Early events in the activation of Fc gamma RIIA in human neutrophils: stimulated insolubilization, translocation to detergent-resistant domains, and degradation of Fc gamma RIIA

The signal transduction mechanisms associated with the ligation of FcgammaRIIA in human neutrophils are as yet only incompletely characterized. In the present study, we have investigated the distribution and fate of FcgammaRIIA following its cross-linking. The results obtained indicate that cross-linking of FcgammaRIIA led, within a few seconds, to its translocation into a nonionic detergent-insoluble fraction. This was followed, within a couple of minutes, by a substantial loss of immunoreactive FcgammaRIIA in the cells. The stimulated degradation of FcgammaRIIA was blocked by the Src kinase inhibitor PP1 but not by wortmannin, ST-638, piceatannol, or cytochalasin B. Cross-linked FcgammaRIIA could be solubilized by saponin (in the presence of Nonidet P-40) and by beta-octylglucoside. Sucrose gradient analysis of the distribution of FcgammaRIIA revealed that its cross-linking led to its translocation into the pellets and not the light buoyant density fractions classically associated with lipid rafts. Disruption of cholesterol-containing membrane microdomains with filipin prevented the degradation of FcgammaRIIA but did not inhibit the stimulation of the pattern of tyrosine phosphorylation or the mobilization of calcium that followed FcgammaRIIA cross-linking. These data suggest that both cholesterol-rich domains and Src kinases are required for the degradation of the activated FcgammaRIIA and provide new insights into the early events following FcgammaRIIA cross-linking.     

Effects of interleukin-18 on natural killer cells: costimulation of activation through Fc receptors for immunoglobulin

The antitumor activity of monoclonal antibodies is mediated by effector cells, such as natural killer (NK) cells, that express Fc receptors for immunoglobulin. Efficacy of monoclonal antibodies, including the CD20 antibody rituximab, could be improved by agents that augment the function of NK cells. Interleukin (IL)-18 is an immunostimulatory cytokine that has antitumor activity in preclinical models. The effects of IL-18 on NK cell function mediated through Fcγ receptors were examined. Human NK cells stimulated with immobilized IgG in vitro secreted IFN-γ as expected; such IFN-γ production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-γ production by NK cells stimulated with immobilized IgG or CD16 antibodies. NK cell IFN-γ production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk and several other kinases involved in CD16 signaling pathways. IL-18 augmented antibody-dependent cellular cytotoxicity (ADCC) of human NK cells against rituximab-coated Raji cells in vitro. IL-18 and rituximab acted synergistically to promote regression of human lymphoma xenografts in SCID mice. Inasmuch as IL-18 costimulates IFN-γ production and ADCC of NK cells activated through Fc receptors in vitro and augments antitumor activity of rituximab in vivo, it is an attractive cytokine to combine with monoclonal antibodies for treatment of human cancer.     

Cross-linking of both Fc gamma RI and Fc gamma RII induces secretion of tumor necrosis factor by human monocytes, requiring high affinity Fc-Fc gamma R interactions. Functional activation of Fc gamma RII by treatment with proteases or neuraminidase

Cross-linking of Fc gamma R on human monocytes with human IgG has been shown to induce secretion of the inflammatory and immunoregulatory cytokine TNF. In the present study we examined the role of both constitutively expressed monocyte Fc gamma R, the 72-kDa high affinity Fc gamma R (Fc gamma RI), and the 40-kDa low affinity receptor (Fc gamma RII), in the induction of TNF secretion. On the basis of preferential binding of the Fc moiety of murine mAb of different isotype, Fc gamma RI and Fc gamma RII were selectively cross-linked by using either solid-phase murine (m)IgG2a, or solid-phase mIgG1, respectively. On freshly isolated, untreated monocytes only cross-linking of Fc gamma RI with solid-phase mIgG2a induced TNF secretion. The interaction between Fc gamma RII and mIgG1 could be enhanced by treatment of monocytes with proteases or with the desialylating enzyme neuraminidase. After treatment of monocytes with these enzymes, TNF secretion was effectively induced by solid-phase mIgG1, apparently through cross-linking of Fc gamma RII. However, mIgG1-induced TNF secretion differed between protease-treated monocytes from high responder individuals and monocytes from low responder individuals, TNF secretion being considerably less in the latter population. Protease-treated monocytes and mononuclear cells from individuals with an inherited defect in cell membrane expression of Fc gamma RI were induced to secrete TNF by solid-phase human IgG, confirming the capacity of Fc gamma RII to induce TNF secretion. It was not possible to induce TNF secretion by cross-linking Fc gamma RI or Fc gamma RII with anti-Fc gamma R mAb and soluble or solid-phase anti-mIgG, indicating that high affinity Fc-Fc gamma R interactions are necessary to induce release of this cytokine.     

Efficiency of platelet adhesion to fibrinogen depends on both cell activation and flow

The kinetics of adhesion of platelets to fibrinogen (Fg) immobilized on polystyrene latex beads (Fg-beads) was determined in suspensions undergoing Couette flow at well-defined homogeneous shear rates. The efficiency of platelet adhesion to Fg-beads was compared for ADP-activated versus "resting" platelets. The effects of the shear rate (100-2000 s(-1)), Fg density on the beads (24-2882 Fg/microm(2)), the concentration of ADP used to activate the platelets, and the presence of soluble fibrinogen were assessed. "Resting" platelets did not specifically adhere to Fg-beads at levels detectable with our methodology. The apparent efficiency of platelet adhesion to Fg-beads readily correlated with the proportion of platelets "quantally" activated by doses of ADP, i.e., only ADP-activated platelets appeared to adhere to Fg-beads, with a maximal adhesion efficiency of 6-10% at shear rates of 100-300 s(-1), decreasing with increasing shear rates up to 2000 s(-1). The adhesion efficiency was found to decrease by only threefold when decreasing the density of Fg at the surface of the beads by 100-fold, with only moderate decreases in the presence of physiologic concentrations of soluble Fg. These adhesive interactions were also compared using activated GPIIbIIIa-coated beads. Our studies provide novel model particles for studying platelet adhesion relevant to hemostasis and thrombosis, and show how the state of activation of the platelet and the local flow conditions regulate Fg-dependent adhesion.     

Immunochromatography on insolubilized antibodies of very low affinity: application to immunoadsorbence of bovine alpha-fetoprotein.

Cross-reactive antibodies were utilized to prepare immunoadsorbents possessing a very low affinity to bovine alpha-fetoprotein (AFP). A goat anti-human AFP serum cross-reactive with bovine AFP was first depleted of antibodies reactive with bovine AFP in immunodiffusion. The remaining antibodies from this serum and gamma-globulin from a sheep antiserum against rabbit AFP, without prior absorption, were coupled to Sepharose. Chromatography of fetal calf serum on these adsorbents resulted in retardation of bovine AFP relative to other proteins. A major part of the AFP eluted from the columns with phosphate-buffered saline. The rest eluted as a sharp peak with a small quantity of 4 or 6 M urea. The elution of AFP with the initial column buffer has made it possible to prepare pure AFP that has not been subjected to the chaotropic elution buffers usually employed in affinity chromatography. Elimination of the washing step and the ease of elution has allowed purification of gram amounts of AFP. The fact that immunoadsorbents prepared from antibodies with no detectable reactivity in immunodiffusion still caused delayed elution in chromatography suggests that this procedure may be useful in search of proteins cross-reactive with a known protein.     

Anti-CD45 inhibition of human B cell proliferation depends on the nature of activation signals and the state of B cell activation. A study with anti-IgM and anti-CDw40 antibodies

The proliferation of human peripheral and tonsillar B cells stimulated with the anti-CDw40 mAb 626.1 and/or anti-IgM antibody (Ab) in the presence of anti-CD45 mAb A.1.1 was investigated. The anti-CD45 mAb suppressed the anti-CDw40-stimulated proliferation of peripheral blood B cells but had no effect on the proliferation of unfractionated tonsillar B cells stimulated similarly. When tonsillar B cells were separated according to their sizes, the anti-CDw40-induced proliferation of small tonsillar B cells was inhibited by the anti-CD45 mAb, whereas large tonsillar B cells were resistant. In contrast, anti-IgM-induced proliferation of human B cells was always significantly inhibited by the anti-CD45 mAb regardless of cell size and tissue origin. The anti-CD45 mAb also inhibited the anti-IgM-induced initial rise in intracellular [Ca2+] and the G0-G1 cell cycle transition of small tonsillar B cells. However, co-stimulation with anti-IgM/anti-CDw40 Ab resulted in the resistance to the anti-CD45 inhibitory effect on proliferation of peripheral blood B cells and the majority of tonsillar B cells. In contrast, B cell proliferation co-stimulated with anti-IgM Ab/and B cell growth factors were always suppressed by the anti-CD45 mAb. These results demonstrate that certain activational signal mechanisms utilized by anti-CDw40/anti-IgM Ab and anti-IgM Ab/B cell growth factors are different in that B cells stimulated with these agents differ in their sensitivity to the anti-CD45 mAb. Moreover, both the activational state of human B cells and the nature of activation signals given determine their response to the inhibitory signals delivered by the anti-CD45 mAb.     

Immune complexes bind preferentially to Fc gamma RIIA (CD32) on apoptotic neutrophils, leading to augmented phagocytosis by macrophages and release of proinflammatory cytokines

Many human inflammatory diseases are associated with tissue deposition of immune complexes and influx of neutrophils. We show that immune complexes bind preferentially to apoptotic neutrophils via FcgammaRIIA (CD32) and that increased binding is associated with clustering of immune complexes on the plasma membrane of the apoptotic cell. Phagocytosis of immune complex-opsonized apoptotic neutrophils by human macrophages was substantially enhanced (4.4-fold increase compared with control apoptotic neutrophils) and stimulated macrophages to release the proinflammatory cytokines TNF-alpha and IL-6. Immune complexes may perturb the normal pathways for clearance of apoptotic neutrophils by augmenting their clearance at the price of proinflammatory cytokine release. This represents a novel mechanism by which immune complexes may modulate the resolution of inflammation.     

Recognition of DNA by single-chain derivatives of the phage 434 repressor: high affinity binding depends on both the contacted and non-contacted base pairs.

Single-chain derivatives of the phage 434 repressor, termed single-chain repressors, contain covalently dimerized DNA-binding domains (DBD) which are connected with a peptide linker in a head-to-tail arrangement. The prototype RR69 contains two wild-type DBDs, while RR*69 contains a wild-type and an engineered DBD. In this latter domain, the DNA- contacting amino acids of thealpha3 helix of the 434 repressor are replaced by the corresponding residues of the related P22 repressor. We have used binding site selection, targeted mutagenesis and binding affinity studies to define the optimum DNA recognition sequence for these single-chain proteins. It is shown that RR69 recognizes DNA sequences containing the consensus boxes of the 434 operators in a palindromic arrangement, and that RR*69 optimally binds to non-palindromic sequences containing a 434 operator box and a TTAA box of which the latter is present in most P22 operators. The spacing of these boxes, as in the 434 operators, is 6 bp. The DNA-binding of both single-chain repressors, similar to that of the 434 repressor, is influenced indirectly by the sequence of the non-contacted, spacer region. Thus, high affinity binding is dependent on both direct and indirect recognition. Nonetheless, the single-chain framework can accommodate certain substitutions to obtain altered DNA-binding specificity and RR*69 represents an example for the combination of altered direct and unchanged indirect readout mechanisms.     

Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII.

The gene encoding the rat glycosylation enzyme beta1-4-N-acetylglucosaminyltransferase III (GnTIII) was cloned and coexpressed in a recombinant production Chinese hamster ovary (CHO) cell line expressing a chimeric mouse/human anti-CD20 IgG1 antibody. The new cell lines expressed high levels of antibody and have growth kinetics similar to that of the parent. Relative QPCR showed the cell lines to express varying levels of mRNA. High-performance liquid chromatography (HPLC) analysis showed the enzyme to have added bisecting N-acetylglucosamine (GlcNAc) residues in most (48% to 71%) of the N-linked oligosaccharides isolated from antibody preparations purified from the cell lines. In an ADCC assay the new antibody preparations promoted killing of CD20-positive target cells at approximately 10- to 20-fold lower concentrations than the parent. This activity was blocked using an anti-Fc gamma RIII antibody, supporting the role of Fc gamma RIII binding in this increase. In addition, cell binding assays showed the modified antibody bound better to Fc gamma RIII-expressing cells. The increase in ADCC activity is therefore likely due to an increased affinity of the modified antibody for the Fc gamma RIII receptor.     

Further evidence for heterogeneity of Fc gamma-receptors on guinea pig splenic B and T lymphocytes: analysis using monoclonal antibodies to two distinct types of Fc gamma-receptor

In our previous paper, we reported that guinea pig splenic lymphocytes expressed two distinct Fc-receptors for homologous IgG (Fc gamma Rs), one monospecific for IgG2 (Fc gamma 2R) and the other bispecific for IgG1 and IgG2 (Fc gamma 1/gamma 2R), when analyzed by EA-rosette assay. These Fc gamma Rs on the cells were further studied by using two monoclonal antibodies toward the Fc gamma Rs on guinea pig peritoneal macrophages (anti-Fc gamma 1/gamma 2R and anti-Fc gamma 2R antibody). The anti-Fc gamma 1/gamma 2R antibody completely inhibited the rosette formation of splenic lymphocytes with IgG1-sensitized sheep erythrocytes [EA(IgG1)]. On the other hand, EA(IgG2)-rosette formation was inhibited partially by anti-Fc gamma 2R but not by anti-Fc gamma 1/gamma 2R antibody. Complete inhibition of the EA (IgG2)-rosette formation was achieved by simultaneous additions of both anti-Fc gamma 2R and anti-Fc gamma 1/gamma 2R antibodies. The binding of IgG2 antibody complexed with ovalbumin to the cells was partially inhibited by either anti-Fc gamma R antibody, and complete inhibition occurred in the presence of both the antibodies, indicating that two types of Fc gamma R, Fc gamma 1/gamma 2R, and Fc gamma 2R, are expressed on the cells. The determination of these Fc gamma Rs on B and T lymphocytes by two-color flow cytometry showed that about 52% of B lymphocytes expressed Fc gamma 1/gamma 2R alone and 32% of the cells expressed both the Fc gamma Rs. On the other hand, about 12% of T lymphocytes was found to express Fc gamma 2R alone and the cells expressing Fc gamma 1/gamma 2R were in the minority (3.8%). T lymphocytes expressing both the Fc gamma Rs were not detected. These results show that guinea pig B lymphocytes bear two types of Fc gamma Rs and are heterogeneous with regard to their Fc gamma Rs and that T lymphocytes express Fc gamma 2R mainly.     

The nonintegrin laminin binding protein (p67 LBP) is expressed on a subset of activated human T lymphocytes and, together with the integrin very late activation antigen-6, mediates avid cellular adherence to laminin.

A search for genes expressed in activated T cells revealed that the nonintegrin, 67-kDa laminin binding protein (p67 LBP) is expressed on the surface of a subset (10-15%) of activated peripheral blood T cells. Surface p67 LBP expression is detectable by FACS using the anti-p67 LBP mAb, MLuC5, within 6 h of T cell activation with phorbol dibutyrate and ionomycin, peaks 18-36 h postactivation, and persists for 7-10 days. The subset of T cells expressing p67 LBP is composed of mature, single-positive cells (85% CD4+8-, 15% CD4-8+) of memory cell phenotype (100% CD45 RO+/CD45 RA-). The p67 LBP+ T cells also express the integrin alpha6 chain (CD49f), which is known to associate with p67 LBP on tumor cells. In addition, the p67 LBP+ T cells express the integrin beta1, which associates with alpha6 in the laminin-specific integrin receptor very late activation Ag (VLA)-6 (alpha6beta1). Expression of an exogenous cDNA encoding the 37-kDa LBP precursor (p37 LBPP) confers p67 LBP surface expression on a p67 LBP-negative Jurkat T cell line (B2.7). Expression of p67 LBP induces B2.7 transfectants to adhere to laminin, but avid laminin binding depends on coexpression of VLA-6. Taken together, these data indicate that p67 LBP is an activation-induced surface structure on memory T cells that, together with VLA-6, mediates cellular adherence to laminin.     

The best of both worlds: cicada males change costly signals to achieve mates while females choose a mate based on both calling and courtship songs

Cicadas usually sing and mate in the higher parts of trees. Studies addressing the effects of different acoustic signals on mate choice in Cicadidae are very limited. We investigated the effects of both acoustical features and morphological traits on mate choice in an East Asian cicada Platypleura kaempferi. Males produce high-rate calling songs that attract females, then produce low-rate courtship songs to secure mating when a female is attracted. Higher calling song rate (CR), shorter single-pulse duration, and shorter pulse period of the calling song, together with lower courtship song rate and longer echeme period of the courtship song, are the most desirable traits used by females to choose a mate. These traits indicate that the more a male can raise the rate of song production, the higher the probability he is sexually selected by the female. No correlation was found between morphological traits and mating success. After mating, a minority of males started emitting calling songs again, but the CR was significantly lower than before mating and none of them attracted a new mate later. This promotes females mating with unmated males. We hypothesize that P. kaempferi may have the best of both worlds due to the unique song modulation and the mechanism of female mate choice: males change energetically, costly acoustic signals to achieve mates, while females choose a mate based on males’ acoustic properties. Our results contribute to better understanding the diversity of mating preference and enrich the mechanism of mate choice in acoustic insects.     

Effect of divalent cations on the affinity and selectivity of alpha4 integrins towards the integrin ligands vascular cell adhesion molecule-1 and mucosal addressin cell adhesion molecule-1: Ca2+ activation of integrin alpha4beta1 confers a distinct ligand specificity

A microtiter plate assay measuring the binding of cells expressing integrins alpha4beta1 or alpha4beta7 to VCAM-1 and MAdCAM-1, expressed as Ig fusion proteins, was used to explore the interplay between the variables of integrin beta-chain, identity and density of ligand, and identity and concentration of activating cations. Both Mn2+ and Mg2+ supported binding of either integrin to either ligand. Ca2+ supported only the binding of alpha4beta1 to VCAM-Ig. Cation concentrations required for half-maximal binding (EC50) ranged from 0.8-280 microM for Mn2+ and 0.8-30 mM for Mg2+, being thus 2-3 logs lower for Mn2+ compared to Mg2+ independent of ligand. EC50 values for binding of alpha4beta1 to VCAM-Ig were 30-45-fold lower compared to MAdCAM-Ig, while alpha4beta7 showed an opposite 3-15-fold selectivity for MAdCAM-Ig over VCAM-Ig. The density of ligand required for adhesion via alpha4beta1 was markedly lower with Mn2+ versus Mg2+, and with VCAM-Ig versus MAdCAM-Ig. These results were interpreted in terms of a coupled equilibrium model, in which binding of activating metal ions and of integrin ligands each stabilizes activated integrin. We conclude that Mn2+ and Mg2+ bind to common regulatory sites with different affinities, producing similar activated states of the integrin. The resulting activated alpha4beta1 binds more strongly to VCAM-Ig versus MAdCAM-Ig by 30-45-fold, while similarly activated alpha4beta7 binds more strongly to MAdCAM-Ig versus VCAM-Ig by 3-15-fold. Inhibition studies showed that Ca2+ also binds to regulatory sites on both integrins. However, the Ca2+-activated state of alpha4beta1 is distinct from that achieved by Mn2+ and Mg2+, possessing increased selectivity for binding to VCAM-1 versus MAdCAM-1.     

Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an activator of the sigB operon: differential activation mechanisms due to ethanol and salt stress

Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenetic factor is the ability to form adherent biofilms. The polysaccharide intercellular adhesin (PIA), which is synthesized by the products of the icaADBC gene cluster, is essential for biofilm accumulation. In the present study, we characterized the gene locus inactivated by Tn917 insertions of two isogenic, icaADBC-independent, biofilm-negative mutants, M15 and M19, of the biofilm-producing bacterium S. epidermidis 1457. The insertion site was the same in both of the mutants and was located in the first gene, rsbU, of an operon highly homologous to the sigB operons of Staphylococcus aureus and Bacillus subtilis. Supplementation of Trypticase soy broth with NaCl (TSB(NaCl)) or ethanol (TSB(EtOH)), both of which are known activators of sigB, led to increased biofilm formation and PIA synthesis by S. epidermidis 1457. Insertion of Tn917 into rsbU, a positive regulator of alternative sigma factor sigma(B), led to a biofilm-negative phenotype and almost undetectable PIA production. Interestingly, in TSB(EtOH), the mutants were enabled to form a biofilm again with phenotypes similar to those of the wild type. In TSB(NaCl), the mutants still displayed a biofilm-negative phenotype. No difference in primary attachment between the mutants and the wild type was observed. Similar phenotypic changes were observed after transfer of the Tn917 insertion of mutant M15 to the independent and biofilm-producing strain S. epidermidis 8400. In 11 clinical S. epidermidis strains, a restriction fragment length polymorphism of the sigB operon was detected which was independent of the presence of the icaADBC locus and a biofilm-positive phenotype. Obviously, different mechanisms are operative in the regulation of PIA expression in stationary phase and under stress induced by salt or ethanol.     

Monoclonal antibodies to human protein C: effects on the biological activity of activated protein C and the thrombin-catalyzed activation of protein C1

Thirteen monoclonal antibodies designated as MFC-1 to MFC-13 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of mice immunized with purified human protein C. Studies were made to determine where the antibodies bound to the molecule of protein C and whether they affected the biological actions of protein C. By using the immunoblotting technique, six of these antibodies were shown to bind to the light chain of protein C, and five to the heavy chain of protein C and also activated protein C. The remaining two antibodies bound to neither the light chain nor the heavy chain, though both antibodies bound to the intact protein C. Antibodies specific for the light chain did not bind to the gamma-carboxyglutamic acid-domain. Two of the antibodies specific for the heavy chain (MFC-13 and -1) inhibited the amidolytic activity of activated protein C. The MFC-13 also inhibited the activity of bovine activated protein C, but not that of human Factor IXa, Factor Xa, or thrombin. In addition to these two antibodies, another one for the heavy chain (MFC-10) and two antibodies for the light chain (MFC-9 and -11) inhibited the inactivation of Factor Va by human activated protein C. One of the antibodies which inhibited the enzyme activity (MFC-1) blocked the inhibition of activated protein C by protein C inhibitor. Another one for the heavy chain (MFC-5) inhibited the activation of protein C by thrombin regardless of the presence or absence of thrombomodulin. Based on these results, we have established the positions of some monoclonal antibody-binding sites on the protein C molecule.     

Differential effects of monoclonal antibodies anti-L3T4 and anti-LFA1 on the antigen-induced proliferation of T-helper-cell clones: correlation between their susceptibility to inhibition and their affinity for antigen

Recognition by specific T helper (TH) cells of antigen presented by antigen-presenting cells (APC) involves, in addition to the antigen-specific receptor, non-antigen-specific molecules such as L3T4 and LFA1. In the present study, we analyzed the relationship between the avidity for antigen presented by APC of three TH cell lines and the participation of L3T4 and LFA1 cell surface antigens. We found a correlation between the avidity of TH cells for the complex GAT/Ia on APC measured by two independent assays and the participation of the cell-adhesion molecules L3T4 as measured by the ability of corresponding monoclonal antibody (MAb) to block the antigen-induced proliferation of TH cells. In contrast to the situation found with cytolytic T-lymphocyte (CTL) clones, we also found a differential inhibiting effect of anti-LFA1 MAb on the GAT-specific proliferation of the three TH clones. The results indicate a direct correlation between the inhibitory effects of anti-LFA1 and anti-L3T4 MAb and the affinity of TH cells for the complex formed by antigen and Ia.     

Efficient induction of CCR9 on T cells requires coactivation of retinoic acid receptors and retinoid X receptors (RXRs): exaggerated T Cell homing to the intestine by RXR activation with organotins

The active vitamin A metabolite retinoic acid (RA) imprints gut-homing specificity on lymphocytes upon activation by inducing the expression of α4β7 integrin and CCR9. RA receptor (RAR) activation is essential for their expression, whereas retinoid X receptor (RXR) activation is not essential for α4β7 expression. However, it remains unclear whether RXR activation affects the RA-dependent CCR9 expression on T cells and their gut homing. The major physiological RA, all-trans-RA, binds to RAR but not to RXR at physiological concentrations. Cell-surface CCR9 expression was often induced on a limited population of murine naive CD4(+) T cells by all-trans-RA or the RAR agonist Am80 alone upon CD3/CD28-mediated activation in vitro, but it was markedly enhanced by adding the RXR agonist PA024 or the RXR-binding environmental chemicals tributyltin and triphenyltin. Accordingly, CD4(+) T cells treated with the combination of all-trans-RA and tributyltin migrated into the small intestine upon adoptive transfer much more efficiently than did those treated with all-trans-RA alone. Furthermore, naive TCR transgenic CD4(+) T cells transferred into wild-type recipients migrated into the small intestinal lamina propria following i.p. injection of Ag, and the migration was enhanced by i.p. injection of PA024. We also show that PA024 markedly enhanced the all-trans-RA-induced CCR9 expression on naturally occurring naive-like regulatory T cells upon activation, resulting in the expression of high levels of α4β7, CCR9, and Foxp3. These results suggest that RXR activation enhances the RAR-dependent expression of CCR9 on T cells and their homing capacity to the small intestine.     

Susceptibility of human dendritic cells (DCs) to measles virus (MV) depends on their activation stages in conjunction with the level of CDw150: role of Toll stimulators in DC maturation and MV amplification

Human CD46 (membrane cofactor protein, or MCP) and CDw150 (signaling lymphocyte activation molecule, or SLAM) serve as receptors for measles virus (MV), which induces marked host immune suppression. Although monocytes express CD46, they are considerably resistant to MV. Once monocytes differentiate into immature myeloid dendritic cells (iDCs) (GM-CSF + IL-4-treated), the cells become susceptible to MV. Therefore, we have identified CD46-adapted and CDw150-adapted strains of MV, and the dynamics of CD46 and CDw150 during monocyte-iDC conversion were examined in conjunction with MV susceptibility. Strikingly, CDw150 was not detected in monocytes and moderately induced in iDCs, while CD46 was constantly expressed in monocyte-to-iDC differentiation. Thus, iDCs were found to become highly permissive to CDw150-adapted MV strains via expression of CDw150. In fact, polyclonal and monoclonal antibodies that specifically blocked the MV receptor function of CD46 or CDw150 cancelled MV replication in iDCs according to the preferential usage of either CD46 or CDw150 in each strain of MV. Next, we showed that DCs that matured via stimulation of their Toll-like receptors (TLRs) 2 and/or 4 exhibited an approximately fivefold increase in CDw150 at the protein level, and concomitantly, higher levels of MV amplification were observed in mixed culture of lymphocytes than in iDCs without TLR2/4 stimuli. Hence, amplification of CDw150-dependent MV strains was augmented in DCs parallel with the levels of CDw150 in the presence of lymphocytes possessing CDw150. TLR-mediated functional potential of DCs may affect the degree of MV amplification through distinct MV strain-specific receptor usage of CDw150 or CD46.