Highly expressed cytoplasmic FcεRIß in human mast cells functions as a negative regulator of the FcRγ-mediated cell activation signal.

BACKGROUND: We recently reported that the interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human mast cell (MC) activation and that FcεRIß functions as an amplifier of FcεRI-mediated activation signal. Some of FcεRIß in cytoplasm appeared not to be co-localized with FcεRIα. The function of FcεRIß in the cytoplasm remains unknown. METHODS: The localization of FcεRIß and FcεRIα in giant papillae specimens from patients with allergic keratoconjunctivitis and of FcεRIß, FcεRIα, and Lyn in cultured human MCs was examined using confocal microscopy. FcεRIß was overexpressed using an adenovirus vector system. Mediators were measured by enzyme immunoassays or enzyme-linked immunosorbent assays. RESULTS: In the subepithelial region, FcεRIß was mainly localized in the cell membrane of MCs. In the perivascular region, FcεRIß expression was scattered throughout the cytoplasm and in the cell membrane of MCs. Overexpression of FcεRIß in MCs mainly increased its cytoplasmic expression and slightly up-regulated cell surface FcεRI expression. However, overexpression of FcεRIß in MCs resulted in down-regulation of the tyrosine phosphorylation levels of FcεRIß and Syk and down-regulation of the Ca(2+) influx soon after FcεRI aggregation and then resulted in down-regulation of degranulation, PGD2 synthesis, and production of a set of cytokines. This negative regulatory effect may be due to inhibition of the redistribution of Lyn to small patches within the plasma membrane. CONCLUSION: Cytoplasmic FcεRIß, which is not co-localized with FcεRIα, may function as a negative regulator, as it can capture important signalling molecules such as Lyn.

Data Build-up for the Construction of Korean Specific Greenhouse Gas Emission Inventory in Livestock Categories.

Many studies on methane (CH4) and nitrous oxide (N2O) emissions from livestock industries have revealed that livestock production directly contributes to greenhouse gas (GHG) emissions through enteric fermentation and manure management, which causes negative impacts on animal environment sustainability. In the present study, three essential values for GHG emission were measured; i.e., i) maximum CH4 producing capacity at mesophilic temperature (37°C) from anaerobically stored manure in livestock category (B0,KM, Korean livestock manure for B0), ii) EF3(s) value representing an emission factor for direct N2O emissions from manure management system S in the country, kg N2O-N kg N(-1), at mesophilic (37°C) and thermophilic (55°C) temperatures, and iii) Nex(T) emissions showing annual N excretion for livestock category T, kg N animal(-1) yr(-1), from different livestock manure. Static incubation with and without aeration was performed to obtain the N2O and CH4 emissions from each sample, respectively. Chemical compositions of pre- and post-incubated manure were analyzed. Contents of total solids (% TS) and volatile solid (% VS), and the ratio of carbon to nitrogen (C/N) decrease significantly in all the samples by C-containing biogas generation, whereas moisture content (%) and pH increased after incubation. A big difference of total nitrogen content was not observed in pre- and post-incubation during CH4 and N2O emissions. CH4 emissions (g CH4 kg VS(-1)) from all the three manures (sows, layers and Korean cattle) were different and high C/N ratio resulted in high CH4 emission. Similarly, N2O emission was found to be affected by % VS, pH, and temperature. The B0,KM values for sows, layers, and Korean cattle obtained at 37°C are 0.0579, 0.0006, and 0.0828 m(3) CH4 kg VS(-1), respectively, which are much less than the default values in IPCC guideline (GL) except the value from Korean cattle. For sows and Korean cattle, Nex(T) values of 7.67 and 28.19 kg N yr(-1), respectively, are 2.5 fold less than those values in IPCC GL as well. However, Nex(T) value of layers 0.63 kg N yr(-1) is very similar to the default value of 0.6 kg N yr(-1) in IPCC GLs for National greenhouse gas inventories for countries such as South Korea/Asia. The EF3(s) value obtained at 37°C and 55°C were found to be far less than the default value.

Association between atopy and variants of the beta subunit of the high-affinity immunoglobulin E receptor.

The beta-subunit of the high-affinity IgE receptor (Fc epsilon RI-beta) on chromosome 11 is maternally linked to atopy, the state of enhanced IgE responsiveness underlying allergic asthma and rhinitis. We have identified a common variant of Fc epsilon RI-beta, lle181Leu within the 4th transmembrane domain. Leu181 shows significant association with positive IgE responses in a random patient sample. Amongst 60 unrelated nuclear families with allergic asthmatic probands, Leu181 is identified in 10 (17%), is maternally inherited in each, and shows a strong association with atopy. Our data indicate that Fc epsilon RI-beta, subject to maternal modification, may be the atopy-causing locus on chromosome 11q.

Oncology in Cambodia.

Cambodia, a country of 14 million inhabitants, was devastated during the Khmer Rouge period and thereafter. The resources of treatment are rare: only one radiotherapy department, renovated in 2003, with an old cobalt machine; few surgeons trained to operate on cancer patients; no hematology; no facilities to use intensive chemotherapy; no nuclear medicine department and no palliative care unit. Cervical cancer incidence is one of the highest in the world, while in men liver cancer ranks first (20% of all male cancers). Cancers are seen at stage 3 or 4 for 70% of patients. There is no prevention program - only a vaccination program against hepatitis B for newborns - and no screening program for cervical cancer or breast cancer. In 2010, oncology, recognized as a full specialty, was created to train the future oncologists on site at the University of Phnom Penh. A new National Cancer Center will be built in 2013 with modern facilities for radiotherapy, medical oncology, hematology and nuclear medicine. Cooperation with foreign countries, especially France, and international organizations has been established and is ongoing. Progress is occurring slowly due to the shortage of money for Cambodian institutions and the lay public.

The interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human mast cell activation.

BACKGROUND: FcεRIß reportedly functions as an amplifier of the FcεRIγ-mediated activation signal using a reconstitution system. However, the amplification mechanisms in human mast cells (MCs) are poorly understood. We previously reported the hyperexpression of FcεRIß of MCs in giant papillae from vernal keratoconjunctivitis patients, compared with that in conjunctivae from nonallergic conjunctivitis patients. Elucidation of the molecular mechanisms of the amplification induced by FcεRIß should provide new targets for novel therapeutic interventions. The aim is to understand in greater details the function of FcεRIß in human MC FcεRI expression and signaling. METHODS: FcεRIß and Lyn expression was reduced using a lentiviral shRNA silencing technique. Localization of Lyn and FcεRIß in cultured MCs was examined by confocal microscopic analysis. Mediators were measured by ELISAs. RESULTS: The diminution of FcεRIß significantly downregulated cell surface FcεRI expression and FcεRI-mediated mediator release/production. The downregulation of FcεRI-mediated degranulation was not only due to the decrease in FcεRI expression. The diminution of FcεRIß inhibited the redistribution of Lyn within the cell membrane following IgE sensitization. The diminution of Lyn in MCs significantly downregulated FcεRI-mediated degranulation. The recombinant cell-penetrating forms of phosphorylated FcεRIß immunoreceptor tyrosine-based activation motif (ITAM) for intracellular delivery disturbed the interaction between Lyn and phosphorylated endogenous FcεRIß ITAM, resulted in inhibiting IgE-dependent histamine release from MCs in vitro and from giant papillae specimens ex vivo. CONCLUSION: The interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human MC activation, and specific inhibition of the interaction may represent a new therapeutic strategy for the treatment of human allergic diseases.

Indirect Estimation of CH4 from Livestock Feeds through TOCs Evaluation.

Thirty-five available feeds were fermented in vitro in order to investigate their soluble total organic carbon (TOCs) and methane (CH4) production rate. A fermentation reactor was designed to capture the CH4 gas emitted and to collect liquor from the reactor during in vitro fermentation. The results showed that CH4 production rate greatly varied among feeds with different ingredients. The lowest CH4-producing feeds were corn gluten feed, brewer's grain, and orchard grass among the energy, protein, and forage feed groups, respectively. Significant differences (p<0.05) were found in digestibility, soluble total organic carbon (TOCs), and CH4 emissions among feeds, during 48 h of in vitro fermentation. Digestibility and TOCs was not found to be related due to different fermentation pattern of each but TOCs production was directly proportional to CH4 production (y = 0.0076x, r(2) = 0.83). From this in vitro study, TOCs production could be used as an indirect index for estimation of CH4 emission from feed ingredients.

Effects of processing of starter diets on performance, nutrient digestibility, rumen biochemical parameters and body measurements of brown swiss dairy calves.

In order to investigate the effect of physical forms of starter diets on performance, weaning age, nutrient digestibility and rumen biochemical factors, 24 female of neonatal Brown Swiss calves (average body weight of 39.5±1.2 kg) were randomly assigned to three treatments. Dietary treatments were mashed (MS), pelleted (PS), and texturized (TS) starter using 8 calves from birth till 90 days of age in each treatment. Diets were formulated to be iso-nitrogenous with 21% crude protein. Based on the experimental results, calves that received PS and TS diets, had significant higher average daily gain (ADG) than those receiving MS (p<0.01). Dry matter intake in calves fed PS and TS was greater than calves fed MS (p<0.05), but there was no significant difference in feed efficiency. Treatments had no effect on initiation of rumination. Weaning age of calves in MS was longer than the other two treatments (p<0.05). Crude protein and organic matter digestibility in MS treated calves were lower than other treatments (p<0.05). No differences were observed in neutral detergent fiber (NDF) and ash digestibility among treatments (p>0.05). Ruminal pH was higher (p<0.01) in MS than the other groups, but ruminal ammonia (g/dl) concentration was not different among the treatments. Body measurements such as body length, pin width, hip width, pin to hip length, size of metacarpus and metatarsus bones, hip height, wither height, stomach size and heart girth were not significantly different among the treatments. Overall, it is concluded that starter diets in the form of pellet and texture can improve performance in neonatal Brown Swiss calves compared to the mashed form.

Association with FcRgamma is essential for activation signal through NKR-P1 (CD161) in natural killer (NK) cells and NK1.1+ T cells.

Natural killer (NK) cells exhibit cytotoxicity against variety of tumor cells and virus-infected cells without prior sensitization and represent unique lymphocytes involved in primary host defense. NKR-P1 is thought to be one of NK receptors mediating activation signals because cross-linking of NKR-P1 activates NK cells to exhibit cytotoxicity and IFN-gamma production. However, molecular mechanism of NK cell activation via NKR-P1 is not well elucidated. In this study, we analyzed the cell surface complex associated with NKR-P1 on NK cells and found that NKR-P1 associates with the FcRgamma chain which is an essential component of Fc receptors for IgG and IgE. The association between FcRgamma and NKR-P1 is independent of Fc receptor complexes. Furthermore, NK cells from FcRgamma-deficient mice did not show cytotoxicity or IFN-gamma production upon NKR-P1 cross-linking. Similarly, NK1.1+ T cells from FcRgamma-deficient mice did not produce IFN-gamma upon NKR-P1 crosslinking. These findings demonstrate that the FcRgamma chain plays an important role in activation of NK cells via the NKR-P1 molecule.

Blockade of transforming growth factor beta/Smad signaling in T cells by overexpression of Smad7 enhances antigen-induced airway inflammation and airway reactivity.

Transforming growth factor (TGF)-beta has been implicated in immunosuppression. However, it remains obscure whether regulation of T cells by TGF-beta contributes to the immunosuppression in vivo. To address this issue, we developed transgenic mice expressing Smad7, an intracellular antagonist of TGF-beta/Smad signaling, selectively in mature T cells using a plasmid construct coding a promoter element (the distal lck promoter) that directs high expression in peripheral T cells. Peripheral T cells were not growth inhibited by TGF-beta in Smad7 transgenic mice. Although Smad7 transgenic mice did not spontaneously show a specific phenotype, antigen-induced airway inflammation and airway reactivity were enhanced in Smad7 transgenic mice associated with high production of both T helper cell type 1 (Th1) and Th2 cytokines. Thus, blockade of TGF-beta/Smad signaling in mature T cells by expression of Smad7 enhanced airway inflammation and airway reactivity, suggesting that regulation of T cells by TGF-beta was crucial for negative regulation of the inflammatory (immune) response. Our findings also implicated TGF-beta/Smad signaling in mature T cells as a regulatory component of allergic asthma.

FcepsilonRI-mediated thymic stromal lymphopoietin production by interleukin-4-primed human mast cells.

A significant increase of mRNA expression of thymic stromal lymphopoietin (TSLP) has been reported in the bronchial mast cells (MCs) of asthmatic subjects; however, the mechanism underlying the upregulation of TSLP mRNA and protein remains unknown. FcepsilonRI-mediated activation of human MCs upregulated TSLP mRNA expression by 5.2+/-2.9-fold, while activation of the MCs using lipopolysaccharide and polyriboinosinic:polyribocytidylic acid failed to upregulate TSLP. Stimulation of MCs with interleukin (IL)-4 alone did not affect the TSLP mRNA expression, while pre-incubation of MCs with IL-4 for 48 h significantly enhanced the FcepsilonRI-mediated TSLP mRNA expression (by 53.7+/-15.9-fold; p<0.05) and the amount of TSLP in the cell pellets increased significantly from 23.4+/-4.3 pg mL(-1) to 121.5+/-3.7 pg mL(-1) (p<0.0001). However, the released TSLP was rapidly degraded by proteases that were released by MCs. We identified the population of cells expressing TSLP in the lungs of 16 asthmatic and 11 control subjects by immunohistochemistry. The percentage of TSLP-positive MCs in the total population of MCs was significantly increased in asthmatic airways (p<0.0001). Thus, MCs are able to store TSLP intracellularly and to produce TSLP following aggregation of FcepsilonRI in the presence of IL-4.

Activation of basophils by stem cell factor: comparison with insulin-like growth factor-I.

BACKGROUND: Basophils are an active participant in the pathogenesis of local inflammation in allergic diseases such as asthma, but it is not fully known how basophil activation is regulated in inflamed tissue. OBJECTIVE: In order to clarify the control mechanisms of basophil activation in chronic inflammation and at remodeling sites, we analyzed the effects of fibroblast-derived cytokines, stem cell factor (SCF), and insulin-like growth factor-I (IGF-I) on basophils. METHODS: The effects of SCF and IGF-I on degranulation and surface activation marker expression by basophils were assessed and compared. RESULTS: SCF enhanced human basophil histamine release elicited by some, but not all, secretagogues; degranulation in response to IgE- or FcepsilonRI-mediated stimulation and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) was enhanced by SCF. SCF slightly enhanced ionophore A23187-induced histamine release by basophils from some donors, but it failed to affect the release elicited by monocyte chemoattractant protein-1 (MCP-1), formylmethionyl-leucyl-phenylalanine (FMLP) or C5a. The repertoire of secretagogues responsive to SCF was similar to that of IGF-I. Expression levels of both CD11b and CD69 markers were significantly enhanced by the combination of SCF and IGF-I. CONCLUSIONS: These results suggest that SCF and IGF-I may modify the activation of basophils in a similar and/or synergistic fashion. Interaction of basophils with these cytokines might be involved in the pathogenesis of local inflammation and the remodeling process in asthma.

Nasal mast cells in perennial allergic rhinitics exhibit increased expression of the Fc epsilonRI, CD40L, IL-4, and IL-13, and can induce IgE synthesis in B cells.

Cross-linking of allergen specific IgE bound to the high affinity IgE receptor (FC epsilonRI) on the surface of mast cells with multivalent allergens results in the release of both pre-formed and newly generated mediators, and in the manifestation of allergic symptoms. The expression of Fc epsilonRI, and the synthesis of IgE are therefore critical for the development of allergic diseases. In this study, we report that nasal mast cells (NMC) from patients with perennial allergic rhinitis (PAR) expressed significantly greater levels of the Fc epsilonRI, CD40L, IL-4, and IL-13 as compared to NMC from patients with chronic infective rhinitis (CIR). The level of Fc epsilonRI expression in NMC of PAR patients strongly correlated with the levels of serum total (r = 0.8, P < 0.003) and specific IgE (r = 0.89, P < 0.0004) antibodies. In addition, stimulation of NMC with IL-4, upregulated the Fc epsilonRIalpha chain expression both at the protein and mRNA levels, as detected by flow cytometry and reverse transcriptase-polymerase chain reaction. Furthermore, NMC from PAR, but not CIR, patients induced IgE synthesis by purified B cells in the presence of Der fII (mite antigen). These results suggest novel and critical roles for mast cells in promoting the allergic reaction through the increased expression of Fc epsilonRI and by enhancing and amplifying the IgE production, within the local microenvironment.

Recombinant soluble form of the human high-affinity immunoglobulin E (IgE) receptor inhibits IgE production through its specific binding to IgE-bearing B cells.

A recombinant soluble form of the alpha subunit of the human high-affinity receptor for IgE (rsFc epsilon RI alpha), one of the potent IgE-binding molecules, was tested for its ability to regulate IL-4-induced IgE synthesis by human lymphocytes. Addition of rsFc epsilon RI alpha to cultures induced a dose-dependent inhibition of the T cell-dependent and independent synthesis of IgE. The suppression of IgE synthesis was observed at the protein and the mRNA levels, and it was IgE class specific. By flow cytometry, specific binding of rsFc epsilon RI alpha was detected on surface IgE-bearing B cells as well as on U266 cells, and it was completely blocked by preincubation with IgE. rsFc epsilon RI alpha bound to the cell surface IgE could be effectively dissociated not only by a large excess of IgE, but also by an anti-rsFc epsilon RI alpha mAb that competes with IgE for the binding to rsFc epsilon RI alpha. This mAb abolished the rsFc epsilon RI alpha-mediated suppression of IgE synthesis. These data suggest that rsFc epsilon RI alpha may have a function in selectively suppressing IgE synthesis through its interaction with the membrane-bound form of IgE.

Resistance of Fc receptor- deficient mice to fatal glomerulonephritis.

Immune complex-mediated inflammation is a common mechanism of various autoimmune diseases. Glomerulonephritis (GN) is one of these diseases, and the main mechanism of the induction of GN has been unclear. We examined the contribution of Fc receptors in the induction of nephrotoxic GN by establishing and analyzing mice deficient in the Fc receptor gamma chain (FcRgamma). Whereas all wild-type mice died from severe glomerulonephritis with hypernitremia by administration of anti-glomerular basement membrane (GBM) antibodies, all FcRgamma-deficient mice survived. Histologically, wild-type mice showed glomerular hypercellularity and thrombotic changes, whereas the renal tissue in FcRgamma-deficient mice was almost intact. Deposition of anti-GBM antibody as well as complement components in the GBM were equally observed in both wild-type and knockout mice. These results demonstrate that the triggering of this type of glomerulonephritis is completely dependent on FcR+ cells.

Sequential requirements of the N-terminal palmitoylation site and SH2 domain of Src family kinases in the initiation and progression of FcepsilonRI signaling.

Initial biochemical signaling originating from high-affinity immunoglobulin E receptor (FcepsilonRI) has been ascribed to Src family kinases. To understand the mechanisms by which individual kinases drive the signaling, we conducted reconstitution experiments: FcepsilonRI signaling in RBL2H3 cells was first suppressed by a membrane-anchored, gain-of-function C-terminal Src kinase and then reconstructed with Src family kinases whose C-terminal negative regulatory sequence was replaced with a c-myc epitope. Those constructs derived from Lyn and Fyn, which are associated with detergent-resistant membranes (DRMs), physically interacted with resting FcepsilonRI and reconstructed clustering-induced signaling that leads to calcium mobilization and ERK1 and -2 activation. c-Src-derived construct, which was excluded from DRMs, failed to interact with FcepsilonRI and to restore the signaling, whereas creation of palmitoylatable Cys3 enabled it to interact with DRMs and with FcepsilonRI and to restore the signaling. Deletion of Src homology 3 (SH3) domain from the Lyn-derived construct did not alter its ability to transduce the series of signaling. Deletion of SH2 domain did not affect its association with DRMs and with FcepsilonRI nor clustering-induced tyrosine phosphorylation of FcepsilonRI beta and gamma subunits, but it almost abrogated the next step of tyrosine phosphorylation of Syk and its recruitment to FcepsilonRI. These findings suggest that Lyn and Fyn could, but c-Src could not, drive FcepsilonRI signaling and that N-terminal palmitoylation and SH2 domain are required in sequence for the initial interaction with FcepsilonRI and for the signal progression to the molecular assembly.

The Fc receptor (FcR) gamma subunit is essential for IgE-binding activity of cell-surface expressed chimeric receptor molecules constructed from human high-affinity IgE receptor (Fc epsilon RI) alpha and FcR gamma subunits.

The Fc receptor (FcR) gamma subunit was originally discovered as a homodimeric subunit of the high-affinity IgE receptor (FcepsilonRI). But it was recently found to be a common signal-generating subunit of Fc receptors including IgG Fc receptors (FcgammaRs) and IgA Fc receptor (FcalphaR), and furthermore to generate a signal also with stimuli through non-immune receptors. In addition, it plays an essential role in cell-surface expression of the FcepsilonRI and the FcgammaRIIIA isoform and also regulates cell-surface expression and ligand-binding affinity of the FcgammaRI. In this report, we addressed the possibility that the FcRgamma could affect the correct folding of the IgE-binding region of the FcepsilonRIalpha subunit by using the chimeric receptor molecules constructed from human FcepsilonRIalpha and FcRgamma. Furthermore, we demonstrated that the seven amino acid residues in the C-terminal region on the extracellular domain of the FcepsilonRlalpha were essential for maintaining the IgE-binding activity of the FcepsilonRIalpha exodomain on the cell membrane and/or may affect the correct folding of the alpha subunit itself within the cell.

Edge contacts of graphene formed by using a controlled plasma treatment.

Despite the fact that the outstanding properties of graphene are well known, the electrical performance of the material is limited by the contact resistance at the metal-graphene interface. In this study, we demonstrate the formation of "edge-contacted" graphene through the use of a controlled plasma processing technique that generates a bond between the graphene edge and the contact metal. This technique controls the edge structure of the bond and significantly reduces the contact resistance. This simple approach requires no additional post-processing and has been proven to be very effective. In addition, controlled pre-plasma processing was applied in order to produce CVD-graphene field effect transistors with an enhanced adhesion and improved carrier mobility. The contact resistance attained by using pre-plasma processing was 270 Ω µm, which is a decrease of 77%.

Differential responsiveness of Langerhans cell subsets of varying phenotypic states in normal human epidermis.

Epidermal Langerhans cell heterogeneity is poorly understood with regard to phenotypic characteristics, such as the expression of human leukocyte antigen (HLA)-DR, integrin, and Fc receptor molecules, as well as functional characteristics, such as the ability to process and present antigens or produce cytokines during various phases of immigration and maturation. Technical limitations of Langerhans cell number have limited functional assays on putative Langerhans cell subsets in in vivo epidermis. Therefore, we used flow cytometry for simultaneous phenotypic and functional assessment at the single-cell level within the Langerhans cell population. Freshly isolated human epidermal cell suspensions were stained with a battery of monoclonal antibodies, including anti-HLA-DR, -CD1a, -CD1c, -CD11c, -Fc gamma RII, and -Fc epsilon RI. Two distinct Langerhans cell subsets were identified by their different levels of HLA-DR expression. The DRHi subset expressed higher amounts of CD11c and exhibited greater cytoplasmic complexity and higher baseline calcium than the DRLo subset (p < or = 0.03 for each). Some subjects also expressed high levels of Fc epsilon RI in the DRHi, CD11cHi subset. To determine whether these phenotypic subsets may exhibit differential signal-transduction functional properties, Langerhans cells were partially enriched over Ficoll-Hypaque and their cytosolic mobilization after the addition of ionomycin was analyzed using the calcium indicator, indo-1, in conjunction with quantitative analysis of HLA-DR expression. By this real-time flow cytometric analysis, a new subpopulation was revealed within the DRLo Langerhans cell subset. This subset increased its cytosolic calcium concentration much more than the other two subsets (change in indo-1 blue:violet emission ratio of 37.33 +/- 2.34 in the Hi Flux DRLo subset versus 13.23 +/- 0.29 in the Lo Flux DRLo subset, and versus 7.6 +/- 2.99 in the Lo Flux DRHi subset). These data indicate that functional, as well as phenotypic, subsets of Langerhans cells exist within normal human epidermis. Their responses to physiologic stimuli may relate to maturational stage or the level of in vivo activation.

Lack of cognate help by CD4+ T cells and anergy of CD8+ T cells are the principal mechanisms for anti-leukocyte function-associated antigen-1/intercellular adhesion molecule-1-induced cardiac allograft tolerance.

Combined treatment with anti-leukocyte function-associated antigen-1 (LFA-1) and anti-intercellular adhesion molecule-1 (ICAM-1) monoclonal antibodies leads to allograft tolerance in murine cardiac transplantation. In the present study, we analyzed the mechanisms for this tolerance induction. In the tolerant mice, proliferative response of splenic T cells against donor-type cardiac myocytes and of CD8+ T cells against donor-type alloantigens was impaired as compared with responses in naive or rejected mice, but was completely restored with exogenous interleukin 2. This suggests that class I-restricted CD8+ T cells of tolerant mice were rendered anergic against donor-type alloantigens in the periphery. In contrast, proliferative response of CD4+ T cells against donor-type alloantigens in vitro was comparable between tolerant and naive mice. When heart and skin grafts from the same donor (BALB/c [H2d]) were simultaneously transplanted to C3H mice (H2k), both were rejected within 29 days, even though the mice were similarly treated with anti-LFA-1 and anti-ICAM-1 monoclonal antibodies. In contrast, when heart graft from BALB/c and skin graft from third-party donor (C57BL/6 [H2b]) were simultaneously transplanted to C3H mice under the same condition, the heart graft was accepted indefinitely and the skin graft was rejected. These findings suggest that the peripheral tolerance against cardiac allografts could be induced by selective inactivation of alloreactive CD8+ T cells resulting from the lack of cognate help by CD4+ T cells.

Distinct aggregation of beta- and gamma-chains of the high-affinity IgE receptor on cross-linking.

The high-affinity IgE receptor (FcepsilonRI) on mast cells and basophils consists of a ligand-binding alpha-chain and two kinds of signaling chains, a beta-chain and disulfide-linked homodimeric gamma-chains. Crosslinking by multivalent antigen results in the aggregation of the bound IgE/alpha-chain complexes at the cell surface, triggering cell activation, and subsequent internalization through coated pits. However, the precise topographical alterations of the signaling beta- and gamma-chains during stimulation remain unclarified despite their importance in ligand binding/signaling coupling. Here we describe the dynamics of FcepsilonRI subunit distribution in rat basophilic leukemia cells during stimulation as revealed by immunofluorescence and immunogold electron microscopy. Immunolocalization of beta- and gamma-chains was homogeneously distributed on the cell surfaces before stimulation, while crosslinking with multivalent antigen, which elicited optimal degranulation, caused a distinct aggregation of these signaling chains on the cell membrane. Moreover, only gamma- but not beta-chains were aggregated during the stimulation that evoked suboptimal secretion. These findings suggest that high-affinity IgE receptor beta- and gamma-chains do not co-aggregate but for the most part form homogenous aggregates of beta-chains or gamma-chains after crosslinking.