Molecular basis and role of differential cytokine production in T helper cell subsets in immunologic disease.
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Cytokines↗
Biomedical subjects
S J Ono
Publications and source records attributed to S J Ono.
HLA-DRA promoter polymorphism and diversity generation within the immune system.
Multiple major histocompatibility complex (MHC) alleles exist at most class I and II loci. Polymorphism of MHC polypeptides may reflect either different levels of selective pressure operating on each molecule or different mutation rates at different loci. To gain further insight into this issue, we sequenced the non-coding promoter region of the HLA-DRA gene from several Epstein-Barr virus-transformed B cell lines and compared the extent of polymorphism found in this region with the known polymorphism of the HLA-DQB promoter. Our results indicate that the HLA-DRA promoter displays a low level of polymorphism while the promoter of HLA-DQB exhibits a nucleotide substitution rate fivefold greater than that of DRA. Moreover, through phylogenetic analysis, the HLA-DRA promoter was found to have diverged much less than the associated alleles of HLA-DRB1 and -DQA1. Taken together, these results suggest that the HLA-DRA promoter is highly conserved and may be under a stronger functional constraint than the promoter regions of other MHC class II genes.
Alleles↗
Polymorphic nucleotides within the human IL-4 promoter that mediate overexpression of the gene.
Atopy, which predisposes individuals to develop asthma, severe systemic anaphylaxis, and atopic dermatitis, is usually associated with dramatically elevated total serum IgE levels and is thought to be controlled by a major susceptibility gene and multiple minor susceptibility genes. A recent sib-pair analysis revealed a tight linkage between markers on 5q31.1 and a major susceptibility gene controlling total serum IgE levels. Due to its location within this cluster and its biologic role in Ig class switching and Th2 cell differentiation, the IL-4 gene has emerged as one major candidate for the atopy gene. In one model, polymorphisms within IL-4 regulatory elements might result in overexpression of the gene, amplifying Th2 cell differentiation and class switching to IgE. In support of this model, we report that the human IL-4 promoter exists in multiple allelic forms that exhibit distinct transcriptional activities in IL-4-positive T cells. A particular allele has an unusually high transcriptional activity. A nucleotide substitution within a recently described OAP40 element located just upstream of an NF-AT site (P sequence) appears to be largely responsible for the increased promotor strength of this particular allelic form of the IL-4 promoter. In EMSAs, this substitution results in a markedly enhanced affinity for sequence-specific complexes exhibiting an AP-1 specificity. The identification of allelic nucleotides, which results in overexpression of the IL-4 gene, provides specific targets for a comprehensive screening of atopic and nonatopic individuals and may provide a clue for genetic predisposition for atopy.
Alleles↗
Biology and genetics of atopic disease.
Several immunological disorders including allergic rhinitis, bronchial asthma, atopic dermatitis, food allergies, urticaria, nonhereditary angioedema, systemic anaphylaxis, and allergic conjunctivitis are associated with a positive family history, and share a positive response in the Prausnitz-Kuster (wheal and flare) reaction. Studies have shown that 20-30% of the population has a strong genetic predisposition for this condition, termed atopy, whose hallmark is a greatly elevated serum IgE concentration. A great deal is known about the cellular interactions that mediate the sensitization, immediate and late-phase reactions that follow encounters with allergen, as well as about the cell surface and signaling events that result in mediator release from inflammatory cells. Less is known of the genes that confer genetic predisposition for atopy; however, a worldwide effort to identify atopy genes is making significant progress.
Animals↗
Inhibition of NF-AT-dependent transcription by NF-kappa B: implications for differential gene expression in T helper cell subsets.
Activation of individual CD4+ T cells results in differential lymphokine expression: interleukin 2 (IL-2) is preferentially produced by T helper type 1 (TH1) cells, which are involved in cell-mediated immune responses, whereas IL-4 is synthesized by TH2 cells, which are essential for humoral immunity. The Ca(2+)-dependent factor NF-ATp plays a key role in the inducible transcription of both these lymphokine genes. However, while IL2 expression requires the contribution of Ca(2+)- and protein kinase C-dependent signals, we report that activation of human IL4 transcription through the Ca(2+)-dependent pathway is diminished by protein kinase C stimulation in Jurkat T cells. This phenomenon is due to mutually exclusive binding of NF-ATp and NF-kappa B to the P sequence, an element located 69 bp upstream of the IL4 transcription initiation site. Human IL4 promoter-mediated transcription is downregulated in Jurkat cells stimulated with the NF-kappa B-activating cytokine tumor necrosis factor alpha and suppressed in RelA-overexpressing cells. In contrast, protein kinase C stimulation or RelA overexpression does not affect the activity of a human IL4 promoter containing a mouse P sequence, which is a higher-affinity site for NF-ATp and a lower-affinity site for RelA. Thus, competition between two general transcriptional activators, RelA and NF-ATp, mediates the inhibitory effect of protein kinase C stimulation on IL4 expression and may contribute to differential gene expression in TH cells.
Animals↗
Modulation of MHC class II expression in human cells by dexamethasone.
The effect of dexamethasone on human MHC class II expression was examined on various cell types including lymphocytes, monocytes, and epithelial cells. Dexamethasone decreased the surface expression of HLA-DR and -DP, but not HLA-DQ, on lymphocytic cell lines that constitutively express these molecules. In addition, dexamethasome down-regulated the mRNA levels of HLA-DRA, but not of HLA-DQB, in Jijoye cells, a human lymphoblastic cell line. Similarly, dexamethasone decreased HLA-DR expression on epithelial and monocytic cell lines that express HLA-DR upon IFN-gamma treatment. In total, these results suggest that dexamethasone inhibits both constitutive and IFN-gamma-inducible MHC class II expression in several cell types. Moreover, these results indicate that the inhibitory effect of dexamethasone on MHC class II expression is selective for HLA-DR and -DP but not HLA-DQ. Possible mechanisms of dexamethasone-mediated regulation of MHC class II expression are discussed.
Antigens, Surface↗
Functional roles of the transcription factor Oct-2A and the high mobility group protein I/Y in HLA-DRA gene expression.
The class II major histocompatibility complex gene HLA-DRA is expressed in B cells, activated T lymphocytes, and in antigen-presenting cells. In addition, HLA-DRA gene expression is inducible in a variety of cell types by interferon-gamma (IFN-gamma). Here we show that the lymphoid-specific transcription factor Oct-2A plays a critical role in HLA-DRA gene expression in class II-positive B cell lines, and that the high mobility group protein (HMG) I/Y binds to multiple sites within the DRA promoter, including the Oct-2A binding site. Coexpression of HMG I/Y and Oct-2 in cell lines lacking Oct-2 results in high levels of HLA-DRA gene expression, and in vitro DNA-binding studies reveal that HMG I/Y stimulates Oct-2A binding to the HLA-DRA promoter. Thus, Oct-2A and HMG I/Y may synergize to activate HLA-DRA expression in B cells. By contrast, Oct-2A is not involved in the IFN-gamma induction of the HLA-DRA gene in HeLa cells, but antisense HMG I/Y dramatically decreases the level of induction. We conclude that distinct sets of transcription factors are involved in the two modes of HLA-DRA expression, and that HMG I/Y may be important for B cell-specific expression, and is essential for IFN-gamma induction.
Base Sequence↗
Expression of the chemokine RANTES by a human bronchial epithelial cell line. Modulation by cytokines and glucocorticoids.
The chemokine RANTES is a potent chemoattractant for eosinophils, lymphocytes, and monocytes, and has been detected recently in the epithelium of human airways mucosa. We have studied, therefore, the expression of RANTES mRNA and protein in the human bronchial epithelial cell line BEAS-2B. Using Northern blot analysis, RANTES mRNA was not detectable in unstimulated BEAS-2B cells. Incubation of cells with TNF-alpha or IFN-gamma induced the expression of RANTES mRNA and protein within 16 h. The combination of TNF-alpha and IFN-gamma displayed a marked synergism in inducing RANTES expression. Pretreatment of cells with the glucocorticoid budesonide (10(-10)-10(-7) M) for 24 h inhibited expression of RANTES mRNA and protein stimulated by either TNF-alpha or TNF-alpha plus IFN-gamma in a concentration- and time-dependent manner. Nonglucocorticoid steroids did not inhibit RANTES mRNA expression. Production of RANTES by epithelium could contribute to the mechanism of selective cellular recruitment occurring in the airways during inflammation, thus playing a relevant role in the pathogenesis of diseases such as asthma, rhinitis, and polyposis. The down-regulation of RANTES production by glucocorticoids in epithelial cells may contribute to the efficacy of these compounds in reducing cellular infiltration and, ultimately, to their anti-inflammatory properties.
Blotting, Northern↗
Mapping of the interaction site of the defective transcription factor in the class II major histocompatibility complex mutant cell line clone-13 to the divergent X2-box.
We have previously described a mutant B lymphoblastoid cell line, Clone-13, that expresses HLA-DQ in the absence of HLA-DR and -DP. Several criteria indicated that the defect in this cell line influences the activity of an isotype-specific transcription factor. Indeed, transient transfection of HLA-DRA and DQB reporter constructs indicated that the affected factor operates via cis-elements located between -141 base pairs and the transcription initiation site. A series of hybrid DRA/DQB reporter constructs was generated to further map the relevant cis-elements in this system. Insertion of oligonucleotides spanning the DQB X-box (but not the DQB-W region or the DQB Y-box) upstream of -141 in a DRA reporter plasmid rescued expression to nearly wild-type levels. Substitution promoters were then generated where the entire X-box, or only the X1- or X2-boxes of HLA-DRA were replaced with the analogous regions of HLA-DQB. The DQB X2-box was able to restore expression to the silent DRA reporter construct. Moreover, replacement of the DQB X2-box with the DRA X2-box markedly diminished the activity of the DQB promoter in the mutant cell. None of the hybrid reporter constructs were defective when transfected into the wild-type, HLA-DR/-DQ positive parental cell line, Jijoye. These studies suggest that the divergent X2-box of the class II major histocompatibility complex promoters plays an important role in influencing differential expression of the human class II isotypes.
B-Lymphocytes↗
L cells expressing DQ molecules of the DR3 and DR4 haplotypes: reactivity patterns with mAbs.
cDNAs coding for the HLA class II DR and DQ alpha and beta chains of the diabetogenic haplotypes DR3 and DR4 were introduced into a mammalian expression vector and transfected into L-cell mouse fibroblasts to produce cells expressing individual human class II molecules. Stable L transfectants were generated expressing each of the DR or DQ isotypes of the cis-encoded alpha and beta chains of the DR3 or DR4 haplotypes, as well as the trans-encoded alpha and beta chains of the DQ molecules of the two haplotypes. However, isotype mismatched combinations (DR alpha/DQ beta or DQ alpha/DR beta) did not result in any stable transfectants. The stable DQ L-cell transfectants obtained, along with homozygous B-cell lines expressing the DQ2 and DQ8 specificities, were tested against a large panel of twentyone anti-HLA class II monoclonal antibodies (mAbs). Their unusual reactivity patterns are described including the failure of most "pan-DQ" mAbs to react with all DQ expressing L-cell transfectants. Interestingly, some mAbs react with certain alpha beta heterodimers expressed on B-LCL but fail to recognize the same heterodimers expressed on the transfectants. This is suggestive of minor structural modifications that class II molecules undergo depending on the cells they are expressed on.
Animals↗
How are class II MHC genes turned on and off?
Fragments of foreign antigen are detected by CD4+ helper T cells via the T cell receptor for antigen in the context of major histocompatibility complex (MHC) class II molecules. Very few cells normally express class II MHC molecules, and these cells play critical roles in antigen presentation and in the thymic selection of T lymphocytes before their exit into the periphery. Because of the central role the class II MHC molecules play in immune system function, it is not surprising that the lack of expression of these molecules results in a severe combined immunodeficiency disorder (called bare lymphocyte syndrome) and that the aberrant expression of the molecules is frequently observed in the target organs of various autoimmune disorders (e.g., multiple sclerosis and rheumatoid arthritis). Because both classes of disease could conceivably be treated by molecular approaches targeted at either restoring or inhibiting expression of class II MHC genes, there has been an intense effort during the past decade to elucidate the regulatory mechanisms of class II MHC genes. An analysis of recent advances in this effort is provided in this review article.
Animals↗
A novel cysteine-rich sequence-specific DNA-binding protein interacts with the conserved X-box motif of the human major histocompatibility complex class II genes via a repeated Cys-His domain and functions as a transcriptional repressor.
The class II major histocompatibility complex (MHC) molecules function in the presentation of processed peptides to helper T cells. As most mammalian cells can endocytose and process foreign antigen, the critical determinant of an antigen-presenting cell is its ability to express class II MHC molecules. Expression of these molecules is usually restricted to cells of the immune system and dysregulated expression is hypothesized to contribute to the pathogenesis of a severe combined immunodeficiency syndrome and certain autoimmune diseases. Human complementary DNA clones encoding a newly identified, cysteine-rich transcription factor, NF-X1, which binds to the conserved X-box motif of class II MHC genes, were obtained, and the primary amino acid sequence deduced. The major open reading frame encodes a polypeptide of 1,104 amino acids with a symmetrical organization. A central cysteine-rich portion encodes the DNA-binding domain, and is subdivided into seven repeated motifs. This motif is similar to but distinct from the LIM domain and the RING finger family, and is reminiscent of known metal-binding regions. The unique arrangement of cysteines indicates that the consensus sequence CX3CXL-XCGX1-5HXCX3CHXGXC represents a novel cysteine-rich motif. Two lines of evidence indicate that the polypeptide encodes a potent and biologically relevant repressor of HLA-DRA transcription: (a) overexpression of NF-X1 from a retroviral construct strongly decreases transcription from the HLA-DRA promoter; and (b) the NF-X1 transcript is markedly induced late after induction with interferon gamma (IFN-gamma), coinciding with postinduction attenuation of HLA-DRA transcription. The NF-X1 protein may therefore play an important role in regulating the duration of an inflammatory response by limiting the period in which class II MHC molecules are induced by IFN-gamma.
Amino Acid Sequence↗
Secretion of IL-4 from human basophils. The relationship between IL-4 mRNA and protein in resting and stimulated basophils.
Purified human basophils have been examined for secretion of IL-4 protein and expression of IL-4 mRNA after stimulation with several secretagogues. In general, these studies used a 15-min preincubation with IL-3, before challenge with secretagogues. Under these conditions, IL-4 release averaged 30 pg/10(6) basophils (range 4-70) after challenge with anti-IgE Ab. FMLP and C5a led to somewhat lower levels of secretion. A direct comparison of basophils at 88 to 99% purity with basophils from the same preparations, but at lower purities, showed that the amount of IL-4 secretion was proportional to the purity of the basophils. The presence of mRNA for IL-4 (as determined by reverse transcriptase-PCR, competitive reverse transcriptase-PCR, or Northern blots) was also strictly related to the purity of the basophils. IL-4 mRNA was also found to be constitutively present and was increased after stimulation. The concentration of polyclonal anti-IgE Ab required for optimal IL-4 release was somewhat less than that required for optimal histamine release. IL-4 secretion was slower (t1/2 of 1.5 h) than histamine release and was inhibited by cycloheximide. In a final series of studies, we found that IL-3 was not required for IL-4 secretion; a short 15-min preincubation with IL-3 resulted in the same or slightly less IL-4 release than no treatment with IL-3. In contrast, an 18-h pretreatment with IL-3 resulted in a nearly tenfold increase in IL-4 secretion. We conclude that human basophils secrete IL-4 in response to several secretagogues and that IL-3 priming is not necessary to observe IL-4 secretion.
Base Sequence↗
Transcription of a subset of human class II major histocompatibility complex genes is regulated by a nucleoprotein complex that contains c-fos or an antigenically related protein.
Transcriptional regulation of the human major histocompatibility complex class II genes requires at least two upstream elements, the X and Y boxes, located in the -50- to -150-base-pair region of all class II promoters. The DRA and DPB promoters contain phorbol ester-responsive elements overlapping the 3' side of their X boxes. Mutation of this sequence down-regulates the efficiency of the DRA promoter, suggesting that a positive regulator(s) binds to this site. In this report, anti-sense c-fos RNA and an anti-c-fos antibody were used to show that the product of the protooncogene c-fos or an antigenically related protein is a component of a complex that binds to the X box and is required for maximal transcription from the DRA and DPB promoters. As c-fos (or its related proteins) cannot bind alone to DNA, these results suggest that it may dimerize with other members of the JUN/AP-1 family, such as hXBP1, to participate in the activation of a subset of class II major histocompatibility complex genes.
Amino Acid Sequence↗
Human X-box-binding protein 1 is required for the transcription of a subset of human class II major histocompatibility genes and forms a heterodimer with c-fos.
A complementary DNA encoding a member of the leucine-zipper class of proteins (human X-box-binding protein, hXBP-1) that binds to the 3' end of the conserved X box (X2) of the HLA-DRA major histocompatibility complex gene was recently described. Further gel-retardation analysis has demonstrated that hXBP-1 also binds to HLA-DPB X2 but not to other X2 sequences. Transient transfection of a mammalian expression vector with the hXBP-1 cDNA inserted in the antisense orientation represses the surface expression of HLA-DR and HLA-DP in Raji cells. Cotransfection of the antisense hXBP-1 vector with a HLA-DRA/chloramphenicol acetyltransferase (but not a HLA-DQB/chloramphenicol acetyltransferase) reporter plasmid decreases chloramphenicol acetyltransferase activity in Raji cells and in gamma-interferon-treated HeLa cells relative to cells cotransfected with a control antisense vector. Moreover, hXBP-1 is shown to form a stable heterodimer with the product of the c-fos protooncogene. These data suggest that the hXBP-1 c-fos heterodimer is critical for the transcription of a subset of the human class II major histocompatibility complex genes and that the regulatory mechanisms for the different class II genes are distinct.
Antisense Elements (Genetics)↗
An isotype-specific trans-acting factor is defective in a mutant B cell line that expresses HLA-DQ, but not -DR or -DP.
The B lymphoblastoid cell line clone 13 (a subclone of the mutant cell line P3JHR-1) has been found to express high levels of HLA-DQ; by contrast, HLA-DR and -DP antigens are not expressed and cannot be induced by interferon gamma. Northern blot analysis using gene-specific probes indicated that the lack of surface expression of the DR and DP antigens is due to a marked decrease in the levels of steady-state RNA for both the alpha and beta chains. Southern blots demonstrated that none of the transcriptionally repressed genes are grossly deleted. Preparations of interspecific transient heterokaryons between clone 13 and the class II antigen-positive murine B cell lymphoma, A20, resulted in reactivation of the DRA gene and surface expression of both the DR and DP molecules. The efficiency of the DRA promoter relative to the DQB promoter is markedly and specifically diminished in clone 13 (P3JHR-1) as compared with the parental cell line, Jijoye, as assayed both by transient expression of appropriate chloramphenicol acetyltransferase gene (CAT) constructs and by in vitro transcription analysis. These data clearly demonstrate the existence of an isotype-specific trans-acting factor, and provide direct evidence that the highly homologous class II genes have distinct regulatory mechanisms.
Antibodies, Monoclonal↗
A single dose of the MHC-linked susceptibility determinant associated with the RT1u haplotype is permissive for insulin-dependent diabetes mellitus in the BB rat.
Syndromes of insulin-dependent diabetes mellitus (IDDM) have been described in the mouse, in the rat and in man. In all three species, the presence of one or more specific alleles of the major histocompatibility complex is a prerequisite for the appearance of the disease. In the BB rat, diabetes is associated with the RT1u haplotype. We have performed a series of intercrosses of diabetic BB rats with normal Lewis and Buffalo rats and examined the offspring of all litters producing at least one diabetic animal. Forty-five of the 250 rats that developed diabetes were heterozygous for the RT1u haplotype by serotyping. Furthermore, the diabetic rats heterozygous by serotyping at the RT1A class I loci were also heterozygous at the RT1B and RT1D loci of the class II region and did not show evidence of a recombinant haplotype when examined by Southern blot analyses using molecular probes for class I and class II genes. Diabetic rats heterozygous or homozygous for RT1u were phenotypically indistinguishable with respect to age of onset and severity of disease. Therefore, in the rat, as in the human, a single dose of the high-risk allele at the major histocompatibility complex is sufficient for the development of IDDM if other susceptibility factors and the appropriate environmental factors are in place.
Animals↗
Interferon-gamma induces transcription and differential expression of MHC genes in rat insulinoma cell line RINm5F.
We have reported that enhanced levels of class I major histocompatibility complex (MHC) antigen are expressed throughout the islets of prediabetic and newly diabetic BB rats and that the endocrine cells of the islet remained class II negative. In this study we investigated the molecular biology of lymphokine-induced expression of the class I and II MHC genes in subclones of the rat insulinoma cell line RINm5F. Treatment of a particular subclone of RINm5F cells (which are normally class II negative, class I low expressors) with crude lymphokine preparation or various doses of recombinant interferon-gamma resulted in enhancement of MHC class I antigen expression but no detectable induction of class II antigen expression. This enhancement of class I antigen expression was a dose-dependent phenomenon and was preceded by a dose-dependent increase in class I-specific RNA. Both class I and II genes were induced at the transcriptional level, as determined by Northern blotting and in vitro nuclear transcription assays, but exhibited strikingly different induction kinetics. Supernatants from concanavalin A-stimulated splenocytes had a similar class I-restricted inductive effect on MHC gene expression. This subclone of RINm5F cells, which exhibits a class I lymphokine response-positive, class II response-negative phenotype, 1) mimics the behavior of beta-cells in the prediabetic and newly diabetic pancreas and 2) represents a valuable system for probing the similarities and differences in the lymphokine-mediated induction pathways for class I and II MHC genes.
Adenoma, Islet Cell↗