Dominant effector genetics in mammalian cells.

We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.

Murine B cell proliferation and protection from apoptosis with an antibody against a 105-kD molecule: unresponsiveness of X-linked immunodeficient B cells.

We established a novel monoclonal antibody, RP/14, that can protect B cells from apoptosis induced by irradiation or dexamethasone. A molecule recognized by RP/14 (the RP antigen) was expressed on B cells with B220bright, IgMdull, and IgDbright. Immunoprecipitation experiments revealed that RP/14 recognized a monomeric protein with an approximate molecular mass of 105 kD. Stimulation of B cells with RP/14 for 48 h induced B cell proliferation and blastogenesis. In contrast to B cells of wild-type mice, X-linked immunodeficient (XID) B cells did not proliferate upon stimulation with RP/14, although the RP antigen was expressed to the same extent as that of wild-type B cells. These results suggest that the RP antigen-mediated signaling pathway is important for rescuing B cells from apoptosis and is deficient in XID B cells.

Receptors for T cell-replacing factor/interleukin 5. Specificity, quantitation, and its implication.

T cell-replacing factor (TRF)/IL-5 is a glycosylated polypeptide that acts as a key factor for B cell growth and differentiation. Since IL-5 action is probably mediated by specific cell surface receptor(s), we have characterized the binding of IL-5 to cells using biosynthetically [35S]methionine-labeled IL-5 and 125I-IL-5 that had been prepared using Bolton-Hunter reagent. The radiolabeled IL-5 binds specifically to BCL1-B20 (in vitro line) (a murine chronic B cell leukemic cell line previously shown to differentiate into IgM-secreting cells in response to IL-5) within 10 min at 37 degrees C. There are two classes of binding sites with high affinity (Kd = 66 pM) and low affinity (Kd = 12 nM) for IL-5 and an average number of binding sites for high affinity and for low affinity were 400 and 7,500 per cell, respectively. The specificity of binding of radiolabeled IL-5 has been confirmed by demonstrating that only unlabeled IL-5 and anti-IL-5 mAb but not by IL-1, IL-2, IL-3, IFN-gamma, and GM-CSF inhibit radiolabeled IL-5 binding to BCL1-B20 cells. Treatment of surface-bound radiolabeled IL-5 with bivalent crosslinkers identified a membrane polypeptide of Mr 46,500 to which IL-5 is crosslinked. A variety of cell types have been surveyed for the capacity to bind specifically radiolabeled IL-5 with high affinity. BCL1 cells MOPC104E (murine myeloma cell line) expressed IL-5-R, whereas BAL. 17 and L10 A (B cell lymphoma) did not. T cell line, mastocytoma cell line, or macrophage tumor cell line did not display detectable levels of IL-5-R. were hardly detectable on normal resting B cells but were expressed on LPS-activated B cells, fitting the function of IL-5 that acts on activated B cells for their differentiation into Ig-secreting cells. Intriguingly, early B cell lines (J-87 and T-88) that grow in the presence of IL-5 expressed significant but low numbers of high-affinity binding sites for IL-5. The biological effects of IL-5 were mediated by high-affinity binding sites. The identification and characterization of IL-5-R should provide new insight into the apparent diverse biological activities of IL-5.

Delayed progression of a murine retrovirus-induced acquired immunodeficiency syndrome in X-linked immunodeficient mice.

The murine acquired immunodeficiency syndrome (MAIDS) caused by defective LP-BM5 murine leukemia virus (MuLV) is a disease that shows severe immunodeficiency with abnormal lymphoproliferation, and hypergammaglobulinemia in susceptible C57BL/6 (B6) mice. To examine the cellular mechanisms of development of MAIDS, we injected LP-BM5 MuLV intraperitoneally into B6 mice bearing the X chromosome-linked immunodeficiency (xid). xid mice lack functionally mature B cells including Ly-1 B cells (also known as B-1 cells). All B6 mice died by 20 wk after LP-BM5 MuLV inoculation. In marked contrast, xid mice have continued to survive without any sign of MAIDS-related symptoms till at least 20 wk after the inoculation. The delayed progression of MAIDS in xid mice appears to depend on xid mutation, according to our experiments using both sexes of (B6.xid x B6)F1 and (B6 x B6.xid)F1 mice. Furthermore, Ly-1 B cells, enriched by a FACS, were shown to integrate the defective genome and appeared to be a major virus-infected B cell population. Our data corroborate that Ly-1 B cells play an important role in the induction and progression of MAIDS.

Transforming growth factor beta induces IgA production and acts additively with interleukin 5 for IgA production.

Effects of transforming growth factor beta (TGF-beta) on IgA production by LPS-stimulated B cells have been studied. TGF-beta itself could augment polyclonal IgA production in concomitant inhibition of polyclonal IgM and IgG1 production. Furthermore, TGF-beta and IL-5 additively augmented IgA production. TGF-beta exerted its activity early in the culture (by 2 d in a 5-d culture) and IL-5 was required late in the culture. Surface IgA- (sIgA-) B cells responded to TGF-beta for the development of IgA-secreting cells. By contrast, sIgA+ B cells, but not sIgA- B cells, responded to IL-5 for IgA production. These results suggest that TGF-beta has a differential role in the induction of IgA production from IL-5 on murine-activated B cells.

Transgenic mice expressing a B cell growth and differentiation factor gene (interleukin 5) develop eosinophilia and autoantibody production.

Interleukin 5 (IL-5) has been suggested to be involved in the growth and differentiation of B cells and eosinophils. Especially, Ly-1+ B cells, which have been considered to produce autoantibodies, are selectively developed by this lymphokine in long-term bone marrow culture. To envisage the possible engagement of IL-5 in the development of these cells in vivo, transgenic mice carrying the mouse IL-5 gene ligated with a metallothionein promoter were generated. Transgenic mice carrying the IL-5 gene exhibited elevated levels of IL-5 in the serum and an increase in the levels of serum IgM and IgA. A massive eosinophilia in peripheral blood, bone marrow, and spleen, and an infiltration of muscle and liver with eosinophils, were observed. When cadmium-containing saline was injected intraperitoneally into transgenic mice, IL-5 production was augmented about five times within 24 h, and a distinctive Ly-1+ B cell population became apparent in the spleen after 5 d. IL-5 receptors were detected on those cells by monoclonal antibodies against IL-5 receptors. Another interesting finding in these transgenic mice was an increase in polyreactive anti-DNA antibodies of IgM class. It is suggested, therefore, that aberrant expression of the IL-5 gene may induce accumulation of Ly-1+ B cells and eosinophils. Furthermore, this IL-5 transgenic mouse can be a model mouse for eosinophilia, and we can determine the role of IL-5 in the differentiation of Ly-1+ B cells and eosinophils by using this mouse.

IL-5 receptor-mediated tyrosine phosphorylation of SH2/SH3-containing proteins and activation of Bruton's tyrosine and Janus 2 kinases.

Interleukin 5 (IL-5) induces proliferation and differentiation of B cells and eosinophils by interacting with its receptor (IL-5R) which consists of two distinct polypeptide chains, alpha and beta (beta c). Although both IL-5R alpha and beta c lack a kinase catalytic domain, IL-5 is capable of inducing tyrosine phosphorylation of cellular proteins. We investigated the role of IL-5R alpha in tyrosine phosphorylation of molecules involved in IL-5 signal transduction, using an IL-5-dependent early B cell line, Y16 and transfectants expressing intact or mutant IL-5R alpha together with intact beta c. The results revealed that the transfectants expressing truncated IL-5R alpha, which entirely lacks a cytoplasmic domain, together with beta c, showed neither protein-tyrosine phosphorylation nor proliferation in response to IL-5. This confirms that IL-5R alpha plays a critical role in protein-tyrosine phosphorylation which triggers cell growth. IL-5 stimulation results in rapid tyrosine phosphorylation of beta c and proteins containing Src homology 2 (SH2) and/or SH3 domains such as phosphatidyl-inositol-3 kinase, Shc, Vav, and HS1, suggesting their involvement in IL-5-mediated signal transduction. IL-5 stimulation significantly enhanced activities of Janus 2 and B cell-specific Bruton's tyrosine kinases (JAK2 and Btk) and increased the tyrosine phosphorylation of JAK2 kinase. These results and recent data on signaling of growth factors taken together, multiple biochemical pathways driven by tyrosine kinases such as JAK2 and Btk are involved in IL-5 signal transduction.

Novel monoclonal antibodies specific for human cardiac myosin light-chain 1: useful tools for analysis of normal and pathological hearts.

To investigate the developmental, physiological and pathophysiological roles of human cardiac myosin light-chain 1 (LC1s), we developed two novel monoclonal antibodies (KA1 and KB1) against human cardiac LC1s and examined LC1s in normal and pathological hearts immunohistochemically. KA1 and KB1 were specific only for atrial LC1 (ALC1) and for both ALC1 and ventricular LC1 (VLC1), respectively, in human hearts. Among human tissues tested, including skeletal muscle, vascular smooth muscle, and liver, KA1 did not crossreact with proteins in any other tissues than atria, whereas KB1 crossreacted with the slow-type LC1 of skeletal muscle. Among adult mammalian hearts of several other species including pig, dog, hamster, and rat, KA1 and KB1 crossreacted only with ALC1 and with both ALC1 and VLC1, respectively. ALC1 was strongly and uniformly observed in human fetal atria and ventricles and in normal adult human atria, but sporadically in normal adult human ventricles. In the overloaded ventricle (dilated cardiomyopathy), ALC1 was highly augmented but not uniform. These results suggest that the fetal VLC1 is immunohistochemically identical to the adult type of ALC1 and that ALC1 is expressed homogeneously in human fetal ventricles and sporadically in normal adult ventricles, and is re-expressed heterogeneously and in an increased amount in the overloaded ventricle.

Regulatory effect of anti-interleukin-5 monoclonal antibody on intestinal worm burden in a primary infection with strongyloides venezuelensis in mice.

The intestinal worm burden in Strongyloides venezuelensis-infected mice was influenced by treatment with anti-interleukin-5 (IL-5) monoclonal antibody (NC17) when NC17 was given to mice 3-7 days before infection. The present study has examined the involvement of IL-5 in susceptibility at different in the development of the parasite in the host. The results show that the number of tissue-migrating larvae recovered from the lungs in a primary infection was not affected by anti-IL-5 monoclonal antibody treatment, whereas intestinal worm counts increased in mice treated with 0.25-1 mg of NC17. In mice treated with 0.1 mg of NC17, adult worm recovery was not significantly different from non-treated controls. Peripheral and tissue eosinophilia were not observed in the early phase of infection (days 4-8). Six days after transfer of lung-stage larvae to NC17-treated mice, adult worm recovery was higher than that of control mice. These results suggest that non-eosinophil response(s), which were dependent on IL-5, were involved in the initial establishment of the intestinal stage of S. venezuelensis in mice. We discuss the mechanisms that control the susceptibility to the parasite from the viewpoint of host defence.

Functional analysis of thymic B cells.

The expression of low- and high-affinity interleukin-5 receptors (IL-5Rs) on thymic B cells and reactivity of thymic B cells to IL-5 was investigated. Thymic B cells consist of two populations (CD5+ and CD5- B cells), as previously described. Three-color FACS analyses using anti-CD5, anti-IgK, and anti-IL-5R mAbs reveal that approximately 60% of both populations (CD5+ and CD5-) in the thymus possess IL-5R, detected by mAb H-7. In Scatchard plot analyses, IL-5Rs on thymic B cells are observed as low affinity receptors; the high-affinity IL-5R, which is known to be expressed on some IL-5-activated splenic B blasts or some IL-5-dependent cell line cells, is not clearly detected on thymic B cells. The reactivity of thymic B cells to IL-5 is found to be significantly lower than that of splenic B cells both in proliferative responses and LPS-induced IgM and IgA antibody responses. These findings are compatible with the expression of the low-affinity IL-5R on thymic B cells. The responsiveness of thymic B cells to either IL-6 or the combination of IL-4, IL-5 and IL-6 is also lower than that of splenic B cells. Furthermore, the thymic B cells are found to induce neonatal tolerance. Therefore, thymic B cells act as antigen-presenting cells in the negative selection of thymocytes, rather than as antibody-producing cells under the influence of foreign antigens and/or regulatory cytokines.

Maintenance of CD5+ B cells at an early developmental stage by interleukin-5: evidence from immunoglobulin gene usage in interleukin-5 transgenic mice.

We have characterized the development and expansion of CD5+ B cells in interleukin-5 (IL-5) transgenic mice in terms of autoantibody production and immunoglobulin gene usage. CD5+IL-5R alpha+ B cells maintained in the presence of IL-5 secreted fewer autoantibodies and had fewer N nucleotides at the 3' end of the D elements compared with CD5- B cells. The reduction in nucleotides, along with the finding that CD5+IL-5R alpha+ B cells in IL-5 transgenic mice use Q52 families more frequently than age-matched control B cells, also suggests that these cells have the characteristics of fetus-type B cells and represent an early stage of B-cell development. All of the VH11 families were expressed with JH1 and the Q52 families were frequently expressed with JH1. Furthermore, JH proximal DQ52 was frequently used in IL-5 transgenic mice. All of these characteristics in terms of immunoglobulin gene usage have been described for CD5+ B cells. These results suggest that IL-5 maintains CD5+ B cells that have a fetus-type of immunoglobulin gene usage. This cytokine could be responsible for prolonging the life span of immature CD5+ B cells, which subsequently mature to CD5- B cells that secrete polyreactive natural antibodies.

Effect of anti-IL-5 monoclonal antibody on allergic bronchial eosinophilia and airway hyperresponsiveness in mice.

The effect of pretreatment with rat anti-murine interleukin-5 (IL-5) antibody on antigen-induced bronchial eosinophilia and bronchial reactivity to acetylcholine in mice were studied. Three inhalations of an antigen by actively sensitized animals resulted in an increase in airway reactivity to acetylcholine. Twenty-four hours after the final inhalation, the number of leukocytes (mononuclear cells and eosinophils) and the amount of IL-5 in BALF increased significantly. Anti-IL-5 monoclonal antibody inhibited the antigen-induced increase of eosinophils with little effect on bronchial hyperreactivity.

Increased expression of atrial myosin light chain 1 in the overloaded human left ventricle: possible expression of fetal type myocytes.

We examined the isoforms of myosin light chain 1 in the human left ventricles using pyrophosphate and sodium dodecyl sulfate polyacrylamide gel electrophoresis, peptide mapping, and immunoblotting with monoclonal antibodies against human atrial light chain 1. The relationship between hemodynamic parameters and light chain 1 isoform composition was compared among groups of patients with hypertrophic cardiomyopathy (n = 8), dilated cardiomyopathy (n = 9) and aortic stenosis (n = 5), and controls (n = 6). (1) The light chain 1, which differed from ventricular light chain 1 found in the normal adult ventricle, was highly expressed in the overload left ventricle, and was identical to atrial and fetal ventricular light chain 1 with respect to the physiochemical and immunological properties. (2) The expression of atrial/fetal light chain 1 was augmented in the subendocardial area in comparison with the mid- or subepicardial areas in the hypertrophied left ventricles. (3) The values (%) of the relative expression of atrial/fetal light chain 1 to total light chains 1 determined by densitometric analysis were significantly higher in patients with dilated cardiomyopathy (40.2 +/- 5.8) and those with aortic stenosis (43.1 +/- 6.2) than in the controls (16.9 +/- 2.5) (p less than 0.01), but there was no significant difference between the patients with hypertrophic cardiomyopathy (28.0 +/- 3.7) and the controls. (4) The values of the ratio significantly correlated with those of peak circumferential wall stress (r = 0.53, p less than 0.005). These results suggest that atrial/fetal light chain 1 is expressed in the left ventricles in response to the increased hemodynamic load.

Stable, stoichiometric delivery of diverse protein functions.

As contemporary "genomics" steadily reveals an increasing number of novel gene sequences, the need for efficient methodologies to functionally characterize these genes in vivo increases significantly. Reliable coupling of target gene expression to a variety of surrogate reporter functions is critical to properly assay novel gene function in complex cell populations. Ideally, independent target and reporter proteins would be derived from a single open reading frame creating a stoichiometric relationship without obscuring subcellular localization. We report here effective strategies for assaying gene function through the stable production of chimeric polyproteins, processed intracellularly by inclusion of an intervening 19-amino-acid sequence from the 2A region of the Foot and Mouth Disease virus. Using drug-resistance and flow cytometry-based assay systems, we demonstrate that diverse protein functions are effectively delivered to various cell types by retroviral constructs as single 2A-cleaved polyproteins. For example, cells infected with a retrovirus encoding a nuclear cell cycle regulator, linked via the 2A-motif to a marker membrane protein, showed a direct correlation between cell cycle arrest and surface marker level. This demonstrates the utility of this methodology for stable and stoichiometric delivery of distinctly localized protein functionalities. In particular, the ability to exploit multiple cellular functions will serve to accelerate the functional characterization of gene products and facilitate novel gene therapy approaches.

Isolated oculomotor nerve palsy following midbrain infarction.

A 62 year-old male patient presented with isolated oculomotor nerve palsy following a small infarction of the medial ventral midbrain, documented by magnetic resonance imaging. There was a 12-year history of hypertension, but no diabetes mellitus. Angiography revealed atherosclerosis of the paramedian mesencephalic arteries. Magnetic resonance imaging may be useful in patients with small brain stem infarctions.

Role of interleukin-5 in local accumulation of eosinophils in mouse allergic peritonitis.

The injection of ragweed pollen extracts into the peritoneal cavity of actively immunized mice caused an accumulation of eosinophils in the cavity. We investigated the role of interleukin-5 (IL-5), one of the T-cell-derived factors, in this in vivo local accumulation. Ciclosporin, an immunosuppressant that mainly suppresses T cell functions, dose-dependently inhibited the in vivo local accumulation. Injections of the neutralizing antibody to IL-5 into the cavity decreased the number of the accumulating eosinophils. When the peritoneal cells of the immunized mice were cultured in the presence of the antigen in vitro, IL-5 activity was detected in the culture supernatant. The results indicate that IL-5 plays an important role in the in vivo local accumulation of eosinophils after an antigen challenge.