN) mediates transmembrane signaling events distinct from FcγRII

To investigate the ability of FcγRIII(PMN), the GPI-anchored isoform of FcγRIII (CD16) in polymorphonuclear leukocytes (PMN), to mediate transmembrane signaling events, we measured changes in membrane potential with DiOC(5) and in intracellular calcium with indo-1. FcγR were ligated by anti-FcγRIII mAb 3G8 (IgG and Fab), anti-FcγRII mAb IV.3 (IgG and Fab), and human IgG aggregates. Cell bound mAbs were also crosslinked by goat F(ab’)(2) anti-mouse IgG. 3G8 IgG elicited a rapid change in [Ca(2+)](i), which was unaffected by EGTA, Vibrio cholerae toxin (CT), or Bordetella pertussis toxin (PT), and was abolished by BAPTA . Univalent receptor binding with 3G8 Fab gave no response but crosslinking with F(aV)2 GAM gave a rapid [Ca2,](i) response. Neither IV.3 Fab, IV.3 IgG, nor crosslinking of IV.3 Fab elicited a calcium signal. PI-PLC-treated PMN with the density of FcγRIII(PMN) reduced to that of FcγRII showed an unattenuated change in [Ca(2+)](i), with a 3G8 stimulus. The effects of IgG aggregates paralleled those of 3G8 mAb. These data indicate that multivalent ligation of FcγRIII(PMN) initiates an increase in [Ca(2+)];, derived from intracellular stores, that is distinct from both the FMLP- and FcγRII-induced responses. Ligand-dependent interaction with FcγRII is not required. Since FcγRIII(PMN) can internalize the FcγRIII-specific probe Con A-opsonized E and lyse anti-FcγRIII heteroantibody-opsonized chick E, this GPI-anchored molecule mediates both signal transduction and integrated cell responses.     

T Cell Development in Mice Lacking All T Cell Receptor ζ Family Members (ζ, η, and FcεRIγ)

The ζ family includes ζ, η, and FcεRIγ (Fcγ). Dimers of the ζ family proteins function as signal transducing subunits of the T cell antigen receptor (TCR), the pre-TCR, and a subset of Fc receptors. In mice lacking ζ/η chains, T cell development is impaired, yet low numbers of CD4⁺ and CD8⁺ T cells develop. This finding suggests either that pre-TCR and TCR complexes lacking a ζ family dimer can promote T cell maturation, or that in the absence of ζ/η, Fcγ serves as a subunit in TCR complexes. To elucidate the role of ζ family dimers in T cell development, we generated mice lacking expression of all of these proteins and compared their phenotype to mice lacking only ζ/η or Fcγ. The data reveal that surface complexes that are expressed in the absence of ζ family dimers are capable of transducing signals required for α/β–T cell development. Strikingly, T cells generated in both ζ/η^−/− and ζ/η^−/−–Fcγ^−/− mice exhibit a memory phenotype and elaborate interferon γ. Finally, examination of different T cell populations reveals that ζ/η and Fcγ have distinct expression patterns that correlate with their thymus dependency. A possible function for the differential expression of ζ family proteins may be to impart distinctive signaling properties to TCR complexes expressed on specific T cell populations.     

Reversal of Proinflammatory Responses by Ligating the Macrophage Fcγ Receptor Type I

Macrophages can respond to a variety of infectious and/or inflammatory stimuli by secreting an array of proinflammatory cytokines, the overproduction of which can result in shock or even death. In this report, we demonstrate that ligation of macrophage Fcγ receptors (FcγR) can lead to a reversal of macrophage proinflammatory responses by inducing an upregulation of interleukin (IL)-10, with a reciprocal inhibition of IL-12 production. IL-10 upregulation was specific to FcγR ligation, since the ligation of the Mac-1 receptor did not alter IL-10 production. The identification of the specific FcγR subtype responsible for IL-10 upregulation was determined in gene knockout mice. Macrophages from mice lacking the FcR γ chain, which is required for assembly and signaling by FcγRI and FcγRIII, failed to upregulate IL-10 in response to immune complexes. However, mice lacking either the FcγRII or the FcγRIII were fully capable of upregulating IL-10 production, implicating FcγRI in this process. The biological consequences of FcγRI ligation were determined in both in vitro and in vivo models of inflammation and sepsis. In all of the models tested, the ligation of FcγR promoted the production of IL-10 and inhibited the secretion of IL-12. This reciprocal alteration in the pattern of macrophage cytokine production illustrates a potentially important role for FcγR-mediated clearance in suppressing macrophage proinflammatory responses.     

FcγRIIB regulates autoantibody responses by limiting marginal zone B cell activation

FcγRIIB is an inhibitory receptor expressed throughout B cell development. Diminished expression or function is associated with lupus in mice and humans, in particular through an effect on autoantibody production and plasma cell (PC) differentiation. Here, we analyzed the effect of B cell–intrinsic FcγRIIB expression on B cell activation and PC differentiation. Loss of FcγRIIB on B cells in Fcgr2b–conditional KO (Fcgr2b-cKO) mice led to a spontaneous increase in autoantibody titers. This increase was most striking for IgG3, suggestive of increased extrafollicular responses. Marginal zone (MZ) B cells had the highest expression of FcγRIIB in both mice and humans. This high expression of FcγRIIB was linked to increased MZ B cell activation, Erk phosphorylation, and calcium flux in the absence of FcγRIIB triggering. We observed a marked increase in IgG3⁺ PCs and B cells during extrafollicular PC responses in Fcgr2b-cKO mice. The increased IgG3 response following immunization of Fcgr2b-cKO mice was lost in MZ-deficient Notch2 Fcgr2b–double KO mice. Importantly, patients with systemic lupus erythematosus (SLE) had a decrease in FcγRIIB expression that was strongest in MZ B cells. Thus, we present a model in which high FcγRIIB expression in MZ B cells prevented their hyperactivation and ensuing autoimmunity.     

Lipid Raft-Dependent FcεRI Ubiquitination Regulates Receptor Endocytosis through the Action of Ubiquitin Binding Adaptors

The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcεRI) expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcεRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcεRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.     

Neonatal Immune Neutropenia due to Isoantibodies against the Granulocyte Receptor FcγRIIIb

IntroductionNeonatal neutropenia is often secondary to sepsis, low birth weight, pregnancy-induced maternal hypertension, and other conditions.Case ReportWe report a case of asymptomatic isoimmune neutropenia in a pair of preterm twins. Genotyping confirmed that the mother was negative for HNA-1a, 1b, and 1c, consistent with an FcγRIIIb deficiency. The father was 1(a+b+c−) and the neonates were 1(a–b+c−). A strongly positive result was observed in the granulocyte immunofluorescence test against paternal neutrophils (IgG antibodies). IgG anti-CD16b isoantibodies were detected in the mother''s breast milk. Neutropenia resolved after 28 days without requiring any specific treatments.DiscussionEven though neonatal alloimmune neutropenia (NAN) is usually benign and self-limiting, some patients pre­sent with delayed separation of the umbilical cord, mild skin infections, omphalitis, or severe infections like pneumonia, sepsis, and meningitis. Thus, it is important to rule out NAN in case of neonatal neutropenia.     

The CD3-γδε and CD3-ζ/η Modules Are Each Essential for Allelic Exclusion at the T Cell Receptor β Locus but Are Both Dispensable for the Initiation of V to (D)J Recombination at the T Cell Receptor–β, –γ, and –δ Loci

The pre–T cell receptor (TCR) associates with CD3-transducing subunits and triggers the selective expansion and maturation of T cell precursors expressing a TCR-β chain. Recent experiments in pre-Tα chain-deficient mice have suggested that the pre-TCR may not be required for signaling allelic exclusion at the TCR-β locus. Using CD3-ε– and CD3-ζ/η–deficient mice harboring a productively rearranged TCR-β transgene, we showed that the CD3-γδε and CD3-ζ/η modules, and by inference the pre-TCR/CD3 complex, are each essential for the establishment of allelic exclusion at the endogenous TCR-β locus. Furthermore, using mutant mice lacking both the CD3-ε and CD3-ζ/η genes, we established that the CD3 gene products are dispensable for the onset of V to (D)J recombination (V, variable; D, diversity; J, joining) at the TCR-β, TCR-γ, and TCR-δ loci. Thus, the CD3 components are differentially involved in the sequential events that make the TCR-β locus first accessible to, and later insulated from, the action of the V(D)J recombinase.     

Rho is Required for the Initiation of Calcium Signaling and Phagocytosis by Fcγ Receptors in Macrophages

Phagocytosis of bacteria by macrophages and neutrophils is an essential component of host defense against infection. The mechanism whereby the interaction of opsonized particles with Fcγ receptors triggers the engulfment of opsonized particles remains incompletely understood, although activation of tyrosine kinases has been recognized as an early step. Recent studies in other systems have demonstrated that tyrosine kinases can in turn signal the activation of small GTPases of the ras superfamily. We therefore investigated the possible role of Rho in Fc receptor–mediated phagocytosis. To this end we microinjected J774 macrophages with C3 exotoxin from Clostridium botulinum, which ADP-ribosylates and inactivates Rho. C3 exotoxin induced the retraction of filopodia, the disappearance of focal complexes, and a global decrease in the F-actin content of J774 cells. In addition, these cells exhibited increased spreading and the formation of vacuolar structures. Importantly, inactivation of Rho resulted in the complete abrogation of phagocytosis. Inhibition of Fcγ receptor–mediated phagocytosis by C3 exotoxin was confirmed in COS cells, which become phagocytic upon transfection of the FcγRIIA receptor. Rho was found to be essential for the accumulation of phosphotyrosine and of F-actin around phagocytic cups and for Fcγ receptor–mediated Ca²⁺ signaling. The clustering of receptors in response to opsonin, an essential step in Fcγ-induced signaling, was the earliest event shown to be inhibited by C3 exotoxin. The effect of the toxin was specific, since clustering and internalization of transferrin receptors were unaffected by microinjection of C3. These data identify a role for small GTPases in Fcγ receptor–mediated phagocytosis by leukocytes.     

A Critical Role for Syk in Signal Transduction and Phagocytosis Mediated by Fcγ Receptors on Macrophages

Receptors on macrophages for the Fc region of IgG (FcγR) mediate a number of responses important for host immunity. Signaling events necessary for these responses are likely initiated by the activation of Src-family and Syk-family tyrosine kinases after FcγR cross-linking. Macrophages derived from Syk-deficient (Syk⁻) mice were defective in phagocytosis of particles bound by FcγRs, as well as in many FcγR-induced signaling events, including tyrosine phosphorylation of a number of cellular substrates and activation of MAP kinases. In contrast, Syk⁻ macrophages exhibited normal responses to another potent macrophage stimulus, lipopolysaccharide. Phagocytosis of latex beads and Escherichia coli bacteria was also not affected. Syk⁻ macrophages exhibited formation of polymerized actin structures opposing particles bound to the cells by FcγRs (actin cups), but failed to proceed to internalization. Interestingly, inhibitors of phosphatidylinositol 3-kinase also blocked FcγR-mediated phagocytosis at this stage. Thus, PI 3-kinase may participate in a Syk-dependent signaling pathway critical for FcγR-mediated phagocytosis. Macrophages derived from mice deficient for the three members of the Src-family of kinases expressed in these cells, Hck, Fgr, and Lyn, exhibited poor Syk activation upon FcγR engagement, accompanied by a delay in FcγR-mediated phagocytosis. These observations demonstrate that Syk is critical for FcγR-mediated phagocytosis, as well as for signal transduction in macrophages. Additionally, our findings provide evidence to support a model of sequential tyrosine kinase activation by FcγR's analogous to models of signaling by the B and T cell antigen receptors.     

Biosynthesis of 5α-Cholestan-3β-ol in Cerebrotendinous Xanthomatosis

Cerebrotendinous Xanthomatosis is a rare, inherited disease characterized by an extraordinary accumulation of cholestanol in all tissues, xanthomatous deposits in the brain, lungs, and Achilles tendons, premature atherosclerosis, and low plasma cholesterol concentrations. In two patients with the disease, the biosynthesis of cholestanol was examined by different techniques. After cholesterol-4-(14)C was injected intravenously into one patient, cholestanol and cholesterol isolated from the bile on 3 different days over the ensuing week contained significant radioactivity. The specific radioactivity-time curves for cholesterol-(14)C and cholestanol-(14)C suggested a precursor product relationship and provided additional evidence for the transformation of cholesterol into cholestanol. The second patient received intravenously a mixture of mevalonate-2-(14)C and stereospecifically labeled mevalonate-3R,4R-(3)H. Again cholesterol and cholestanol were isolated from the bile, and the (3)H/(14)C ratio in both sterols was almost the same. This experiment again demonstrated that the biosynthetic path of cholestanol proceeded through cholesterol and not directly from earlier 5alpha-H-saturated precursors. These two independent lines of evidence indicate that the extraordinary deposition of cholestanol in Cerebrotendinous Xanthomatosis arises from cholesterol presumably through the accentuation of the normal biosynthetic pathway.     

Inhibition of β-Lactamases by β-Lactam Antibiotics

The inhibitory properties of a selected number of beta-lactam antibiotics were studied, with the use of three distinct types of beta-lactamases. The three enzymes were found to be distinguishable on the basis of their susceptibility to inhibition. Not one of the potential inhibitors tested was found to be a potent inhibitor of all three enzymes, but nafcillin possessed the broadest inhibitory activity. The enzyme isolated from Enterobacter cloacae was found to be the most susceptible. In some cases, the degree of inhibition varied with the time of incubation, and, depending upon the time chosen, widely different observations could be made. It is suggested that, in studies such as these, every consideration should be given to the period of incubation and to the concentration of inhibitor employed. Mixtures of inhibitor and cephaloridine did not always act synergistically against growing bacteria, and a number of reasons for failure are suggested.     

Apoptosis of Fashigh CD4+ Synovial T Cells by Borrelia-reactive Fas-ligandhigh γδ T Cells in Lyme Arthritis

The function of the minor subset of T lymphocytes bearing the γδ T cell antigen receptor is uncertain. Although some γδ T cells react to microbial products, responsiveness has only rarely been demonstrated toward a bacterial antigen from a naturally occurring human infection. Synovial fluid lymphocytes from patients with Lyme arthritis contain a large proportion of γδ cells that proliferate in response to the causative spirochete, Borrelia burgdorferi. Furthermore, synovial γδ T cell clones express elevated and sustained levels of the ligand for Fas (APO-1, CD95) compared to αβ T cells, and induce apoptosis of Fas^high CD4⁺ synovial lymphocytes. The findings suggest that γδ T cells contribute to defense in human infections, as well as manifest an immunoregulatory function at inflammatory sites by a Fas-dependent process.     

Hemoglobin Hasharon (α247 his(CD5)β2): a hemoglobin found in low concentration

Hemoglobin Hasharon (alpha(2) (47 his)(CD5)beta(2)) was found to comprise only 16-19% of hemolysates of carriers. These heterozygotes appeared to have mild, compensated, hemolytic anemia. Hb Hasharon was more heat-labile than hemoglobins A, S, or C. Its specific activity was higher than that of Hb A after administration of (59)Fe to two carriers. When hemoglobin synthesis by bone marrow cells was studied in vitro, about 18% of incorporated leucine appeared in the Hb Hasharon fraction. It is suggested that Hb Hasharon is unstable in vivo, and that mild hemolytic anemia and a relatively small decrease in its concentration in hemolysates result from its denaturation within red cells. Decreased synthesis, which appears to be the major cause of the small amount of abnormal hemoglobin, may protect heterozygotes from clinically significant hemolytic anemia.     

Effective Erythropoiesis Induced by 5β-Pregnane-3β-Hydroxy-20-One in Squirrel Monkeys

The erythropoietic effect of 5beta-pregnane-3beta-hydroxy-20-one, a naturally occurring steroid metabolite of progesterone, was evaluated in the squirrel monkey by ferrokinetic studies. red cell survival, and blood volume measurements. The intramuscular administration of this steroid in pharmacologic doses shortened the (59)Fe plasma clearance and increased the plasma iron turnover, thereby indicating an increase in erythropoiesis. A normal (59)Fe red cell uptake was observed, and the bone marrow maturation time was not altered. Red cell survival was the same in the treated and control groups. After five weekly injections of the steroid, the monkeys increased their red cell mass by 57%. A significant increase in white blood cells and a slight elevation of platelet counts in the treated monkeys also suggest a possible direct stimulation of hemopoietic stem cells by the steroid metabolite.These observations indicate that some steroid metabolites can stimulate an early increase in iron turnover (within 48 h) that is not secondary to hemolysis. The increased red cell mass indicates effective erythropoiesis in primates.