Non-eosinophilic airway hyper-reactivity in mice, induced by IFN-γ producing CD4(+) and CD8(+) lung T cells, is responsive to steroid treatment.

Non-eosinophilic asthma is characterized by infiltration of neutrophils into the lung and variable responsiveness to glucocorticoids. The pathophysiological mechanisms have not been characterized in detail. Here, we present an experimental asthma model in mice associated with non-eosinophilic airway inflammation and airway hyper-responsiveness (AHR). For this, BALB/c mice were sensitized by biolistic DNA immunization with a plasmid encoding the model antigen ß-galactosidase (pFascin-ßGal mice). For comparison, eosinophilic airway inflammation was induced by subcutaneous injection of ßGal protein (ßGal mice). Intranasal challenge of mice in both groups induced AHR to a comparable extent as well as recruitment of inflammatory cells into the airways. In contrast to ßGal mice, which exhibited extensive eosinophilic infiltration in the lung, goblet cell hyperplasia and polarization of CD4(+) T cells into Th2 and Th17 cells, pFascin-ßGal mice showed considerable neutrophilia, but no goblet cell hyperplasia and a predominance of Th1 and Tc1 cells in the airways. Depletion studies in pFascin-ßGal mice revealed that CD4(+) and CD8(+) cells cooperated to induce maximum inflammation, but that neutrophilic infiltration was not a prerequisite for AHR induction. Treatment of pFascin-ßGal mice with dexamethasone before intranasal challenge did not affect neutrophilic infiltration, but significantly reduced AHR, infiltration of monocytes and lymphocytes as well as content of IFN-γ in the bronchoalveolar fluid. Our results suggest that non-eosinophilic asthma associated predominantly with Th1/Tc1 cells is susceptible to glucocorticoid treatment. pFascin-ßGal mice might represent a mouse model to study pathophysiological mechanisms proceeding in the subgroup of asthmatics with non-eosinophilic asthma that respond to inhaled steroids.

Antigen dose-dependent suppression of murine IgE responses is mediated by CD4(-)CD8(-) double-negative T cells.

BACKGROUND: The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. OBJECTIVE: The aim of the study was to identify immune cells that are involved in antigen dose-dependent regulation of IgE formation. METHODS: Wild-type mice as well as T helper (Th)1-deficient IL-12p40(-/-) and IFN-gamma(-/-) mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen-dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT-PCR analysis. Splenocytes or T cell subpopulations were transferred into naïve mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. RESULTS: Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL-4, IL-5 and IL-13, but no induction of IFN-gamma production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen- and isotype-specific manner. Neither CD4(+) nor CD8(+) T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4(-)CD8(-) double-negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. CONCLUSION: Our data demonstrate that CD4(-)CD8(-) dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.

Sm protein-Sm site RNA interactions within the inner ring of the spliceosomal snRNP core structure.

Seven Sm proteins, E, F, G, D1, D2, D3 and B/B', assemble in a stepwise manner onto the single-stranded Sm site element (PuAU(4-6)GPu) of the U1, U2, U4 and U5 spliceosomal snRNAs, resulting in a doughnut-shaped core RNP structure. Here we show by UV cross-linking experiments using an Sm site RNA oligonucleotide (AAUUUUUGA) that several Sm proteins contact the Sm site RNA, with the most efficient cross-links observed for the G and B/B' proteins. Site-specific photo-cross-linking revealed that the G and B/B' proteins contact distinct uridines (in the first and third positions, respectively) in a highly position-specific manner. Amino acids involved in contacting the RNA are located at equivalent regions in both proteins, namely in loop L3 of the Sm1 motif, which has been predicted to jut into the hole of the Sm ring. Our results thus provide the first evidence that, within the core snRNP, multiple Sm protein-Sm site RNA contacts occur on the inner surface of the heptameric Sm protein ring.

Variations in normal color vision. I. Cone-opponent axes.

Early postreceptoral color vision is thought to be organized in terms of two principal axes corresponding to opposing L- and M-cone signals (LvsM) or to S-cone signals opposed by a combination of L- and M-cone signals (SvsLM). These cone-opponent axes are now widely used in studies of color vision, but in most cases the corresponding stimulus variations are defined only theoretically, based on a standard observer. We examined the range and implications of interobserver variations in the cone-opponent axes. We used chromatic adaptation to empirically define the LvsM and SvsLM axes and used both thresholds and color contrast adaptation to determine sensitivity to the axes. We also examined the axis variations implied by individual differences in the color matching data of Stiles and Burch [Opt. Acta 6, 1 (1959)]. The axes estimated for individuals can differ measurably from the nominal standard-observer axes and can influence the interpretation of postreceptoral color organization (e.g., regarding interactions between the two axes). Thus, like luminance sensitivity, individual differences in chromatic sensitivity may be important to consider in studies of the cone-opponent axes.

Variations in normal color vision. II. Unique hues.

We examined individual differences in the color appearance of nonspectral lights and asked how they might be related to individual differences in sensitivity to chromatic stimuli. Observers set unique hues for moderately saturated equiluminant stimuli by varying their hue angle within a plane defined by the LvsM and SvsLM cone-opponent axes that are thought to characterize early postreceptoral color coding. Unique red settings were close to the +L pole of the LvsM axis, while green, blue, and yellow settings clustered along directions intermediate to the LvsM and SvsLM axes and thus corresponded to particular ratios of LvsM to SvsLM activity. Interobserver differences in the unique hues were substantial. However, no relationship was found between hue settings and relative sensitivity to the LvsM and SvsLM axes. Moreover, interobserver variations in different unique hues were uncorrelated and were thus inconsistent with a common underlying factor such as relative sensitivity or changes in the spectral sensitivities of the cones. Thus for the moderately saturated lights we tested, the unique hues appear largely unconstrained by normal individual differences in the cone-opponent axes. In turn, this suggests that the perceived hue for these stimuli does not depend on fixed (common) physiological weightings of the cone-opponent axes or on fixed (common) color signals in the environment.

Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease of spinal motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. SMN is part of a macromolecular complex that contains the SMN-interacting protein 1 (SIP1) and spliceosomal Sm proteins. Although it is clear that SIP1 as a component of this complex is essential for spliceosomal uridine-rich small ribonucleoprotein (U snRNP) assembly, the role of SMN and its functional interactions with SIP1 and Sm proteins are poorly understood. Here we show that the central region of SMN comprising a tudor domain facilitates direct binding to Sm proteins. Strikingly, the SMA-causing missense mutation E134K within the tudor domain severely reduced the ability of SMN to interact with Sm proteins. Moreover, antibodies directed against the tudor domain prevent Sm protein binding to SMN and abolish assembly of U snRNPs in vivo. Thus, our data show that SMN is an essential U snRNP assembly factor and establish a direct correlation between defects in the biogenesis of U snRNPs and SMA.

Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner.

The association of Sm proteins with U small nuclear RNA (snRNA) requires the single-stranded Sm site (PuAU(4-6)GPu) but also is influenced by nonconserved flanking RNA structural elements. Here we demonstrate that a nonameric Sm site RNA oligonucleotide sufficed for sequence-specific assembly of a minimal core ribonucleoprotein (RNP), which contained all seven Sm proteins. The minimal core RNP displayed several conserved biochemical features of native U snRNP core particles, including a similar morphology in electron micrographs. This minimal system allowed us to study in detail the RNA requirements for Sm protein-Sm site interactions as well as the kinetics of core RNP assembly. In addition to the uridine bases, the 2' hydroxyl moieties were important for stable RNP formation, indicating that both the sugar backbone and the bases are intimately involved in RNA-protein interactions. Moreover, our data imply that an initial phase of core RNP assembly is mediated by a high affinity of the Sm proteins for the single-stranded uridine tract but that the presence of the conserved adenosine (PuAU.) is essential to commit the RNP particle to thermodynamic stability. Comparison of intact U4 and U5 snRNAs with the Sm site oligonucleotide in core RNP assembly revealed that the regions flanking the Sm site within the U snRNAs facilitate the kinetics of core RNP assembly by increasing the rate of Sm protein association and by decreasing the activation energy.

Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs.

The U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) involved in pre-mRNA splicing contain seven Sm proteins (B/B', D1, D2, D3, E, F, and G) in common, which assemble around the Sm site present in four of the major spliceosomal small nuclear RNAs (snRNAs). These proteins share a common sequence motif in two segments, Sm1 and Sm2, separated by a short variable linker. Crystal structures of two Sm protein complexes, D3B and D1D2, show that these proteins have a common fold containing an N-terminal helix followed by a strongly bent five-stranded antiparallel beta sheet, and the D1D2 and D3B dimers superpose closely in their core regions, including the dimer interfaces. The crystal structures suggest that the seven Sm proteins could form a closed ring and the snRNAs may be bound in the positively charged central hole.

An unusual chemical reactivity of Sm site adenosines strongly correlates with proper assembly of core U snRNP particles.

The small nuclear ribonucleoprotein particles (snRNP) U1, U2, U4, and U5 contain a common set of eight Sm proteins that bind to the conserved single-stranded 5'-PuAU3-6GPu-3' (Sm binding site) region of their constituent U snRNA (small nuclear RNA), forming the Sm core RNP. Using native and in vitro reconstituted U1 snRNPs, accessibility of the RNA within the Sm core RNP to chemical structure probes was analyzed. Hydroxyl radical footprinting of in vitro reconstituted U1 snRNP demonstrated that riboses within a large continuous RNA region, including the Sm binding site, were protected. This protection was dependent on the binding of the Sm proteins. In contrast with the riboses, the phosphate groups within the Sm core site were accessible to modifying reagents. The invariant adenosine residue at the 5' end, as well as an adenosine two nucleotides downstream of the Sm binding site, showed an unexpected reactivity with dimethyl sulfate. This novel reactivity could be attributed to N7-methylation of the adenosine and was not observed in naked RNA, indicating that it is an intrinsic property of the RNA- protein interactions within the Sm core RNP. Further, this reactivity was observed concomitantly with formation of the Sm subcore intermediate during Sm core RNP assembly. As the Sm subcore can be viewed as the commitment complex in this assembly pathway, these results suggest that the peculiar reactivity of the Sm site adenosine bases may be diagnostic for proper assembly of the Sm core RNP. Consistent with this idea, a strong correlation was found between the unusual N7-A methylation sensitivity of the Sm core RNP and its ability to be imported into the nucleus of Xenopus laevis oocytes.

A major, novel systemic lupus erythematosus autoantibody class recognizes the E, F, and G Sm snRNP proteins as an E-F-G complex but not in their denatured states.

OBJECTIVE: To determine whether the E, F, and G Sm proteins present antigenic determinants recognized by systemic lupus erythematosus (SLE) patient sera, and if so, whether the antigenicity depends on the native conformations of the polypeptides and/or is E-F-G complex restricted. METHODS: Radioimmunoprecipitation, epitope tagging, expression polymerase chain reaction, in vitro translation, in vitro reconstitution, and immunoblotting. RESULTS: Most of the anti-Sm SLE patient sera tested reacted with one or more of the E, F, and G proteins in immunoprecipitation studies but not on immunoblots. All sera, however, highly efficiently immunoprecipitated the E-F-G complex. This complex recognition was detected exclusively in anti-Sm patient sera but not in patient sera with other serotypes. CONCLUSION: We demonstrate the presence of a novel and abundant anti-Sm autoantibody class in SLE patient sera which exclusively or predominantly recognizes conformational Sm epitopes present on the E-F-G complex but not on the denatured proteins. This complex recognition is highly specific for sera of the anti-Sm serotype and may be relevant for clinical diagnosis as well as for understanding the etiology of anti-Sm autoantibody production.

The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro.

Stable association of the eight common Sm proteins with U1, U2, U4 or U5 snRNA to produce a spliceosomal snRNP core structure is required for snRNP biogenesis, including cap hypermethylation and nuclear transport. Here, the assembly of snRNP core particles was investigated in vitro using both native HeLa and in vitro generated Sm proteins. Several RNA-free, heteromeric protein complexes were identified, including E.F.G, B/B'.D3 and D1.D2.E.F.G. While the E.F.G complex alone did not stably bind to U1 snRNA, these proteins together with D1 and D2 were necessary and sufficient to form a stable U1 snRNP subcore particle. The subcore could be chased into a core particle by the subsequent addition of the B/B'.D3 protein complex even in the presence of free competitor U1 snRNA. Trimethylation of U1 snRNA's 5' cap, while not observed for the subcore, occurred in the stepwise-assembled U1 snRNP core particle, providing evidence for the involvement of the B/B' and D3 proteins in the hypermethylation reaction. Taken together, these results suggest that the various protein heterooligomers, as well as the snRNP subcore particle, are functional intermediates in the snRNP core assembly pathway.

snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein-protein interactions.

The spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 share eight proteins B', B, D1, D2, D3, E, F and G which form the structural core of the snRNPs. This class of common proteins plays an essential role in the biogenesis of the snRNPs. In addition, these proteins represent the major targets for the so-called anti-Sm auto-antibodies which are diagnostic for systemic lupus erythematosus (SLE). We have characterized the proteins F and G from HeLa cells by cDNA cloning, and, thus, all human Sm protein sequences are now available for comparison. Similar to the D, B/B' and E proteins, the F and G proteins do not possess any of the known RNA binding motifs, suggesting that other types of RNA-protein interactions occur in the snRNP core. Strikingly, the eight human Sm proteins possess mutual homology in two regions, 32 and 14 amino acids long, that we term Sm motifs 1 and 2. The Sm motifs are evolutionarily highly conserved in all of the putative homologues of the human Sm proteins identified in the data base. These results suggest that the Sm proteins may have arisen from a single common ancestor. Several hypothetical proteins, mainly of plant origin, that clearly contain the conserved Sm motifs but exhibit only comparatively low overall homology to one of the human Sm proteins, were identified in the data base. This suggests that the Sm motifs may also be shared by non-spliceosomal proteins. Further, we provide experimental evidence that the Sm motifs are involved, at least in part, in Sm protein-protein interactions. Specifically, we show by co-immunoprecipitation analyses of in vitro translated B' and D3 that the Sm motifs are essential for complex formation between B' and D3. Our finding that the Sm proteins share conserved sequence motifs may help to explain the frequent occurrence in patient sera of anti-Sm antibodies that cross-react with multiple Sm proteins and may ultimately further our understanding of how the snRNPs act as auto-antigens and immunogens in SLE.

cDNA cloning of the Sm proteins D2 and D3 from human small nuclear ribonucleoproteins: evidence for a direct D1-D2 interaction.

The major small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5 share a set of common proteins denoted B/B', D1, D2, D3, E, F, and G which play an important part in the biogenesis of the snRNPs. In addition, there is a link between the common proteins and autoimmunity; the three D proteins, together with B/B', are the major autoantigens for the so-called anti-Sm antibodies often produced by patients suffering from systemic lupus erythematosus. Here we describe the characterization of the human proteins D2 and D3 by cDNA cloning and immunological methods. D2 and D3 are encoded by distinct genes and are 118 and 126 amino acids in length, respectively. Both proteins prepared by in vitro translation exhibit Sm epitopes and can be precipitated by anti-Sm autoantibodies. They react differently with various patient sera, in a manner consistent with the reaction pattern on immunoblots of the D proteins isolated from HeLa cells. D1 and D2 synthesized in vitro form specific complexes, a result that is significant for the assembly pathway of the various core proteins into an snRNP's core ribonucleoprotein structure. The D3 protein is homologous to the human D1 protein, showing an overall amino acid sequence identity of 29%, including two regions with over 60% identity. D2 has less than 15% sequence identity with D1 and D3. A data bank search revealed a striking similarity (with more than 40% sequence identity) between human D3 and a Saccharomyces cerevisiae gene, previously published as the 5' flanking gene of yeast pep3 [Preston, R.A., Manolson, M., Becherer, K., Weidenhammer, E., Kirkpatrick, D., Wright, R. & Jones, E. (1991) Mol. Cell. Biol. 11, 5801-5812], suggesting that this gene encodes the yeast homologue of the human D3 protein.

Improved method for identification of proteins using two-dimensional electrophoresis with immobilized pH gradient isoelectric focusing.

Recent advances in protein sequence analysis now permit the determination of partial N-terminal and internal primary structure from low picomole quantities of protein. The major remaining hurdles to sequence analysis of small amounts of protein are the identification, isolation, and handling of microgram and submicrogram quantities of protein. The technique of two-dimensional electrophoresis using immobilized pH gradient isoelectric focusing circumvents many of these problems. However, poor correlation between the first and second dimension have prevented use of this technique for the identification of some proteins which can only be assayed prior to the denaturing conditions used in the second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis procedure. An improved method is presented which allows correlation of the native biological activity (first dimension) to a silver stained protein (second dimension) with a high degree of confidence.