Cross-linking of IgG receptors inhibits membrane immunoglobulin-stimulated calcium influx in B lymphocytes

By cross-linking membrane immunoglobulins (mIg), the antigenic stimulation of B lymphocytes induces an increase in intracellular free calcium levels ([Ca2+]i) because of a combination of release from intracellular stores and transmembrane influx. It has been suggested that both events are linked, as in a number of other cases of receptor-induced increase in [Ca2+]i. Conversely, in B lymphocytes, type II receptors for the Fc fragment of IgG (Fc gamma RII) inhibit mIg-mediated signaling. Thus, we have investigated at the level of single cells if these receptors could act on specific phases of mIg Ca2+ signaling. Lipopolysaccharide-activated murine B splenocytes and B lymphoma cells transfected with intact or truncated Fc gamma RII-cDNA were used to determine the domains of Fc gamma RII implicated in the inhibition of the Ca2+ signal. [Ca2+]i was measured in single fura-2-loaded cells by microfluorometry. The phases of release from intracellular stores and of transmembrane influx were discriminated by using manganese, which quenches fura-2, in the external medium as a tracer for bivalent cation entry. The role of membrane potential was studied by recording [Ca2+]i in cells voltage-clamped using the perforated patch-clamp method. Cross-linking of mIgM or mIgG with F(ab')2 fragments of anti-Ig antibodies induced a sustained rise in [Ca2+]i due to an extremely fast and transitory release of Ca2+ from intracellular stores and a long lasting transmembrane Ca2+ influx. The phase of influx, but not that of release, was inhibited by membrane depolarization. The increase in [Ca2+]i occurred after a delay inversely related to the dose of ligand. Co-cross-linking mIgs and Fc gamma RII with intact anti-Ig antibodies only triggered transitory release of Ca2+ from intracellular stores but no Ca2+ influx, even when the cell was voltage-clamped at negative membrane potentials. These transitory Ca2+ rises had similar amplitudes and delays to those induced by cross-linking mIgs alone. Thus, our data show that Fc gamma RII does not mediate an overall inhibition of mIg signaling but specifically affects transmembrane Ca2+ influx without affecting the release of Ca2+ from intracellular stores. Furthermore, this inhibition is not mediated by cell depolarization. Thus, Fc gamma RII represents a tool to dissociate physiologically the phases of release and transmembrane influx of Ca2+ triggered through antigen receptors.     

A naturally occurring mutation in Fc gamma RIIA: a Q to K127 change confers unique IgG binding properties to the R131 allelic form of the receptor

Fc gamma RIIa is widely expressed on hematopoietic cells. There are two known allelic polymorphic forms of Fc gamma RIIa, Fc gamma RIIa-R131 and Fc gamma RIIa-H131, which differ in the amino acid at position 131 in the second lg-like domain. In contrast to Fc gamma RIIa-R131, Fc gamma RIIa-H131 binds hlgG2 but not mIgG1, and this differential binding has clinical implications for host defense, autoimmune disease, immunohematologic disease, and response to therapeutic monoclonal antibodies. We identified a novel Fc gamma RIIA genotype in a healthy individual homozygous for Fc gamma RIIA R/R131 in whom a C to A substitution at codon 127 changes glutamine (Q) to lysine (K) in one of the two Fc gamma RIIA genes. This individual's homozygosity for Fc gamma RIIA-R/R131 leads to the prediction that the receptors on her cells would not bind hIgG2. Monocyte and neutrophil phagocytosis of hIgG2-opsonized erythrocytes was significantly higher (P < .05) for cells from this K/Q127, R/R131 individual than for Q/Q127, R/R131 donors. Platelet aggregation stimulated by an mIgG1 anti-CD9 antibody in this individual was significantly different (P < .05) from Q/Q127, H/R131 and Q/Q127, H/H131 donors and similar to Q/Q127, R/R131. Our data show that the K127/R131 receptors have a unique phenotype, binding both hIgG2 and mIgG1. Further functionally significant mutations in human Fc gamma receptors and possible novel mechanisms for inherited differences in disease susceptibility should be sought with unbiased screening methods.     

Anti-Fc gamma receptor III autoantibody is associated with soluble receptor in rheumatoid arthritis serum and synovial fluid

We sought to detect anti-Fc gamma receptor (Fc gamma R) autoantibodies and soluble Fc gamma R in the serum and synovial fluid (SF) from patients with rheumatoid arthritis (RA) and to correlate these serological abnormalities to the polymorphonuclear cell (PMN) activation state. Paired samples of blood and SF were obtained from 33 RA patients as well as blood from 25 normal adults from SF from 20 non-RA patients. Anti-Fc gamma R autoantibodies were assessed by an enzyme-linked immunosorbent assay (ELISA) using recombinant human Fc gamma R as the substrate. Soluble Fc gamma RIII was determined by an ELISA based on the combination of two monoclonal antibodies (MAb). The mean fluorescence intensity (MFI) of complement receptor 1 (CD35) and 3 (CD11b) and Fc gamma RIII (CD16) was evaluated by flow cytometry on the membrane of PMN. IgM anti-Fc gamma RIII activity was present in seven RA sera and five SF, and IgG in eight RA sera and six SF. The average concentration of soluble Fc gamma RIII was 1.80 +/- 3.50 micrograms/ml in RA patients and 0.33 +/- 0.06 micrograms/ml in normal adults (P < 0.05). This was elevated in the SF of 15 RA, while normal in that of all the non-RA patients. There was an inverse correlation between the CD16 MFI on the PMN and the serum/SF soluble Fc gamma RIII level, whereas the density of CD35 and CD11b was markedly augmented. Anti-Fc gamma RIII activity exists in RA patients, associated with soluble Fc gamma RIII. PMN activation could be due to these autoantibodies and thereby obviate the clearance of immune complexes.     

Structure and function of human IgA Fc receptors (Fc alpha R)

Receptors for the Fc region of IgA are expressed by many human cell types, especially phagocytes located in mucosal areas, where IgA is the prevalent antibody isotype. Binding of IgA-opsonized particles (e.g., bacteria, viruses) to Fc alpha R may trigger a plethora of cell-mediated immune effector functions designed to rid the body of the foreign invader. The IgA receptor present on myeloid cells such as neutrophils, eosinophils, and monocytes (Fc alpha RI or CD89) is a transmembrane glycoprotein that binds both IgA isotypes with similar affinity. Genetic characterization showed Fc alpha RI to be a more distantly related member of the Ig receptor gene family. Recently, Fc alpha RI was found to associate with the FcR gamma-chain signaling molecule through a unique charge-based mechanism. Fc alpha RI is, thus, connected to the intracellular machinery via the ITAM signaling motifs located within the cytoplasmic tail of FcR gamma-chain. Evidence exists in support of receptors for IgA (distinct from Fc alpha RI) on human T and B cells. IgA Fc receptors may, therefore, play a role in both the induction and control of an efficient (mucosal) immune response.     

FcR-mediated inhibition of cell activation and other forms of coinhibition

The tripartite inactivation model proposed that coaggregation of the B cell antigen receptor (BCR) with the Fc receptor (FcR) by antigen and specific IgG antibody complexes explained the Fc-dependent inhibition of immune responses by antibody. This model has since been substantiated by many observations and its impact on studies of immune regulation has been threefold: (1) IgG antibody, via Fc gamma RIIB, mediates inhibition of cell activation in many cell types, demonstrating the general importance of this mechanism in immune regulation; (2) Fc gamma RIIB was the first receptor described that regulates immune responses by coinhibition, that is, regulation as a result of interaction between activating receptors (BCR, TCR, Fc epsilon RI, Fc gamma RIII, Fc gamma RIIA) and inhibitory receptors (Fc gamma RIIB, CTLA4, CD5, CD22, p58/70/140 KIR, gp49B1/gp91, Ly49A/C/E/F/G, NKG2-A/B, APCR, Fas (CD95), TGF beta-R, TNF-R, IFN gamma-R, and others). The list of coinhibitors is expanding, just as the list of costimulators has grown. Tolerance through multiple coinhibitors implies that Signal 1 alone is not tolerogenic; and (3) Studies of Fc gamma RIIB coinhibitory mechanisms have pointed the way to potential general inhibitory signaling pathways used by many receptors, involving the competing effects of various kinases and phosphatases, and other competitive events. Investigations of Fc gamma RIIB physiologic function and of other coinhibitory receptors, together with recent biochemical analyses, give an initial understanding of the biology of these inhibitory receptory receptors. Paradoxes within and between theoretical constructs, functional observations, and mechanistic studies point to critical questions for future study.     

Differential interaction of Crkl with Cbl or C3G, Hef-1, and gamma subunit immunoreceptor tyrosine-based activation motif in signaling of myeloid high affinity Fc receptor for IgG (Fc gamma RI)

Cbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl-Crkl and Crkl-C3G interactions following Fc gamma RI aggregation in U937IF cells. Fc gamma RI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing gamma subunit of Fc gamma RI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and Fc gamma RI gamma after Fc gamma RI activation and the constitutive association between C3G and Crkl provide the first evidence that a Fc gamma RI gamma-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.     

Stimulation of Fc(alpha) receptors induces tyrosine phosphorylation of phospholipase C-gamma(1), phosphatidylinositol phosphate hydrolysis, and Ca2+ mobilization in rat and human mesangial cells

Although knowledge of IgA Fc receptor (Fc(alpha)R) structure and gene organization has progressed in the past few years, signal transduction pathways elicited by its activation have hardly been studied. Previously, we have demonstrated that mesangial cells (MC) possess Fc(alpha)R stimulation triggers several biologic responses. In this work, we studied the early biochemical signals triggered by Fc(alpha)R stimulation in MC. MC incubation with aggregated IgA (AIgA) elicited a dose-dependent increase in cytosolic Ca2+ ([Ca2+]i). The response was rapid and transient, and slowly fell to the original baseline. Ca2+ mobilization was dependent on the Fc region of the IgA, because Fc, but neither Fab fragment nor carbohydrates, inhibited the [Ca2+] rise. The initial induction of [Ca2+]i rise was due to Ca2+ mobilization from inositol trisphosphate (IP3)-sensitive intracellular stores, while sustained levels were maintained through extracellular Ca2+ influx. Stimulation of Fc(alpha)R also resulted in production of IP3, temporally correlated with Ca2+ mobilization. Protein tyrosine kinase inhibitors abolished [Ca2+]i rise, indicating that tyrosine phosphorylation of some substrates is required for Ca2+ mobilization. Stimulation through Fc(alpha)R gave rise to a marked increase in tyrosine phosphorylation of several proteins, including the 147-kDa band, similar in size to phospholipase C-gamma(1) (PLC-gamma(1)). Tyrosine phosphorylation of PLC-gamma(1) reached a maximum 30 s after stimulation, as determined by immunoprecipitation and Western blot. Collectively, these results indicate that the Fc(alpha)R signaling pathway in MC involves PLC-(gamma(1) activation, IP3 formation, and Ca2+ mobilization, and is linked to activation of tyrosine kinases.     

Presence of m3 subtype muscarinic acetylcholine receptors and receptor-mediated increases in the cytoplasmic concentration of Ca2+ in Jurkat, a human leukemic helper T lymphocyte line

Recent studies have demonstrated the presence and the regulatory function of several neurotransmitters in the immune system. In the present study, we examined the presence of acetylcholine receptors, using pharmacological and molecular biological assays, and their transmembrane control and functions, using a biochemical assay, in a cloned human leukemic helper T lymphoma cell line, Jurkat. Several muscarinic agonists, such as acetylcholine, carbachol, muscarine, and oxotremorine-M (Oxo-M), at 100 microM caused a transient elevation of the free cytosolic Ca2+ concentration ([Ca2+]i), in contrast to the tonic elevation of [Ca2+]i induced by 10 micrograms/ml phytohemagglutinin (PHA). It appeared that the elevation induced by Oxo-M, the most potent [Ca2+]i elevator, was more effectively inhibited by p-fluorohexahydrosiladifenidol hydrochloride (p-F-HHSiD) and 4-diphenylacetoxy-N-methylpiperidine methiodine than by pirenzepine and 11-2[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido[2,3-b] [1,4]benzodiazepine-6-one (AF-DX 116), suggesting that a pharmacological M3 subtype of muscarinic receptors is involved in the elevation of [Ca2+]i. Northern blot analysis showed that the m3 type of receptors are expressed in Jurkat cells. Scatchard analysis of [3H]quinuclidinyl benzilate binding to intact cells indicated a Kd of 14.1 nM and a Bmax of 45,370 binding sites/cell. [3H]Quinuclidinyl benzilate binding to cell membranes was also inhibited by p-F-HHSiD rather than by pirenzepine and AF-DX 116. Oxo-M induced formation of inositol trisphosphate, and 5'-O-(2-thio)diphosphate inhibited the formation. Cholera toxin treatment inhibited the PHA-induced [Ca2+]i rise but did not affect the Oxo-M-induced rise. Neither pertussis nor butulinus (type C) toxin affected the rise induced by Oxo-M or PHA. Thus, bacterial toxin-insensitive GTP-binding proteins seem to be involved in the Oxo-M-induced increase in [Ca2+]i. Treatment with 12-O-tetradecanoylphorbol 13-acetate abolished the Oxo-M-induced [Ca2+]i rise but did not affect that induced by PHA. m3 Muscarinic receptors thus appear to cause Ca2+ mobilization from intracellular stores via bacteria toxin-insensitive GTP-binding proteins, phospholipase C activation, and inositol trisphosphate formation in Jurkat cells. Protein kinase C seems to negatively modulate the m3 receptor system.     

Influence of tyrosine phosphorylation on protein interaction with FcgammaRIIa

The cytoplasmic tail of Fc(gamma)RIIa present on human neutrophils shares with other antigen receptors a common amino acid sequence called ITAM (Immunoreceptor Tyrosine-based Activation Motif). After receptor ligation, the tyrosine residues within this motif become phosphorylated. We prepared a recombinant fusion protein of the cytoplasmic tail of Fc(gamma)RIIa (containing the ITAM) with glutathione-S-Transferase (GST-CT) to characterize the phosphorylation of Fc(gamma)RIIa and its ability to interact with other proteins involved in signal transduction. The GST-CT became phosphorylated in the presence of Lyn, Hck and Syk (immunoprecipitated from human neutrophils), but not in the presence of Fgr. Of the active kinases, only Lyn (mainly present in the membrane fraction) was found to associate with the GST-CT in the absence of ATP. This association was also observed in immunoprecipitates of Fc(gamma)RIIa from resting neutrophils, suggesting that Lyn might be the kinase responsible for the initial Fc(gamma)RIIa phosphorylation. Moreover, we observed specific association of Syk and the p85 subunit of PI 3-kinase after incubation of the GST-CT with neutrophil cytosol. This interaction was dependent on tyrosine phosphorylation of the GST-CT. Substitution of 269Tyr by Phe almost completely abolished tyrosine phosphorylation of the fusion protein. Substitution of either 253Tyr or 269Tyr eliminated Syk binding, but only 253Tyr appeared to be essential for p85 binding. We hypothesize that, upon activation, the membrane-associated Lyn is responsible for the initial tyrosine phosphorylation of Fc(gamma)RIIa, thus creating a docking site for Syk and PI 3-kinase.     

The cytoplasmic and the transmembrane domains are not sufficient for class I MHC signal transduction

Class I MHC molecules deliver activation signals to T cells. To analyze the role of the cytoplasmic and the transmembrane (TM) domains of class I MHC molecules in T cell activation, Jurkat cells were transfected with genes for truncated class I MHC molecules which had only four intracytoplasmic amino acids and no potential phosphorylation sites or native molecules or both. Cross-linking either the native or the truncated molecules induced IL-2 production even under limiting stimulation conditions of low engagement of the stimulating mAb. Moreover, direct comparison of transfected truncated and native class I MHC molecules expressed on the same cell revealed significant stimulation induced by cross-linking the truncated molecules, despite low expression. In addition, truncated class I MHC molecules were as able to synergize with CD3, CD2, or CD28 initiated IL-2 production as native molecules. In further experiments, hybrid constructs made of the extracellular portion of the murine CD8 alpha chain and of the TM and the intracytoplasmic domains of H-2Kk class I MHC molecule were transfected into Jurkat T cells. The expression of the transfected hybrid molecules was comparable to that of the native HLA-B7 molecules. Cross-linking the intact monomorphic HLA-A,B,C epitope or the polymorphic HLA-B7 epitope induced IL-2 production upon costimulation with PMA. In contrast, cross-linking the hybrid molecules generated neither an increase in intracellular calcium concentration ([Ca2+]i) nor stimulated IL-2 production. By contrast, cross-linking intact murine class I MHC molecules induced [Ca2+]i, signal and IL-2 production in transfected Jurkat cells. The data therefore indicate that unlike many other signaling molecules, signaling via class I MHC molecules does not involve the cytoplasmic and the TM portions of the molecule, but rather class I MHC signal transduction is likely to be mediated by the extracellular domain of the molecule.     

Reconstitution of CD3 zeta coupling to calcium mobilization via genetic complementation

The integrity of the T cell receptor complex (CD3-TCR) transduction machinery is central to T cell development and to T cell effector function. Molecular dissection of the multimeric CD3-TCR complex revealed that at least two associated polypeptides, CD3 zeta and CD3 epsilon, autonomously couple antigenic recognition event to early and late events of the intracytoplasmic activation cascade. A 18-amino acid motif based on a tandem YXXL stretch, the activation receptor homology sequence 1 (ARH-1) motif, is necessary and sufficient to the transducing properties of both CD3 zeta and CD3 epsilon. Stimulation of chimeric molecules made of ecto- and transmembrane domains of various cell surface proteins and intracytoplasmic domains of CD3 epsilon or CD3 zeta leads to an increase in the intracellular Ca2+ concentration ([Ca2+]i) in Jurkat cells. We describe here that a similar CD25/zeta chimeric molecule was unable to induce a detectable [Ca2+]i rise upon CD25 cross-linking once expressed in the murine thymoma BW-. A Ca2+ influx could, however, be triggered in BW- cells by thapsigargin, i.e. following depletion of Ca2+ stores. Somatic cell hybrids made from BW- and either thymocytes or mature lymph node T cells reconstituted the coupling of CD3 zeta to the Ca2+ signal via an ARH-1 motif-dependent pathway. However, pervanadate-induced Ca2+ mobilization, a phenomenon attributed to tyrosine phosphorylation, was impaired in BW-cells and reconstituted in hybridomas. In contrast to the Ca2+ response, IL-2 production was induced in both BW- and hybrids cells, which questions the functional relevance of [Ca2+]i augmentation in T cell activation. In conclusion, the properties of the BW- thymoma, which define a novel group of CD3 zeta transduction cell mutants, as well as its complementation by somatic cell fusion demonstrate that this cell line represents a useful model to dissect the signaling pathway that couples CD3 zeta to Ca2+ mobilization by genetic reconstitution.     

The same tyrosine-based inhibition motif, in the intracytoplasmic domain of Fc gamma RIIB, regulates negatively BCR-, TCR-, and FcR-dependent cell activation

The cell-triggering properties of BCR, TCR and FcR depend on structurally related immunoreceptor tyrosine-based activation motifs (ITAMs). Fc gamma RIIB have no ITAM and do not trigger cell activation. When coaggregated to BCR, they inhibit B cell activation. We show here that, when coaggregated to these receptors, Fc gamma RIIB inhibit Fc epsilon RI-, Fc gamma RIIA-, and TCR-dependent cell activation. Inhibition also affected cell activation by single ITAMs, in isolated FcR or TCR subunits. The same tyrosine-based inhibitory motif (ITIM), which is highly conserved in murine and human Fc gamma RIIB and that was previously shown to inhibit BCR-dependent B cell activation, was required to regulate TCR- and FcR-dependent cell activation. Our findings endow Fc gamma RIIB, and thus IgG antibodies, with general immunoregulatory properties susceptible to act on all ITAM-containing receptors.     

Paired immunoglobulin-like receptor (PIR)-A is involved in activating mast cells through its association with Fc receptor gamma chain

Paired immunoglobulin-like receptor (PIR)-A and PIR-B possess similar ectodomains with six immunoglobulin-like loops, but have distinct transmembrane and cytoplasmic domains. PIR-B bears immunoreceptor tyrosine-based inhibitory motif (ITIM) sequences in its cytoplasmic domain that recruit Src homology (SH)2 domain-containing tyrosine phosphatases SHP-1 and SHP-2, leading to inhibition of B and mast cell activation. In contrast, the PIR-A protein has a charged Arg residue in its transmembrane region and a short cytoplasmic domain that lacks ITIM sequences. Here we show that Fc receptor gamma chain, containing an immunoreceptor tyrosine-based activation motif (ITAM), associates with PIR-A. Cross-linking of this PIR-A complex results in mast cell activation such as calcium mobilization in an ITAM-dependent manner. Thus, our data provide evidence for the existence of two opposite signaling pathways upon PIR aggregation. PIR-A induces the stimulatory signal by using ITAM in the associated gamma chain, whereas PIR-B mediates the inhibitory signal through its ITIMs.     

Expression of the Fc receptor for IgG (Fc gamma RII/CDw32) by human circulating T and B lymphocytes

Tissue-specific isoforms of the human FcR for IgG Fc gamma RII (CDw32) have previously been described by using mAb. These mAb were shown to exhibit different patterns of reactivity with lymphocytes. Among human PBL, Fc gamma RII has been detected on B cells but not T cells when assessed by flow cytometry and microscopy with the use of mAb KB61 and 41H16. Although KB61 and 41H16 were found to react with B cells, the mAb IV.3, CIKM5, and 2E1 did not react with any PBL subset. In this study, we show that KB61 and 41H16 react strongly with the majority (93-96%) of B cells (CD20+), and weakly with a proportion (18-42%) of T cells (CD3+), including 10 to 14% of CD4+ and 27 to 69% of CD8+ cells. In addition, mRNA for Fc gamma RII was detected in purified CD3+CD8high+ lymphocytes by polymerase chain reaction. KB61 and 41H16 also reacted with a majority of CD3-CD16/CD56+ cells, and CD3-CD20- cells. These findings indicate that a subset of T cells and non-T/non-B cells express Fc gamma RII, and are of interest in the light of previous studies which postulate that human Fc gamma R+ cells and Fc gamma RII+ murine T cells suppress the B cell immune response.     

Tyrosine-containing activation motif-dependent phagocytosis in mast cells

FcR capable of triggering cell activation share with BCR and TCR a conserved intracytoplasmic tyrosine-containing activation motif (TAM). Besides cell activation, these receptors trigger other biologic responses, such as endocytosis of soluble ligands. Murine mast cells express two types of FcR that, when aggregated by antibodies and multivalent Ag, trigger the release of inflammatory mediators and cytokines. These are high affinity receptors for IgE (Fc epsilon RI) and low affinity receptors for IgG (Fc gamma RIII). They comprise each an IgE- or IgG-binding alpha-subunit and two TAM-containing subunits that associate with both receptors: a beta-subunit and a homodimeric gamma-subunit that can associate also with the other subunits of the TCR. Herein, we focused on biologic activities triggered in mast cells via the TAM of the gamma-subunits. Using rat basophilic leukemia (RBL) cells stably transfected with cDNA-encoding murine Fc gamma RIII alpha, we found that murine Fc gamma RIII trigger the phagocytosis of antibody-coated erythrocytes. Using RBL transfectants expressing Fc gamma RIII with a deletion of the intracytoplasmic domain of Fc gamma RIII alpha or chimeric receptors having the extracellular and transmembrane domains of Fc gamma RII and the intracytoplasmic domain of Fc gamma RIII alpha, we showed that intracytoplasmic sequences of Fc gamma RIII alpha are neither necessary nor sufficient for Fc gamma RIII to trigger phagocytosis. Using RBL transfectants expressing chimeric receptors having the extracellular and transmembrane domains of Fc gamma RII and the TAM-containing intracytoplasmic domain of murine Fc gamma RIII gamma, we demonstrated that intracytoplasmic sequences of Fc gamma RIII gamma are sufficient to trigger phagocytosis. Using RBL transfectants expressing the same Fc gamma RII-III gamma chimeras, in the TAM of which one, the other, or both tyrosine residues were mutated, we established that tyrosines in the TAM sequence are required for phagocytosis. Our results endow TAM gamma with previously unknown triggering capacities and Fc gamma RIII with new biologic properties.     

GPI-anchored complement regulatory proteins in seminal plasma. An analysis of their physical condition and the mechanisms of their binding to exogenous cells

We analyzed and compared the properties of three glycosylphosphatidylinositol (GPI)-anchored proteins. CD59, CD55 (both C regulators), and CDw52, and of the transmembrane C regulator CD46 in seminal plasma (SP). We demonstrated previously that anchor-intact SP CD59 is present on the membranes of vesicles (prostasomes) and that cells acquire this protein during incubation with SP. We now report that this acquisition is due partly to adherence of prostasomes to cells and partly to a second mechanism which may involve micellar intermediates. Using fluorescent labeling, ultracentrifugation, and density gradient centrifugation, virtually all CD46 was present on prostasomes whereas CD59, CD55, AND CDw52 were also detected in a form which remained in the 200,000 g supernatant and equilibrated at higher density than prostasomes in gradients. All three GPI-linked proteins eluted at high molecular mass during size exclusion chromatography of this nonprostasome fraction. As documented by videomicroscopy and biochemical analysis, cells acquired new copies of the GPI-linked proteins during incubation with the nonprostasome fraction as well as with prostasomes. These data demonstrate the presence in SP of a stable population of membrane-free, GPI-linked proteins available for transfer to cells. Binding of these proteins to spermatozoa and pathogens in SP may confer new properties on their membranes including increased resistance to C attack. Finally, our data raise the possibility that lipid-associated GPI-linked proteins may be suitable for therapeutic applications.     

Morphine inhibits signal transduction subsequent to activation of protein kinase C in mouse splenocytes

Morphine administered as a subcutaneous implant was previously reported to inhibit the mitogen-induced initial increases in cytoplasmic free calcium concentrations ([Ca2+]i) in mouse splenocytes. The present studies were initiated to determine whether morphine affects signal transduction subsequent to activation of protein kinase C (PKC) in immune cells. Administration of morphine significantly inhibited the phorbol myristate acetate (PMA)-stimulated increase in interleukin-2 receptor (IL-2R) expression in both CD4+ and CD8+ mouse T cells. In contrast, morphine treatment had no effect on PMA/calcium ionophore (A23187)-induced increase in IL-2 secretion, suggesting a selective inhibition of IL-2R expression. Simultaneous administration of morphine and the opiate antagonist naltrexone blocked the effect of morphine on CD4+ cells. The inhibition of PMA-stimulated IL-2R expression was not reproduced by incubating splenocytes with morphine (10(-8)-10(-5) M). These results suggest that this effect of morphine was mediated through opiate-receptors, but not directly via opiate receptors located on T cells. Moreover, adrenalectomy abolished this effect of morphine in CD4+ but not CD8+ T cells, suggesting that the inhibitory effect of morphine on IL-2R expression in CD4+ T cells may be mediated through a morphine-induced increase in corticosteroid levels. Thus, opiate-induced immunosuppression may involve an inhibition of post-PKC events, especially IL-2R expression, as well as impairment of earlier events in the activation of immune cells such as the increase in [Ca2+]i.     

Fc gamma receptor II dependency of enhanced presentation of major histocompatibility complex class II peptides by a B cell lymphoma

Here we show that the B cell lymphoma A20.292 is capable of enhanced antigen presentation to CD4+ T cells in the presence of specific antibodies. This enhancement was inhibited by anti-Fc gamma receptor (R) antibodies, suggesting that it might be due to preferential uptake of the antigen/antibody complex through the Fc gamma RII receptor. However, immunoprecipitation studies revealed that the FcR of A20.292 cells was of the B cell type, Fc gamma RIIb1, which is not thought to be able to internalize antigen/antibody complexes via clathrin-coated pits. It was considered unlikely that A20.292 had an altered form of the B cell Fc gamma R (RIIb1) receptor that enabled internalization, since similar enhancing effects were also observed using an Fc gamma RII cell line that had been transfected with Fc gamma RIIb1. To reconcile these findings with the expression of Fc gamma RIIb1, it is postulated that immune complexes are concentrated on the cell surface by the Fc gamma RIIb1 and are thus available for preferential uptake by random fluid-phase endocytosis. This results in more efficient generation of the epitopes recognized by these T cell hybridomas.     

Urokinase-type plasminogen activator receptor reversibly dissociates from complement receptor type 3 (alpha M beta 2' CD11b/CD18) during neutrophil polarization

Previous studies have shown that the leukocyte integrin CR3 (CD11b/CD18) is physically associated with the urokinase-type plasminogen activator receptor (uPAR;CD87), a glycosyl-phosphatidylinositol (GPI)-linked protein, in resting neutrophil membranes. We now show that uPAR-to-CR3 interactions are reversible, correlating with cell shape. Neutrophils were first labeled with fluorescein conjugates of anti-CR3 F(ab')2 fragments followed by capping using a second-step F(ab')2 directed against murine F(ab')2s. Cells were then probed using rhodamine-conjugated anti-uPAR F(ab')2s. Although uPAR co-caps with CR3 on resting cells, uPAR was found to dissociate or "uncap" coincident with spontaneous cell polarization for migration. CR3 caps transformed into uropods while uPAR accumulated at lamellipodia of polarized cells. Capping was unnecessary for the observed distribution of CR3 and uPAR since the anti-CR3 and anti-uPAR F(ab')2s traffic to the uropod and lamellipodium, respectively, during polarization of uncapped cells. These receptors reassociate when cells return to a spherical morphology. In contrast to uPAR, Fc gamma RIIIB did not dissociate from CR3 caps during cell polarization. Resonance energy transfer (RET) microscopy was used to image the spatial distribution of RET and to follow the kinetics of association and dissociation. Initial levels of RET dramatically fell during cell polarization, but did not change on cells fixed with paraformaldehyde. Receptor reassociation was a biphasic process with initial reassociation about the perimeter of a cap, followed by a plateau and a slower rise in RET within a cap. We suggest that cells regulate receptor-receptor associations depending upon their physiologic activities.