Search

The high affinity IgE receptor, FcepsilonRI, is a key regulatory molecule in the allergic reaction. By screening for cis-acting elements over the entire region of the human FcepsilonRI beta-chain gene, a sequence located in the fourth intron was revealed to serve as a repressor element. This element was recognized by a transcription factor, myeloid zinc finger protein 1 (MZF-1). Introduction of MZF-1 antisense inhibited the suppressive effect of the element on the beta-chain promoter and increased the mRNA for the beta-chain in KU812 cells, indicating that MZF-1 repressed human FcepsilonRI beta-chain gene expression via the element in the fourth intron. Furthermore, it was suggested that a cofactor binding with MZF-1, whose expression level was different among the cell types, was required for transcriptional repression by MZF-1.

We found a novel polymorphism, -66T/C, in the promoter region of human FcepsilonRIalpha, the specific component of the high affinity receptor for IgE (FcepsilonRI), which is essential for the cell surface expression of FcepsilonRI and the binding of IgE Ab. When the effect of the single nucleotide replacement on the promoter function was analyzed, the transcription activity of the T allele promoter was found to be higher than that of the C allele promoter, and was markedly up-regulated by the overexpression of GATA-1 when compared with the C allele promoter. This is probably because the promoter with T at -66 has an additional GATA-1-binding motif in the region, which may assure higher affinity of the transcription factor to the promoter. In accordance with this, EMSA actually indicated that GATA-1 bound to the T allele probe (-80/-59) with the affinity higher than that to the C allele probe. Statistical analysis suggested that a significant portion of nonallergic individuals has heterozygous -66T/C genotype, while most of allergic individuals have homozygous -66T/T genotype in Japanese population. Our findings for the first time demonstrate the presence of FcepsilonRIalpha polymorphism related to the allergic diseases.

The beta chain, a component of the high-affinity receptor for IgE (FcepsilonRI), plays an important role in IgE-mediated allergic reaction. The beta chain accelerates the function of FcepsilonRI by amplification of its surface expression and of signal transduction in effector cells such as mast cells and basophils. Two regulatory regions, +60/+66 and +70/+76, for the human beta chain gene that are required for the cell-type-specific transcriptional activation were identified by transient reporter assay. Electrophoretic mobility shift assay demonstrated that Oct-1 binds both the regions, among which the +70/+76 Oct-1 motif was critical for the transactivation of the beta promoter responsive to Oct-1 overexpression. Regulation of beta chain gene expression is discussed.

MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST-SLAP-130-SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells., (Copyright 2003 Federation of European Biochemical Societies)

The alpha-chain of the high affinity receptor for IgE (Fc epsilon RI) is essential for cell surface expression of Fc epsilon RI and binding of the IgE Ab. The human alpha-chain gene possesses two promoters: the proximal promoter, which is highly conserved with that of rodent; and the distal promoter, the structure and role of which are largely unknown. Transcriptional regulation of the alpha-chain distal promoter was investigated in this study. Transient reporter assay revealed critical region for transcription activity located within -27/-17. EMSA identified Elf-1, YY1, and PU.1 as transcription factors binding to this region. In contrast to the proximal promoter, which was trans-activated by YY1 and PU.1, these transcription factors exhibited repressive function on this promoter. Addition of IL-4 caused a marked increase in transcription from the distal promoter and subsequently increased the intracellular production of the alpha-chain. These results indicate that IL-4-dependent up-regulation of the human alpha-chain was due to enhancement of distal promoter activity and suggests that the two promoters have different regulatory mechanisms for alpha-chain expression.

In immune complex (IC) diseases, FcR are essential molecules facilitating polymorphonuclear cell (PMN) recruitment and effector functions at the IC site. Although FcR-dependent initial tethering and FcR/integrin-dependent PMN accumulation were postulated, their underlying mechanisms remain unclear. We here addressed potential mechanisms involved in PMN recruitment in acute IC glomerulonephritis (nephrotoxic nephritis). Since some renal cells may be recruited from bone marrow (BM) lineages, reconstitution studies with BM chimeras and PMN transfer between wild-type (WT) and FcR-deficient mice (gamma(-/-)) were performed. Severe glomerular damage was induced in WT and W gamma chimeras (BM from WT to irradiated gamma(-/-)), while it was absent in gamma(-/-) and gamma W chimeras (gamma(-/-) BM to WT). Moreover, WT PMN transfer, but not gamma(-/-) PMN, reconstituted the disease in gamma(-/-), indicating that FcR on resident cells is not a prerequisite for PMN recruitment in this disease. Surprisingly, transferred WT PMN were recruited coincidentally with NF-kappa B activation and TNF-alpha overexpression even in glomeruli with preformed IC (nephrotoxic Ab administered 3 days previously), suggesting that PMN can initially be recruited via its own FcR without previous chemoattractant release. Furthermore, H(2)O(2) inhibition by catalase attenuated the acute WT PMN recruitment and the induction of NF-kappa B and TNF-alpha much more than integrin (CD18) blockade, indicating a role for the respiratory burst before integrin-dependent accumulation. In coculture experiments with IC-stimulated PMN and glomeruli, PMN caused acute glomerular TNF-alpha expression predominantly via FcR-mediated H(2)O(2) production. In conclusion, glomerular IC, even preformed, can cause PMN recruitment and injury through PMN FcR-mediated respiratory burst during initial PMN tethering to IC.

IL-3, IL-5, and GM-CSF exert various overlapping functions in basophils. We investigated the receptor expression profiles and concentration-dependent effects of IL-3, IL-5, and GM-CSF on several basophil functions in comparison with their effects on eosinophils. The order of the receptor expression levels was IL-3Ralpha>IL-5Ralpha>GM-CSFRalpha in basophils and IL-5Ralpha>or=GM-CSFRalpha>IL-3Ralpha in eosinophils. Compared with eosinophils, basophils expressed a much higher level of IL-3Ralpha and similar levels of IL-5Ralpha and GM-CSFRalpha. The order of potency was IL-3>IL-5=GM-CSF for degranulation, survival, and CD11b expression in basophils, and IL-5=GM-CSF>or=IL-3 for survival and CD11b expression in eosinophils. However, IL-3 induced CD69 expression preferentially in basophils. Our results indicate that IL-3 is the most potent activator of human basophils, and that the rank order of potency of hemopoietic growth factors virtually corresponded to their receptor expression levels in both cell types.

YY1 is a ubiquitously expressed multifunctional transcription factor that is involved in both positive and negative regulation of gene expression as well as initiation of transcription. Here, we isolated cDNA encoding a full-length open reading frame (ORF) of rat YY1. Rat YY1 is composed of 411 amino acid residues and its amino acid sequence is 97.6% identical to that of mouse YY1 and 97.8% identical to that of human YY1. The transactivating abilities of wild-type rat YY1 and four truncated mutant forms of YY1 were examined by transient reporter assays. When residues 114-193, which sequence includes a portion of the activation region and most of the Gly/Lys-rich region, were lacking, transactivation activity decreased somewhat, but the further deletion in the activation region (of residues 56-113) did not cause further decrease of the activity. On the other hand, N-terminus of the activation region (1-78/100-106) did not have transactivation activity by itself as well as synergistic activity with an erythroid specific transcription factor GATA-1.

Purpose: The inhibitory activity of transforming growth factor-beta2 (TGF-beta2) on corneal endothelial cell proliferation is thought to be a cause of the limited regenerative capacity of corneal endothelial cells that may be related to impaired corneal transparency when many corneal endothelial cells are lost due to various stresses. We determined whether Smad7, an intracellular antagonist of TGF-beta signaling, regulated the inhibitory activity of TGF-beta2 or aqueous humor on corneal endothelial cell proliferation., Methods: The effect of Smad7 on TGF-beta2- or aqueous humor-mediated inhibition of corneal endothelial cell proliferation was evaluated using thymidine uptake assay with cultured rabbit corneal endothelial cells infected with adenovirus carrying Smad7. Expression of Smad or cell cycle-related proteins was detected by immunoblotting. In addition, a small scrape wound was made across a monolayer of Smad7-expressing cultured rabbit corneal endothelial cells to examine the effect of Smad7 on the wound-healing process in vitro., Results: Overexpression of Smad7 abolished the inhibitory effect of TGF-beta2 or aqueous humor on the proliferation of cultured rabbit corneal endothelial cells associated with the inhibition of phosphorylation of Smad2 and downregulation of p27kip1. Smad7-overexpressing cultured rabbit corneal endothelial cells exhibited shorter wound closure time in the presence of aqueous humor than LacZ-expressing cells., Conclusion: Overexpression of Smad7 suppressed the inhibitory effect of TGF-beta2 or aqueous humor on corneal endothelial cell proliferation and accelerated corneal endothelial wound closure in vitro. Modification of Smad7 expression in corneal endothelial cells may thus have applicability in the treatment of wounded corneal endothelium.

Fc alpha R (CD89), the FcR for IgA, is expressed exclusively in myeloid cells, including monocytes/macrophages, neutrophils, and eosinophils, and is thought to mediate IgA-triggered cellular functions in immunity. Here we demonstrate that the Fc alpha R 5'-flanking region from -102 to -64 relative to the ATG translation initiation codon is essential for promoter activity and contains two functional binding motifs for C/EBP and Ets family members at -74 and -92, respectively. EMSAs and cotransfection experiments show that C/EBP alpha acts as a major activator of the Fc alpha R promoter at least in immature myeloid cells. In addition, we found two additional functional targets of C/EBP alpha at -139 and -127. On the other hand, the Fc alpha R Ets binding motif could bind Elf-1 and mediate the trans-activation by cotransfected Elf-1, but a major component of the complex forming on this site appears to be an unidentified Ets-like nuclear protein that is preferentially detected in cells of hemopoietic origin. Furthermore, separation of the C/EBP and Ets binding sites reduces Fc alpha R promoter activity, suggesting some functional interaction between these factors. As the in vivo role of Fc alpha R is still incompletely defined, these findings reveal the features controlling the Fc alpha R promoter in myeloid lineage and provide a foundation for clarifying regulatory mechanisms of Fc alpha R gene expression associated with its potential roles.

More than 10 splice variants of the Fc receptor for IgA (Fc alpha R, CD89) have been identified in human myeloid cells. In this study, we quantified Fc alpha R splice transcripts Delta EC2 and Delta 66 EC2, which lack the entire and a part of the homologous immunoglobulin-like extracellular domain 2 (EC2), respectively. Tumor necrosis factor-alpha was found to specifically increase the ratio of Delta EC2 to the wild type CD89 in neutrophils and conversely decrease the Delta EC2 ratio in monocytes. We also observed a significant decrease in the neutrophil Delta EC2/CD89 ratio in pneumonia patients. These results suggest that Delta EC2 is differentially regulated and could be involved in immunoregulation of IgA-mediated host defense.

Background: Although the ability of basophils to release mediators, called releasability, may be an important aspect which influences the proinflammatory role of these cells, clinical approaches aiming at the depletion of the releasability have not been established. We examined whether the desensitization procedure in Ca(2+)-containing physiological conditions can make basophils completely unresponsive to IgE-mediated stimulation, and whether basophil desensitization is affected by the surface IgE levels., Methods: Human peripheral blood basophils were cultured with low concentrations of anti-IgE antibody or recombinant mite allergen. Following culture, cells were stimulated and their histamine release was measured., Results: Culturing with mite allergen or anti-IgE antibody below threshold concentrations induced potent desensitization in basophils. The desensitizing effect of anti-IgE was dose- and time-dependent; IgE-dependent releasability was completely suppressed when basophils were incubated with a near-threshold concentration of anti-IgE for > or= 4 h. In the continuous presence of subthreshold doses of anti-IgE, basophils remained desensitized even after 3 days. Basophils which had undergone an increase in surface IgE levels after 24-hour culture with IgE demonstrated enhanced desensitization., Conclusions: Near-threshold stimulation in physiological medium can affect basophils, thereby inducing complete and sustained deprivation of releasability without triggering degranulation. Basophil desensitization is regulated by their surface IgE levels. Induction of full desensitization may represent a potentially important therapeutic strategy for IgE-mediated allergic diseases in which basophils play pathogenic roles., (Copyright 2003 S. Karger AG, Basel)

The FcR beta-chain, a subunit of two related multisubunit receptor complexes, the FcepsilonRI and FcgammaRIII, amplifies the mast cell response and is necessary for the cell surface expression of FcepsilonRI in mouse. The transient reporter assay indicated that -69/+4 region is required for cell type-specific transcriptional regulation of mouse beta-chain gene. EMSA using Abs against transcription factors or competitive oligonucleotides demonstrated that -58/-40 region (containing overlapping three GATA-1 sites, -53/-48, -46/-51, and -42/-47) and -31/-26 region (containing one GATA-1 site) are recognized by GATA-1. The promoter activity of beta-chain was decreased by nucleotide replacements of the GATA-1 sites in mouse mast cell line PT18. Furthermore, exogenously produced GATA-1 up-regulated the promoter activity in CV-1 cells, which are negative in the beta-chain production and the up-regulation was apparently suppressed by GATA-1 site mutations. These results indicate that cell type-specific transcription of mouse beta-chain gene is regulated by GATA-1.

Lesional skin of patients with atopic dermatitis (AD) is histologically characterized by hypertrophy of the skin, and the infiltration of a large number of eosinophils and T cells into the dermis. Recent studies have indicated that Th2 cells play a crucial role in the pathogenesis of AD skin. Chemokines and their receptors are implicated in the development of symptoms of various skin diseases such as AD and psoriasis vulgaris (psoriasis). We have examined the in situ expression of a typical Th2-type chemokine, thymus- and activation-regulated chemokine (TARC), and its receptor (CCR4) using immunohistochemical techniques. TARC was found to be highly expressed in the basal epidermis of the lesional skin of AD patients and only slightly in the non-lesional skin. On the other hand, no positive cells were seen in the lesional skin of psoriasis. Consistently, CCR4+ cells were present predominantly in the lesional skin of AD patients, but not in the non-lesional skin. In contrast, in the lesional skin of psoriasis patients, cells positive for CCR5, which is expressed on Th1 cells, were abundantly present. Interestingly, psoralen plus ultraviolet A therapy reduced the number of CCR4+ cells in the AD skin lesions. These results suggest that Th2-type cytokines such as TARC are involved in the pathogenesis of skin lesions in AD patients through the preferential recruitment of Th2 cells.

FcR provides a critical link between ligands and effector cells in immune complex diseases. Emerging evidence reveals that angiotensin (Ang)II exerts a wide variety of cellular effects and contributes to the pathogenesis of inflammatory diseases. In anti-glomerular basement membrane Ab-induced glomerulonephritis (GN), we have previously noted that FcR-deficient mice (gamma(-/-)) surviving from lethal initial damage still developed mesangial proliferative GN, which was drastically prevented by an AngII type 1 receptor (AT1) blocker. We further examined the mechanisms by which renin-Ang system (RAS) participates in this immune disease. Using bone marrow chimeras between gamma(-/-) and AT1(-/-) mice, we found that glomerular injury in gamma(-/-) mice was associated with CD4(+) T cell infiltration depending on renal AT1-stimulation. Based on findings in cutaneous delayed-type hypersensitivity, we showed that AngII-activated renal resident cells are responsible for the recruitment of effector T cells. We next examined the chemotactic activity of AngII-stimulated mesangial cells, as potential mechanisms coupling RAS and cellular immunity. Chemotactic activity for T cells and Th1-associated chemokine (IFN-gamma-inducible protein-10 and macrophage-inflammatory protein 1alpha) expression was markedly reduced in mesangial cells from AT1(-/-) mice. Moreover, this activity was mainly through calcineurin-dependent NF-AT. Although IFN-gamma-inducible protein-10 was NF-kappaB-dependent, macrophage-inflammatory protein 1alpha was dominantly regulated by NF-AT. Furthermore, AT1-dependent NF-AT activation was observed in injured glomeruli by Southwestern histochemistry. In conclusion, our data indicate that local RAS activation, partly via the local NF-AT pathway, enhances the susceptibility to T cell-mediated injury in anti-glomerular basement membrane Ab-induced GN. This novel mechanism affords a rationale for the use of drugs interfering with RAS in immune renal diseases.

FcgammaRIIB1 molecules serve as negative feedback regulator for B cell Ag receptor-elicited activation of B cells; thus, any impaired FcgammaRIIB1 function may possibly be related to aberrant B cell activation. We earlier found deletion polymorphism in the Fcgr2b promoter region among mouse strains in which systemic autoimmune disease-prone NZB, BXSB, MRL, and autoimmune diabetes-prone nonobese diabetic, but not NZW, BALB/c, and C57BL/6 mice have two identical deletion sites, consisting of 13 and 3 nucleotides. In this study, we established congenic C57BL/6 mice for NZB-type Fcgr2b allele and found that NZB-type allele down-regulates FcgammaRIIB1 expression levels in germinal center B cells and up-regulates IgG Ab responses. We did luciferase reporter assays to determine whether NZB-type deletion polymorphism affects transcriptional regulation of Fcgr2b gene. Although NZW- and BALB/c-derived segments from position -302 to +585 of Fcgr2b upstream region produced significant levels of luciferase activities, only a limited activity was detected in the NZB-derived sequence. EMSA and Southwestern analysis revealed that defect in transcription activity in the NZB-derived segment is likely due to absence of transactivation by AP-4, which binds to the polymorphic 13 nucleotide deletion site. Our data imply that because of the deficient AP-4 binding, the NZB-type Fcgr2b allele polymorphism results in up-regulation of IgG Ab responses through down-regulation of FcgammaRIIB1 expression levels in germinal center B cells, and that such polymorphism may possibly form the basis of autoimmune susceptibility in combination with other background contributing genes.

We previously showed that silver stimulates degranulation and leukotriene (LT) C(4) production in rat basophilic leukemia mast cells and now show that silver induces these events by a mechanism that differs from the FcepsilonRI-mediated response. In common with FcepsilonRI cross-linking, silver induced tyrosine phosphorylation of extracellular signal-regulated kinases and furthermore, PD98059, a specific inhibitor of extracellular signal-regulated kinase kinase dose-dependently inhibited the silver-induced LTC(4) production. In contrast to FcepsilonRI cross-linking, silver had no effect on the production of IL-4 and TNF-alpha, indicating that different mechanisms are involved in the activation by these two stimuli. In line with this, silver had no or only marginal effect on the tyrosine phosphorylation of FcepsilonRIbeta, Lyn, Syk, and linker for activation of T cells, the early and crucial events in FcepsilonRI signaling. Silver induced calcium signals that were involved in the metal-induced degranulation, but not LTC(4) production. Unlike Ag, the silver-induced calcium signals were resistant to the depletion of thapsigargin-sensitive calcium stores and the inhibition of tyrosine kinases and phospholipase Cgamma. These findings indicate that silver activates mast cells by bypassing the early signaling events required for the induction of calcium influx. Our data strongly suggest the existence of an alternative pathway bypassing the early signaling events in mast cell activation and indicate that silver may be useful for analyses of such alternative mechanisms.

Transcriptional regulation of the gene encoding human high affinity IgE receptor (Fc epsilon RI) alpha-chain was analyzed. Previously, we reported that GATA-1 and Elf-1 recognition sites were necessary for cell type-specific activation of the alpha-chain gene promoter. More detailed analysis revealed that other transcription factors bound the regions close to the Elf-1 recognition site and there was a more complex mechanism for the regulation of the promoter activity. On the other hand, during a course of studies to find cis-elements over this gene, CAGCTG sequence in the first intron was revealed to serve as an enhancer. A complex composed of USF1 and USF2 activated the human alpha-chain gene expression via this intronic element. Furthermore, we found two novel exons at 18.4 and 12.6kb upstream from the reported first exon and discovered an additional distal promoter.

Background: Transforming growth factor beta (TGF-beta) has been suggested to play an important role in the development of airway remodeling in asthma; this suggestion is based on evidence that expression levels of TGF-beta are correlated with unique parameters of airway remodeling, such as thickness of basement membrane. However, the relevant studies were inconclusive because they were unable to demonstrate active signaling mediated by the cytokine in the airways of asthmatic individuals., Objective: We sought to determine whether TGF-beta signaling was active in the airways of asthmatic subjects and, if so, whether it was correlated with clinicopathologic features associated with the development of airway remodeling in asthma., Methods: We examined the phosphorylation status of Smad2 in bronchial biopsy samples obtained from 40 asthmatic subjects as a marker of active TGF-beta signaling, and we studied its correlation with basement membrane thickness., Results: Expression levels of phosphorylated Smad2 in bronchial biopsy specimens from asthmatic subjects were higher than those in specimens from normal subjects, and they were correlated with basement membrane thickness in asthma., Conclusion: The findings provide evidence that TGF-beta signaling was active in asthmatic airways and that the activity was associated with the development of airway remodeling in asthma.

Purpose: To investigate the concentrations of Fc epsilon receptor I (Fc epsilon RI)-bindable immunoglobulin E (IgE) in the tear fluid and the proportion of IgE-saturated cells among Fc epsilon RI-positive cells in giant papillae from vernal keratoconjunctivitis (VKC) patients., Methods: The tear fluids and giant papillae were obtained from 8 VKC patients with their informed consent. To detect the quantitative difference between Fc epsilon RI-bindable IgE and total IgE in the tear fluid, we used a new enzyme-linked immunosorbent assay system we have developed. Next, to estimate the proportion of IgE-saturated cells among Fc epsilon RI-positive cells, we used two distinct monoclonal antibodies for Fc epsilon RI for the immunohistochemistry. One antibody recognizes all Fc epsilon RI regardless of whether it is with or without receptor-bound IgE. The other does not recognize IgE-bound Fc epsilon RI., Results: The quantitative difference between Fc epsilon RI-bindable IgE and total IgE were detected in the tear fluid of VKC patients. Fc epsilon RI-positive cells were significantly increased in the giant papillae of VKC compared with normal conjunctivae. The proportion of IgE-saturated cells among Fc epsilon RI-positive cells in giant papillae was higher than that in normal conjunctivae., Conclusion: These results suggest that F epsilon RI-bindable IgE may be a critical factor to estimate the severity of VKC.

Lipoprotein glomerulopathy (LPG) is a unique renal disease characterized by intraglomerular lipoprotein thrombi associated with severe proteinuria and frequent progression to renal failure. The histologic hallmark of LPG is the presence of laminated thrombi, consisting of lipid droplet, within the lumina of dilated glomerular capillaries. The findings of thrombi consisting of lipoproteins raised the possibilities that LPG might be related to a primary abnormality in lipid metabolism. However, the precise pathogenic basis of LPG remains unresolved. It was herein found that chronic graft-versus-host disease (GVHD) induced by the transfer of Ia-incompatible spleen cells from B6.C-H2(bm12) into coisogenic C57BL/6 mice with deficiency of Fc receptor gamma chain (FcRgamma) resulted in glomerulopathy that resembled LPG. The uptake of acetylated LDL was partially decreased in peritoneal macrophages isolated from FcRgamma-deficient mice compared with wild-type mice, suggesting that partial impairment of modified LDL uptake might contribute to the development of LPG associated with chronic GVHD in FcRgamma-deficient mice. LPG has been suggested to be a disorder of primary abnormality in lipid metabolism; these findings would therefore provide novel insight into the disease process.

Background: The biological functions of basophils are precisely regulated by various cytokines in vitro, but little is known about surface markers that are upregulated during the cytokine-mediated activation process., Objective: It has been well established that CD69, CD44, and CD54 represent "activation markers" for cytokine-mediated eosinophil activation. The objective of this study was to elucidate the expression and regulation of these molecules in human basophils in vitro as well as in vivo., Methods: Basophils were purified from venous blood by means of density gradient centrifugation followed by negative selection. Surface expression was analyzed by means of flow cytometry. We also studied the expression of CD69, CD44, and CD54 on basophils in bronchoalveolar lavage fluid and blood specimens from patients with asthma., Results: CD44 and CD54 were constitutively expressed on basophils and moderately upregulated by IL-3. On the other hand, CD69 expression was only weakly observed in freshly isolated basophils, but IL-3 induced extremely high levels of expression. Surface CD69 appeared rather slowly in comparison with CD63 and CD11b, and the induction of expression was completed within 24 hours. Basophil CD69 had no functional relevance, but it did have biological relevance. Whole blood basophils from asthmatic individuals expressed significantly higher levels of CD69 than did those from normal individuals. Furthermore, bronchoalveolar lavage fluid basophils showed higher levels of CD69 expression than did blood basophils from the same donors., Conclusion: CD69 expression on basophils was preferentially and strongly upregulated by IL-3. CD69 on basophils might be useful as an in vitro as well as in vivo marker of activation of these cells by IL-3.

Toll-like receptor 2 (TLR2) and TLR4 play important roles in the early innate immune response to microbial challenge. To clarify the functional roles of TLRs 2 and 4 in mast cells, we examined bone marrow-derived mast cells (BMMCs) from TLR2 or TLR4 gene-targeted mice. Peptidoglycan (PGN) from Staphylococcus aureus stimulated mast cells in a TLR2-dependent manner to produce TNF-alpha, IL-4, IL-5, IL-6, and IL-13, but not IL-1beta. In contrast, LPS from Escherichia coli stimulated mast cells in a TLR4-dependent manner to produce TNF-alpha, IL-1beta, IL-6, and IL-13, but not IL-4 nor IL-5. Furthermore, TLR2- but not TLR4-dependent mast cell stimulation resulted in mast cell degranulation and Ca2+ mobilization. In a mast cell-dependent model of acute sepsis, TLR4 deficiency of BMMCs in mice resulted in significantly higher mortality because of defective neutrophil recruitment and production of proinflammatory cytokines in the peritoneal cavity. Intradermal injection of PGN led to increased vasodilatation and inflammation through TLR2-dependent activation of mast cells in the skin. Taken together, these results suggest that direct activation of mast cells via TLR2 or TLR4 by respective microligands contributes to innate and allergic immune responses.

Transcriptional regulation of the gene-encoding human Fc epsilon RI alpha-chain was analyzed in detail. EMSA revealed that either YY1 or PU.1 bound to the region close to that recognized by Elf-1. The alpha-chain promoter activity was up-regulated approximately 2-fold by exogenously expressed YY1 or PU.1 and approximately 7-fold by GATA-1, respectively, in KU812 cells. In contrast, coexpression of GATA-1 with either of PU.1 or YY1 dramatically activated the promoter approximately 41- or approximately 27-fold, respectively. Especially synergic activation by GATA-1 and PU.1 was surprising, because these transcription factors are known to inhibit the respective transactivating activities of each other. These up-regulating effects of PU.1 and YY1 with GATA-1 were inhibited by overexpression of Elf-1, indicating that Elf-1 serves as a repressor for the alpha-chain gene expression. Transcriptional regulation of the alpha-chain gene through four transcriptional factors is discussed.

The transcriptional regulatory region of Elf-1 was analyzed by the combination of a yeast one-hybrid system and site-directed mutagenesis. This analysis enabled us to map an activation region between 85-175 of Elf-1.

Induction of alphaEbeta7 expression on T cells by transforming growth factor (TGF)-beta is thought to be important for intestinal intraepithelial T lymphocyte (IEL) entry into the epithelial compartment. However, there has been no in vivo evidence that up-regulation of alphaEbeta7 expression on T cells by TGF-beta is critical for the selective localization of intestinal IEL in the epithelial area. We have recently established transgenic mice expressing Smad7 under the control of a distal lck promoter where TGF-beta/Smad signaling is specifically blocked in mature T cells. Here we showed that TGF-beta-mediated up-regulation of alphaEbeta7 was impaired on T cells isolated from the Smad7 transgenic mice associated with reduced numbers of intestinal IEL when compared with that in wild-type littermates. These results indicated that failure to induce alphaEbeta7 on T cells by TGF-beta resulted in reduced numbers of intestinal IEL, suggesting the importance of alphaEbeta7 expression by TGF-beta in selective localization of intestinal IEL.

Mucosal mast cells strategically located at the optimal site interact with invading bacteria. Presence of VacA, the virulent Helicobacter pylori cytotoxin, is correlated with the severity of H. pylori-induced gastritis. To examine the mechanisms of inflammation in H. pylori-induced gastritis, we administered VacA to the mice. Inoculation of VacA resulted in epithelium vacuolization and marked infiltrations of mast cells and mononuclear cells into the mucosal epithelium within 24 h. In an in vitro study using bone marrow-derived mast cells, VacA directly bound and showed a chemotactic activity to the mast cell. In addition, VacA induced bone marrow-derived mast cells to produce proinflammatory cytokines, TNF-alpha, macrophage-inflammatory protein-1alpha, IL-1beta, IL-6, IL-10, and IL-13 in a dose-dependent manner without causing degranulation. The present study suggests that early activation of mast cells by VacA may be the host early response to clear the bacteria and also may contribute to the pathogenesis of H. pylori-induced gastritis.

Background: Platelet adhesion on components of the extracellular matrix and platelet activation by those components are crucial for the arrest of posttraumatic bleeding, but they can also harm tissue by occluding diseased vessels. Recent studies have shown that the activation of platelets by collagen is mediated through the same pathway used by immune receptors, with an immunoreceptor tyrosine-based activation motif on the Fc receptor gamma chain (FcRgamma) playing a pivotal role., Methods and Results: We examined the role of collagen-stimulated platelets in the development of injury-induced neointimal formation by using mice deficient in FcRgamma. The left femoral arteries of 8- to 12-week-old FcRgamma-deficient mice (n=16) and C57BL/6 (wild-type) mice (n=16) were injured by a straight spring wire (0.35-mm diameter). Segments of the injured and uninjured femoral arteries were excised at 7 days and 28 days after the vascular injury. Arterial segments were examined by immunohistochemistry and electron microscopy. Two hours after injury, electron microscopy showed marked decreases in platelet adhesion and neutrophil attachment to the vascular wall surface in FcRgamma-knockout mice compared with wild-type mice. At 7 days after injury, staining with anti-neutrophil antibody showed fewer neutrophils in FcRgamma-knockout mice than in wild-type mice. Computer-aided morphometry performed to measure the neointimal area, intima/media ratio, and stenotic area at 28 days after injury showed a significantly smaller ratio and area in FcRgamma-knockout mice than in wild-type mice (for neointimal area, 16 635 +/- 1406 versus 31 483 +/- 2309 microm2, respectively; for intima/media ratio, 1.25 +/- 0.40 versus 2.68 +/- 0.04, respectively; and for stenotic area, 26.8 +/- 2.1% versus 49.3 +/- 4.1%, respectively)., Conclusions: These results demonstrate that FcRgamma may play important roles in the initiation and generation of neointimal hyperplasia after balloon injury through the activation of platelets by collagen.

In the kidney, proteins filtered through glomeruli are reabsorbed by endocytosis along the proximal tubules to avoid renal loss of large amounts of proteins. Recently, neonatal Fc receptor (FcRn), which is involved in the transport of IgG across several epithelial and endothelial cells, was reported to be expressed in renal proximal tubular epithelial cells (RPTECs). However, there has been no direct evidence for receptor-mediated endocytosis of IgG in human RPTECs. To explore physiological roles of FcRn in the proximal tubules, we used the human RPTECs to examine IgG transport. FcRn was expressed in RPTECs and physically associated with beta(2)-microglobulin, preserving the capacity of specific pH-dependent IgG binding. Human IgG was bound to the cell surface of RPTECs in a pH-dependent manner. The human IgG transport assay revealed that receptor-mediated transepithelial transport of intact IgG in RPTECs is bidirectional and that it requires the formation of acidified intracellular compartments. With the use of double immunofluorescence, the internalized human IgG was marked in cytoplasm of RPTECs and colocalized with FcRn. These data define the mechanisms of FcRn-associated IgG transport in RPTEC monolayers. It was suggested that the intact pathway for human IgG transepithelial transport may avoid lysosomal degradation of IgG.

Background: Interleukin (IL)-13 is a T-cell-derived cytokine that shares several functions with IL-4, including the induction of immunoglobulin E synthesis. Recent studies suggest that cytokines expressed locally in the skin play several critical roles in atopic dermatitis (AD), however, little is known about the role of IL-13 in AD lesions., Objectives: The present study was designed to characterize the involvement of IL-13 in AD in the skin and peripheral blood mononuclear cells (PBMC)., Methods: Using lesional and nonlesional skin from adult AD patients and normal skin from healthy volunteers, we performed RT-PCR, in situ RT and immunostaining to determine the IL-13 expression at the mRNA and protein levels. The actual numbers of IL-13 expressing cells in biopsy specimens were counted under the microscope. IL-13 mRNA expression in PBMC from AD patients and healthy volunteers was examined by RT-PCR analysis., Results: IL-13 mRNA expression was detected by RT-PCR in lesional and nonlesional skin and in PBMC from AD patients, but not in normal skin or PBMC from healthy volunteers. In AD lesional skin, numerous IL-13 mRNA-positive cells were demonstrated by in situ RT, and similar numbers of IL-13-positive cells were also detected immunohistochemically. Smaller numbers of IL-13-positive cells were observed in AD nonlesional skin and in normal skin. The differences in the numbers of IL-13-expressing cells between lesional and nonlesional skin were statistically significant. Double immunostaining revealed that IL-13 was produced in approximately 40% of T cells and 20% of mast cells in AD lesional skin, suggesting that T cells and mast cells are major sources of IL-13 in AD lesions., Conclusion: IL-13 may play a local as well as a systemic role in the development of AD lesions., (Copyright 2002 S. Karger AG, Basel)