A highly conserved c-fms gene intronic element controls macrophage-specific and regulated expression.

The c-fms gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c-fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSs) were identified within mouse intron 2. Sequences of these DHSs were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fms intronic regulatory element (FIRE), which is even more highly conserved than the c-fms proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Sp1, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high- and low-level c-fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fms gene.

The role of high-mobility group I(Y) proteins in expression of IL-2 and T cell proliferation.

The high-mobility group I(Y) (HMGI(Y)) family of proteins plays an important architectural role in chromatin and have been implicated in the control of inducible gene expression. We have previously shown that expression of HMGI antisense RNA in Jurkat T cells inhibits the activity of the IL-2 promoter. Here we have investigated the role of HMGI(Y) in controlling IL-2 promoter-reporter constructs as well as the endogenous IL-2 gene in both Jurkat T cells and human PBL. We found that the IL-2 promoter has numerous binding sites for HMGI(Y), which overlap or are adjacent to the known transcription factor binding sites. HMGI(Y) modulates binding to the IL-2 promoter of at least three transcription factor families, AP-1, NF-AT and NF-kappaB. By using a mutant HMGI that cannot bind to DNA but can still interact with the transcription factors, we found that DNA binding by HMGI was not essential for the promotion of transcription factor binding. However, the non-DNA binding mutant acts as a dominant negative protein in transfection assays, suggesting that the formation of functional HMGI(Y)-containing complexes requires DNA binding as well as protein:protein interactions. The alteration of HMGI(Y) levels affects IL-2 promoter activity not only in Jurkat T cells but also in PBL. Importantly, we also show here that expression of the endogenous IL-2 gene as well as proliferation of PBL are affected by changes in HMGI(Y) levels. These results demonstrate a major role for HMGI(Y) in IL-2 expression and hence T cell proliferation.

A role for the architectural transcription factors HMGI(Y) in cytokine gene transcription in T cells.

The ability of CD4- T helper (Th) cells to differentiate into two phenotypes distinguished by their cytokine profile is a major determinant of the type of immune response elicited by bacterial, viral or parasitic infections. The development of Th1 cells is associated with delayed-type hypersensitivity and cell-mediated immune responses while Th2 responses are associated with humoral immunity and allergic inflammation. While these phenotypes exist at the extremes of the immune response and are associated with pathological conditions, there is an enormous plasticity that allows reversibility and the development of a wide array of cytokine profiles. There has been considerable interest in determining the signals and transcription factors that govern the differential production of the Th1 and Th2 cytokines. There are now several candidate transcription factors that may play a role in skewing the cytokine profile in a distinct direction. Because of the plasticity of the system, these transcription factors must be able to respond to environmental signals in a very subtle manner and not simply be on/off switches for expression of the cytokine genes. The architectural transcription factor high mobility group (HMG) I(Y) is a modulator of the function of many of the transcription factors that control cytokine gene transcription. HMGI(Y) appears to play either a positive or negative role depending on the cytokine promoter and its ratio to other transcription factors. It is proposed that HMGI(Y) may have a role in regulating the production of cytokines in favour of a given immune response.

High mobility group protein I(Y) is required for function and for c-Rel binding to CD28 response elements within the GM-CSF and IL-2 promoters.

CD28 response elements (CD28REs) within cytokine promoters are variant NF-kappaB-binding sites and are essential for transcription in response to CD28 receptor activation in T cells. We show that the CK-1 element (CD28RE) within the GM-CSF promoter binds the RelA and c-Rel transcription factors in response to CD28 activation. We further show that the high mobility group protein HMG I(Y) can bind to the CD28REs of both GM-CSF and IL-2 and that this binding is critical for c-Rel, but not RelA, binding. A second NF-kappaB site in the GM-CSF promoter that binds p50 and RelA, but neither c-Rel nor HMG I(Y), failed to respond to CD28 activation. Expression of HMG I or c-Rel antisense RNA inhibited CD28 activation of the IL-2 and GM-CSF promoters, implying that HMG I(Y) enhancement of c-Rel binding plays an important role in the activity of the CD28REs.

Costimulation of cytokine gene expression in T cells by the human T leukemia/lymphotropic virus type 1 trans activator Tax.

Many cell signals such as CD28 and CD4 binding can costimulate cytokine gene expression in activated T cells. We have found that the human T leukemia/lymphotropic virus type 1 viral protein Tax can also strongly costimulate expression of interleukin-2 (IL-2), IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA in T cells activated with the phorbol ester phorbol myristate acetate (PMA) and calcium ionophore, which can mimic activation through the antigen specific T-cell receptor. Reporter constructs also showed strong synergy between both stimuli and showed that Tax and the PMA-Ca2+ ionophore act through different regions of the IL-2 and GM-CSF genes. Furthermore, the Tax-responsive regions (TxRR) from both GM-CSF and IL-2 respond to costimulation through the CD28 surface receptor. The GM-CSF and IL-2 TxRRs showed significantly higher levels of NF-kappaB/rel binding, following induction by Tax, compared with that of the PMA-Ca2+ ionophore with only Tax capable of inducing c-Rel binding to a Consensus kappaB element within the GM-CSF TxRR. Tax protein mutants, however, showed that a pathway(s) other than NF-kappaB/rel induction could also cooperate with the PMA-Ca2+ ionophore to activate the GM-CSF and IL-2 genes. This high-level costimulation by Tax, through multiple pathways, may be important in the early stages of leukemia and in the nervous system disorder tropical spastic paraparesis.

GM-CSF and IL-2 share common control mechanisms in response to costimulatory signals in T cells.

Antigen complexed with major histocompatibility complex class I or II molecules on the surface of antigen presenting cells interacts with the T cell receptor (TCR) on the surface of T cells and initiates an activation cascade. So called costimulatory signals, mediated by other cell surface interactions or soluble cytokines produced by antigen presenting cells, are also required for complete T cell activation. High levels of cytokine gene expression in T cells also required both TCR and costimulatory signals. The granulocyte-macrophage colony-stimulating factor requires sequences in the promoter as well as a powerful enhancer located 3kb upstream to respond to TCR-like signals. These promoter and enhancer regions are mainly activated by the transcription factor nuclear factor of activated T cells (NFAT). The activation of NFAT by TCR signals has been well described for interleukin-2 (IL-2) and IL-4 gene transcription in T cells. Costimulatory signals, such as activation of the CD28 cell surface molecule on T cells, lead to activation through a distinct region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter. This region is termed the CK-1 or CD28RE and appears to bind specific members of the NF-kappa B family of transcription factors. Human T leukemia virus type 1 (HTLV-1) infects T cells and can lead to increase GM-CSF expression. We have found that the HTLV-1 transactivator protein, tax, acts as a costimulatory signal for GM-CSF and IL-2 gene transcription, in that it can cooperate with TCR signals to mediate high level gene expression. Tax activates the GM-CSF promoter through the CK-1/CD28RE region and also activates nuclear factor-kappa B binding to this region. However, other transcription factors or coactivators of NF-kappa B are required for tax activation but these remain to be identified. The CK-1/CD28RE of GM-CSF shows a high degree of similarity to the IL-2 CD28RE and the IL-3 gene also contains a related region. This observation, together with the fact that both GM-CSF and IL-2 respond to TCR signals via NFAT, implies a high degree of conservation in the regulation of cytokine gene expression in T cells.

Immunodiagnosis of human sparganosis mansoni by micro-chemiluminescence enzyme-linked immunosorbent assay.

We compared a microcolorimetric enzyme-linked immunosorbent assay (colorimetric ELISA) and a microchemiluminescence enzyme-linked immunosorbent assay (chemiluminescence ELISA) for the detection of specific immunoglobulin G (IgG) in the serum of 9 patients with sparganosis mansoni and 9 healthy controls. The chemiluminescence ELISA was able to measure serum levels of specific IgG over a far wider range than the colorimetric assay, and its detection limit was at least 10-fold lower. An additional 5 sera from sparganosis patients and 5 more from healthy controls, together with sera from 28 patients with other parasitic diseases, were also examined by the chemiluminescence ELISA. All 14 patients with sparaganosis mansoni showed high levels of chemiluminescence (21,302 +/- 18,907 counts per second [cps]). All sera from the 14 healthy controls (1580 +/- 569 cps) and sera from 27 of the 28 patients with other parasites (4 with taeniasis saginata [1767 +/- 501 cps], 11 with diphyllobothriasis latum [1479 +/- 501 cps], 13 with cysticercosis cellulosae [2376 +/- 1437 cps]) showed chemiluminescence levels lower than those of any of the sparganosis mansoni patients. The exception was a patient with cysticercosis (5980 cps), who may have had a dual infection with Cysticercus cellulosae and Sparganum mansoni. Thus, the chemiluminescence ELISA demonstrated high sensitivity and specificity for human sparganosis mansoni.

Effect of bovine respiratory syncytial virus infection on hypersensitivity to inhaled Micropolyspora faeni.

Respiratory syncytial virus causes mild-to-severe respiratory disease in human infants and young children; a closely related bovine respiratory syncytial virus causes a similar disease pattern in calves. Increased disease severity in atopic children suggests that allergic reactivity may enhance the severity of RSV-induced disease. To examine the association between bovine respiratory syncytial virus (BRSV) infection and allergic reactivity two groups of calves were exposed to aerosolized Micropolyspora faeni (Mf) during an experimental BRSV infection. One group exposed to Mf concurrent with BRSV was challenge-exposed to Mf while infected a second time with BRSV, while the other similarly sensitized and infected group was mock challenged. A control group was exposed only to Mf aerosol and another control group was infected with virus but not exposed to Mf aerosol. Parameters examined included: clinical signs, Mf-specific IgG and IgE, BRSV-specific antibody and IgE, leukotrienes C4 and B4 prostaglandins E2, F2 alpha and D2, and lung pathology. While the initial BRSV infection failed to enhance sensitization to inhaled Mf, a second BRSV infection exacerbated clinical signs resulting from Mf aerosol. Consideration of eicosanoid and antibody profiles together with clinical signs suggests that mechanisms of both type I and type III hypersensitivity were operative during Mf challenge of sensitized calves.

HTLV-1 tax activation of the GM-CSF and G-CSF promoters requires the interaction of NF-kB with other transcription factor families.

The trans-activator protein, tax, from the human T leukemia virus type 1 (HTLV-1) trans-activates both viral and cellular genes. It has previously been shown that granulocyte macrophage-colony stimulating factor (GM-CSF) is constitutively expressed in HTLV-1 infected cells and in cells artificially expressing tax. We show here that the GM-CSF promoter is tax responsive in fibroblasts and T cells, whereas the granulocyte (G)-CSF promoter is tax responsive only in fibroblasts. The tax protein can activate cellular genes through a least two families of transcription factors; the NF-kB/rel and CREB/ATF families. We have used mutant tax proteins to show that the activation of NF-kB proteins is essential for tax trans-activation of both the GM-CSF and G-CSF promoters. The ability of tax to activate CREB/ATF proteins is also essential for GM-CSF transactivation. We have identified a 44 bp region of the GM-CSF promoter that contains tax responsive elements. This region contains a classical NF-kB site, a CK-1 element that can bind the NF-kB p65 protein, as well as a putative ATF binding site. The tax response of the G-CSF promoter requires not only the conserved CK-1 sequence but also an adjacent NF-IL6 binding site that may explain the cell restricted function of the G-CSF promoter.

Bovine respiratory syncytial virus fusion protein gene: sequence analysis of cDNA and expression using a baculovirus vector.

The nucleotide sequence of bovine respiratory syncytial virus (RSV), ATCC strain A51908 fusion (F) glycoprotein gene cDNA was determined. The amino acid sequence deduced was then compared to those of two different isolates of bovine RSV, strains RB 94 and 391-2, and the A and B subtypes of human RSV, strains 18537 and A2. The bovine RSV F protein is highly conserved between the three isolates, A51908 has 97% amino acid identity to RB 94, and 99% identity to 391-2. The F proteins of both the A and B types of human RSV are 81% identical to that of A51908. The cDNA clone was expressed using a baculovirus vector and the expressed recombinant F protein produced in SF9 cells was characterized by Western blot analysis. The recombinant F protein was post-translationally cleaved into the active form and reacted with serum from bovine RSV-infected calves.

Immunoglobulin E responses and lung pathology resulting from aerosol exposure of calves to respiratory syncytial virus and Micropolyspora faeni.

Bovine respiratory syncytial virus (BRSV) infection of calves has been associated with a type-I hypersensitivity syndrome not unlike respiratory syncytial virus-associated pulmonary symptomatology in humans. To study the mechanism of pulmonary pathology in calves and define the relationship with both viral-specific IgE response and IgE titers to concurrent aerosol of Micropolyspora faeni (Mf) we subjected groups of calves to inhalation of Mf during acute BRSV infection. The calves were divided into 4 groups: exposed to virus only (group 1); exposed to aerosolized Mf over a 24-day period and then challenged with Mf during BRSV infection (group 2); similarly exposed to aerosolized Mf and then challenged with Mf without BRSV infection (group 3) and exposed to aerosolized Mf, infected with virus but not challenged with Mf (group 4). All calves were followed for appearance of IgE-specific responses to both BRSV and Mf, clinical disease expression, and pulmonary pathology including viral and allergen localization by immunohistology. Our data indicate an association of BRSV-specific IgE with increased development of lung pathology and clinical disease expression and an enhancing effect of aerosolized Mf on induction of virus-specific IgE. The presence of BRSV-specific IgE was solely in calves with the most significant macroscopic and microscopic pneumonic lesions.