A small, nonpeptidyl mimic of granulocyte-colony-stimulating factor [see commetns].

A nonpeptidyl small molecule SB 247464, capable of activating granulocyte-colony-stimulating factor (G-CSF) signal transduction pathways, was identified in a high-throughput assay in cultured cells. Like G-CSF, SB 247464 induced tyrosine phosphorylation of multiple signaling proteins and stimulated primary murine bone marrow cells to form granulocytic colonies in vitro. It also elevated peripheral blood neutrophil counts in mice. The extracellular domain of the murine G-CSF receptor was required for the activity of SB 247464, suggesting that the compound acts by oligomerizing receptor chains. The results indicate that a small molecule can activate a receptor that normally binds a relatively large protein ligand.

Hydrazinonaphthalene and azonaphthalene thrombopoietin mimics are nonpeptidyl promoters of megakaryocytopoiesis.

High-throughput screening for the induction of a luciferase reporter gene in a thrombopoietin (TPO)-responsive cell line resulted in the identification of 4-diazo-3-hydroxy-1-naphthalenesulfonic acids as TPO mimics. Modification of the core structure and adjustment of unwanted functionality resulted in the development of (5-oxo-1,5-dihydropyrazol-4-ylidene)hydrazines which exhibited efficacies equivalent to those of TPO in several cell-based assays designed to measure thrombopoietic activity. Furthermore, these compounds elicited biochemical responses in TPO-receptor-expressing cells similar to those in TPO itself, including kinase activation and protein phosphorylation. Potencies for the best compounds were high for such low molecular weight compounds (MW < 500) with EC(50) values in the region of 1-20 nM.

Detection of enhanced cytotoxic T lymphocyte response with spleenic dendritic cells enriched by a simple sequential adherence method.

Dendritic cells (DC), which are present in many tissues, play a critical role in the initiation of the immune response by presenting antigens to T and B lymphocytes. DC are present in tissues as a minority cell type as compared to other APCs. Although clearly distinguished by morphology and surface markers, dendritic cells are difficult to isolate and multiple step procedures including Percoll/Hypaque or BSA gradient centrifugation have been used. Here we describe a simple method for isolating an enriched population of dendritic cells from mouse spleen by sequential adherence to petri dishes. The purity of dendritic cells enriched thereby was found to be above 70%. The identity of these cells was confirmed to be DC by morphology, presence of surface markers, and their functions, e.g., strong allogeneic MLR reaction and induction of antigen-specific cytotoxic T lymphocyte response.

Functional characterization of Con A-responsive Lyt2-positive mouse small intestinal intraepithelial lymphocytes.

The surface phenotypes of resting vs Con A-stimulated intraepithelial lymphocytes (IEL) from mouse small intestine were directly determined by immunofluorescence with double labelling. Both Thy 1.2+, Lyt 2+ and Thy 1.2+ Lyt 2- IEL underwent blastogenesis and expressed interleukin-2 (IL-2) receptors. The Lyt 2+ subsets of IEL (which represent 80% of the total cells) were isolated by panning and shown to proliferate in response to Con A and IL-2, although the frequency of responsive precursors was dramatically lower than that seen in the splenic Lyt 2+ T-cell population (1 in 500 vs 1 in 8, respectively). Con A-stimulated Lyt 2+ IEL produced lymphokines supporting the growth of the interleukin-3 (IL-3)-dependent cell line DA-1, and of the FDC-P2 cell line that proliferates in response to both IL-3 and GM-CSF. The results therefore support the possibility that Lyt 2+ IEL act as inducers of local cell-mediated immune reactions by producing haematopoietic lymphokines.

Limit dilution analysis of mast cell precursor frequency in the gut epithelium of normal and Trichinella spiralis infected mice.

Previous studies have established that the gut nematode Trichinella spiralis induces a dramatic thymus dependent intestinal mastocytosis which peaks within 6 to 12 days after primary oral infection. It is not known, however, if the increase in gut mast cells results from the influx of mast cells or their precursors, or from the expansion and differentiation of mast cell precursors (MCP) that are normally present in the small intestinal epithelium. In the present study, the number of mucosal MCP in the intraepithelial lymphocyte (IEL) population and in bone marrow (BM) cells from normal and 4 day T. spiralis infected mice was compared by culturing the cells at limiting dilutions in medium containing interleukin-3 (IL-3). While the MCP frequency in IEL from infected mice was found to be significantly increased in comparison with that found in normal mice, the numbers of MCP in BM from the two groups were equivalent. Resident intraepithelial mucosal MCP therefore undergo a local expansion before the occurrence of an overt T dependent intestinal mastocytosis. This finding lends support to the view that local mucosal T cells are involved in regulating mast cell numbers in response to intestinal helminth infection.

Functional properties of lymphocytes isolated from murine small intestinal epithelium.

Intraepithelial lymphocytes (IEL) were isolated from murine small intestine using a modification of previously published procedures. Analysis of IEL by immunofluorescence using monoclonal antibodies showed they were predominantly Lyt-2+ cells, with relatively few B cells or macrophages present. IEL cultured at sufficiently high cell densities proliferated in response to concanavalin A (Con A), phytohaemagglutin (PHA) and lipopolysaccharide (LPS). IEL were also capable of recognizing alloantigens in an in vivo graft-versus-host assay and in an in vitro mixed lymphocyte reaction. These studies therefore confirm that murine IEL contain cells with immunologic properties characteristic of typical T lymphocytes.

Lymphokine production by mitogen and antigen activated mouse intraepithelial lymphocytes.

Although most intraepithelial lymphocytes (IEL) in mouse small intestine bear surface markers classically associated with T lymphocytes, the T-cell nature of these cells remains controversial. In the present study IEL from normal mice, or from mice infected with the gut nematode Trichinella spiralis, were therefore tested for their ability to produce T-cell-derived lymphokines in response to in vitro stimulation with concanavalin A (Con A) or with specific worm antigens. The data show that Con A-stimulated IEL produce minimal amounts of IL-2, and intermediate levels of IFN-gamma and IL-3 in comparison to the levels produced by spleen T cells. The FDC-P2 cell line, which proliferates in response to both IL-3 and GM-CSF, was identified as the most sensitive and reproducible indicator of lymphokine activity in supernatants from mitogen-stimulated IEL from normal mice. IEL isolated from mice infected with T. spiralis also produced high levels of FDC-P2 growth factors when challenged in vitro with Trichinella-derived antigens; however, normal IEL did not respond to this stimulus. The data thus provide evidence that antigen-sensitive T cells can arise in (or migrate to) the gut epithelium during gut infection.

Identification of a novel inositol bisphosphate isomer formed in chemoattractant stimulated human polymorphonuclear leukocytes.

Analysis of inositol phosphate formation in chemoattractant-stimulated human polymorphonuclear leukocytes demonstrated the production of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,4-bisphosphate and another inositol bisphosphate isomer not detected in unstimulated cells. Studies in cell sonicates provided evidence that the previously unidentified inositol bisphosphate isomer is produced via the degradation of inositol 1,3,4-trisphosphate. This unidentified inositol bisphosphate peak was purified by high pressure liquid chromatography, and base hydrolyzed to form a mixture of inositol monophosphate isomers. Based on these studies, the unidentified peak was identified as inositol 3,4-bisphosphate. Identification of this isomer defines a new metabolic product derived from the initial inositol 1,4,5-trisphosphate formation, and also suggests another substrate for the inositol 1-phosphatase.

Signal transduction in cells following binding of chemoattractants to membrane receptors.

Binding of chemoattractants to specific cell surface receptors on human polymorphonuclear leukocytes (PMNs) initiates a variety of biologic responses, including directed migration (chemotaxis), release of superoxide anions, and lysosomal enzyme secretion. Chemoattractant receptors belong to a large class of receptors which utilize the hydrolysis of polyphosphoinositides to initiate Ca2+ mobilization and cellular activation. Receptor occupancy leads to phospholipase C-mediated hydrolysis of polyphosphoinositol 4,5-bisphosphate (PIP2) yielding inositol 1,4,5-trisphosphate (IP3) and 1,2 sn-diacylglycerol (DAG). These products synergize to initiate cell activation via calcium mobilization (IP3) and protein kinase C activation (DAG). Pertussis toxin, which ADP-ribosylates and inactivates some GTP binding proteins (G proteins), abolishes all chemoattractant-induced responses, including Ca2+ mobilization, IP3 and DAG production, enzyme secretion, superoxide production and chemotaxis. Direct evidence for chemoattractant receptor: G protein coupling was obtained using PMN membrane preparations which contain a Ca2+-sensitive phospholipase C. Hydrolysis of polyphosphoinositides at resting intracellular Ca2+ levels (100 nm) was only observed when the membranes were stimulated with the chemoattractant N-formyl-methyl-leucyl-phenylalanine (fMet-Leu-Phe) in the presence of GTP. Myeloid cells contain two distinct pertussis toxin substrates of similar molecular weight (40 and 41 kD). The 41 kD substrate resembles Gi, whereas a 40 kD substrate is physically associated with a partially purified fMet-Leu-Phe receptor preparation and may therefore represent a novel G protein involved in chemoattractant-stimulated responses. Metabolism of 1,4,5-IP3 to inositol proceeds via two distinct pathways in PMNs: (1) degradation to 1,4-IP2 and 4-IP1 or (2) conversion to 1,3,4,5-IP4, 1,3,4-IP3, 3,4-IP2 and 3-IP1. Initial formation (0-30 s) of 1,4,5-IP3 and DAG occurs at ambient intracellular Ca2+ levels, whereas formation of 1,3,4-IP3 and a second sustained phase of DAG production (30 s-10 min) require elevated cytosolic Ca2+ influx. The later peak of DAG, which is not derived from phosphoinositides, appears to be required for stimulation of respiratory burst activity. Products formed during activation can feed back to attenuate chemoattractant receptor-mediated stimulation of phospholipase C by uncoupling receptor-G protein-phospholipase C interaction.

Influenza A virus-specific H-2d restricted cross-reactive cytotoxic T lymphocyte epitope(s) detected in the hemagglutinin HA2 subunit of A/Udorn/72.

BALB/c mice immunized with a vaccinia virus recombinant expressing the influenza A virus (A/Udorn/72; subtype H3) hemagglutinin HA2 (H3HA2) induced a strong CD8+ cytotoxic T lymphocyte (CTL) response which was H-2d-restricted. Two peptides, derived from the primary sequence of the H3HA2 and consistent with the predictive motif for Kd-restricted epitopes, were tested for their ability to be presented to the CTLs by P815 cells. Peptides corresponding to the amino acids 93SYNAELLVAL102 and 181GYKDWILWI189 of the HA2 primary sequence sensitized target cells for lysis by the HA2-specific CTLs. Secondary in vitro stimulation with dendritic cells as a source of antigen presenting cells treated with peptide 93-102, or infected with recombinant vaccinia virus (HA2-VACC), induced type A cross-reactive CTLs from A/Udorn/72 immune spleen cells. This CD8+ CTL epitope overlaps with an H3HA2 I-Ad-restricted T helper epitope 96-104 recently reported by others. The H3HA2 epitope described here is the first CTL epitope in the influenza hemagglutinin which is found in all three subtypes of influenza A virus.

T cell subclasses in fetal human ileum.

Lymphocytes within fetal human ileum were studied by immunocytochemistry to determine the appearance of T cells in human small intestine and the role of enteric antigen in the accumulation of cells of the suppressor/cytotoxic phenotype in the gut epithelium. In fetal human gut epithelium, cells bearing the pan T cell marker UCHT1 (CD3) were present in all of the specimens studied (11-19 weeks gestation). Of these, UCHT4+ (CD8, suppressor/cytotoxic phenotype) predominated over the leu3a+ (CD4, helper/inducer phenotype), although the differences were not as marked as in postnatal gut. UCHT1+ cells were also present in the lamina propria, frequently as small aggregates beneath the epithelium.

Influenza virus infection elicits class II major histocompatibility complex-restricted T cells specific for an epitope identified in the NS1 non-structural protein.

A T cell epitope of the influenza virus NS1 molecule was identified and shown to be a determinant used in class II major histocompatibility complex-restricted T cell responses to infectious virus. An I-Ed-restricted BALB/c mouse T hybridoma clone recognizing influenza virus A/Puerto Rico/8/34 (PR8; subtype H1N1) but not A/Udorn/72 (subtype H3N2) secreted lymphokines in response to purified recombinant NS1 or fusion proteins containing amino acids 1 to 81 or 1 to 42 of NS1. As expected for recognition of a non-virion protein, the clone failed to respond to u.v.-inactivated virus. The antigenic determinant was localized by synthetic peptides to amino acids 13 to 32 of NS1, explaining the lack of recognition of A/Udorn/72 virus which has an alanine to valine substitution at position 23 within the determinant. A single intranasal dose of infectious PR8 virus was found to elicit T cells that responded to peptide NS1 13-32, suggesting that this determinant is a significant target of T cells in normal infections. To stimulate helper T cell responses similar to those achieved with infectious virus, influenza virus vaccines may therefore have to include NS1 in addition to virion components.

Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant-activated human polymorphonuclear leukocytes.

Binding of chemoattractants to specific cell surface receptors on polymorphonuclear leukocytes (PMNs) initiates a series of biochemical responses leading to cellular activation. A critical early biochemical event in chemoattractant (CTX) receptor-mediated signal transduction is the phosphodiesteric cleavage of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), with concomitant production of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer, and the protein kinase C activator, 1,2-diacylglycerol (DAG). The following lines of experimental evidence collectively suggest that CTX receptors are coupled to phospholipase C via a guanine nucleotide binding (G) protein. Receptor-mediated hydrolysis of PIP2 in PMN plasma membrane preparations requires both fMet-Leu-Phe and GTP, and incubation of intact PMNs with pertussis toxin (which ADP ribosylates and inactivates some G proteins) eliminates the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in isolated plasma membranes. Studies with both PMN particulate fractions and with partially purified fMet-Leu-Phe receptor preparations indicate that guanine nucleotides regulate CTX receptor affinity. Finally, fMet-Leu-Phe stimulates high-affinity binding of GTP gamma S to PMN membranes as well as GTPase activity. A G alpha subunit has been identified in phagocyte membranes which is different from other G alpha subunits on the basis of molecular weight and differential sensitivity to ribosylation by bacterial toxins. Thus, a novel G protein may be involved in coupling CTX receptors to phospholipase C. Studies in intact and sonicated PMNs demonstrate that metabolism of 1,4,5-IP3 proceeds via two distinct pathways: 1) sequential dephosphorylation to 1,4-IP2, 4-IP1 and inositol, or 2) ATP-dependent conversion to inositol 1,3,4,5-tetrakisphosphate (IP4) followed by sequential dephosphorylation to 1,3,4-IP3, 3,4-IP2, 3-IP1 and inositol. Receptor-mediated hydrolysis of PIP2 occurs at ambient intracellular Ca2+ levels; but metabolism of 1,4,5-IP3 via the IP4 pathway requires elevated cytosolic Ca2+ levels associated with cellular activation. Thus, the two pathways for 1,4,5-IP3 metabolism may serve different metabolic functions. Additionally, inositol phosphate production appears to be controlled by protein kinase C, as phorbol myristate acetate (PMA) abrogates PIP2 hydrolysis by interfering with the ability of the activated G protein to stimulate phospholipase C. This implies a physiologic mechanism for terminating biologic responses via protein kinase C mediated feedback inhibition of PIP2 hydrolysis.

Role of a guanine nucleotide regulatory protein in the activation of phospholipase C by different chemoattractants.

It is well established that formyl peptide chemoattractants can activate a phospholipase C in leukocytes via a pertussis toxin (PT)-sensitive guanine nucleotide regulatory (G) protein. Whether this pathway is similarly used by chemoattractant receptors as a class has been unclear. We now report that lipid and peptide chemoattractants in direct comparative studies induced similar amounts of initial (less than or equal to 15 sec) inositol trisphosphate (IP3) release in human polymorphonuclear leukocytes, but the response to lipid chemoattractants was more transient. Production of IP3 by all chemotactic factors was inhibited by treatment of the cells with PT, indicating that chemotactic factor receptors as a class are coupled to phospholipase C via a G protein that is a substrate for ADP ribosylation by PT. The peptide and lipid factors had comparable chemotactic activity, which was also inhibitable by PT. However, transient activation of phospholipase C is apparently an insufficient signal for full cellular activation, since the lipid chemotactic factor leukotriene B4 and platelet-activating factor were poor stimuli for O2- production and lysosomal enzyme secretion compared with N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). Nonetheless, treatment with PT inhibited O2- production and enzyme secretion in response to all chemoattractants, but as previously noted, did not affect Ca2+ ionophores, lectins, or phorbol myristate acetate. Formyl peptide and lipid chemotactic factors induced similar levels of Ca2+ mobilization when monitored by Quin 2 or chlortetracycline (CTC) fluorescence. Although these responses to fMet-Leu-Phe were blocked by PT, the Quin 2 and initial CTC response to the lipid factors were only partially susceptible. Thus, the lipid factors apparently utilize an additional PT-resistant mechanism for redistributing intracellular Ca2+. This latter process requires extracellular Ca2+ and may be independent of the PT-sensitive G protein.

Calcium influx stimulates a second pathway for sustained diacylglycerol production in leukocytes activated by chemoattractants.

Metabolic pathways involved in the activation of polymorphonuclear leukocytes (PMNs) were characterized by using chemoattractants with equivalent chemotactic activity but widely disparate ability to stimulate superoxide production [N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) much greater than leukotriene B4]. Leukotriene B4 stimulated a low level of superoxide production that plateaued at 60 sec, whereas with fMet-Leu-Phe the response continued to increase for 5 min. Both agents produced equivalent initial rises in diacylglycerol (acyl2Gro) (less than or equal to 30 sec); however, only fMet-Leu-Phe induced a second increase of acyl2Gro peaking at ca. 120 sec. Both chemoattractants also caused an equivalent initial (less than or equal to 10 sec) rise in intracellular calcium; however, the elevation induced by fMet-Leu-Phe was more sustained. We sought to determine the biochemical mechanisms underlying these discrepancies. Superoxide production and the second phase of acyl2Gro generation were both inhibited ca. 56% by depleting extracellular calcium or ca. 79% by buffering intracellular calcium. Cytochalasin B greatly enhanced the respiratory burst, acyl2Gro production, and calcium influx, but not inositolphospholipid turnover in PMNs stimulated with chemoattractants. These data indicate that sequential metabolic pathways activate the respiratory burst in PMNs stimulated by chemoattractants. The response is initiated by inositolpolyphospholipid hydrolysis, which results in rapid (less than or equal to 5 sec) calcium mobilization from intracellular stores and acyl2Gro release (peak at ca. 30 sec). To fully activate the respiratory burst, the chemoattractant must also trigger calcium influx, which leads to a sustained cytosolic calcium elevation. This supports a prolonged new phase of acyl2Gro production that is independent of inositolphospholipid hydrolysis and is correlated with superoxide production.

Regulation of inositol phosphate metabolism in chemoattractant-stimulated human polymorphonuclear leukocytes. Definition of distinct dephosphorylation pathways for IP3 isomers.

The metabolism of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer was studied in myo-[3H]inositol labeled, chemoattractant-stimulated human polymorphonuclear neutrophils (PMNs), and in PMN lysates. It was determined that 1,4,5-IP3 is metabolized in vitro by two distinct pathways: 1) by sequential dephosphorylation to 1,4-IP2, 4-IP1, and inositol or 2) by ATP dependent conversion to 1,3,4,5-IP4, followed by dephosphorylation to form 1,3,4-IP3, 3,4-IP2, 3-IP1, and inositol. In PMNs stimulated with 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), 1,4-IP2, 1,4,5-IP3, and IP4, were elevated by 5 s; whereas production of 1,3,4-IP3, 3,4-IP2, and IP1 occurred only after an initial lag (approximately 15 s). The predominant IP1 isomer formed in fMet-Leu-Phe-stimulated cells was 4-IP1. Production of 1,3,4-IP3 and 3,4-IP2 was markedly reduced (17 and 35% of control, respectively) in fMet-Leu-Phe-stimulated cells pretreated to prevent a rise in intracellular calcium ([Ca2+]i). PMNs were also stimulated with leukotriene B4 (LTB4) since this agent is a poor activator of the respiratory burst compared to fMet-Leu-Phe. Peak levels (5 s) of 1,4,5-IP3 were equivalent after stimulation with 0.1 microM fMet-Leu-Phe versus 0.1 microM LTB4 (320 +/- 38% versus 378 +/- 38% of control values, respectively; n = 5); however, at 30 s, 1,4,5-IP3 remained elevated only in fMet-Leu-Phe-stimulated cells. Similarly, elevation of [Ca2+]i was more prolonged in response to 0.1 microM fMet-Leu-Phe (greater than 3 min) versus LTB4 (1 min). Thus, signal transduction in PMNs may be modulated by both the duration of the initial 1,4,5-IP3 signal and by the metabolic pathway(s) utilized to convert this IP3 isomer to other, potentially active inositol phosphate products.

SH2-Containing protein tyrosine phosphatase-1 (SHP-1) association with Jak2 in UT-7/Epo cells.

We have investigated the interaction of the SH2-containing protein tyrosine phosphatase-1 (SHP-1) and Jak2 in an erythropoietin (Epo)-dependent human leukemia cell line, UT-7/Epo, using reciprocal immunoprecipitation and immunoblotting. The Epo-induced kinetics and dose response on phosphorylated Jak2 in anti-SHP-1 precipitates of UT-7/Epo cell lysates were similar to those in direct anti-Jak2 precipitates, suggesting that Jak2 coprecipitated with SHP-1. Furthermore, immunoblotting with anti-Jak2 and anti-SHP-1 antibodies indicated that SHP-1 appeared to be constitutively associated with non-tyrosine-phosphorylated Jak2 in UT-7/Epo cells in the absence of Epo and without phosphorylation of the Epo receptor (EpoR). Competition studies with C-terminal SHP-1 and Jak2 peptides decreased the amounts of SHP-1 and Jak2 detected in immunoprecipitates supporting the specific coprecipitation of SHP-1 and Jak2. In the presence of a recombinant GST-fusion protein containing both the N-terminal and C-terminal SH2 domains of SHP-1, anti-GST precipitated the fusion protein but not cellular Jak2. These studies suggest that SHP-1 and Jak2 are constitutively associated in UT-7/EPO cells. The association is not dependent upon Epo and is not mediated via SHP-1 SH2 binding. Sequential double immunoprecipitation demonstrated that only a small portion of intracellular Jak2 and SHP-1 molecules are constitutively associated. This partial association pattern may allow a more flexible and diverse regulation of Jak2 and SHP-1 activities. Whether Jak2 and SHP-1 are directly associated with each other or are part of a larger complex needs further investigation.

Prophylaxis with respiratory syncytial virus F-specific humanized monoclonal antibody delays and moderately suppresses the native antibody response but does not impair immunity to late rechallenge.

Respiratory syncytial virus (RSV) is the most significant viral cause of lower respiratory tract disease in infants and children. This study tested the hypothesis that a humanized murine monoclonal antibody (MAb) would protect against RSV infection in mice and have minimal suppressive effect upon the immune response because it is directed against a single epitope. A humanized murine MAb (RSHZ19) was tested for both prophylaxis and treatment of RSV infection in BALB/c mice and compared with a polyclonal product. Mice were rechallenged when passively administered antibody was undetectable (day 104). RSHZ19 reduced virus titer and protected against illness when used in prophylaxis and effected rapid virus clearance when used as treatment. Polyclonal antibody was also an effective prophylaxis but required 200 times the dose in total protein. Peak neutralizing antibody responses were delayed and somewhat suppressed in the prophylactically treated groups, but mice were protected against infection on rechallenge. Secondary antibody response to rechallenge in passively immunized mice was equal to that in untreated mice.