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.

Differences in pathogenicity of herpes simplex virus serotypes 1 and 2 may be observed by histopathology and high-resolution magnetic resonance imaging in a murine encephalitis model.

The mouse model for herpes simplex-induced encephalitis (HSE) is an established preclinical tool for evaluating the efficacy of new therapeutic interventions. We evaluated the utility of high-resolution in vivo MRI in observing the progression of experimental HSE during the first week postinfection. Female BALB/c mice were inoculated intracerebrally with HSV-1 or HSV-2 by microinjection. Each animal was evaluated daily by high-resolution (4.7 Tesla) T(2) weighted MRI and clinical disease scoring (neurological and behavioral). Lesions induced by a high dose of HSV-1 (1000 PFU) were detectable by MRI without administration of contrast agent whereas for low dose HSV-1 (100 PFU), administration of contrast agent was necessary to visualize the lesions in the brain. The correlation between the MRI and histologic results was excellent. No HSV-2 induced lesions were observed by MRI. Although both HSV serotypes caused similar clinical disease, significant type differences were found by histologic and MRI examinations. HSV-1 caused necrotizing meningoencephalitis, whereas HSV-2 induced mostly meningitis. The data indicate that in vivo high-resolution MRI may be useful to longitudinally evaluate HSV-1-related pathology in a mouse model of HSE and potentially could be used for monitoring the efficacy of anti-infective therapeutic approaches.

Rapid mobilization of murine hematopoietic stem cells with enhanced engraftment properties and evaluation of hematopoietic progenitor cell mobilization in rhesus monkeys by a single injection of SB-251353, a specific truncated form of the human CXC chemokine GRObeta.

SB-251353 is an N-terminal truncated form of the human CXC chemokine GRObeta. Recombinant SB-251353 was profiled in murine and rhesus monkey peripheral blood stem cell mobilization and transplantation models. SB-251353 rapidly and transiently mobilized hematopoietic stem cells and neutrophils into the peripheral blood after a single subcutaneous injection. Transplantation of equivalent numbers of hematopoietic stem cells mobilized by SB-251353 into lethally irradiated mice resulted in faster neutrophil and platelet recovery than stem cells mobilized by granulocyte colony-stimulating factor (G-CSF). A single injection of SB-251353 in combination with 4 days of G-CSF administration resulted in augmented stem and progenitor cell mobilization 5-fold greater than G-CSF alone. Augmented stem cell mobilization could also be demonstrated in mice when a single injection of SB-251353 was administered with only one-day treatment with G-CSF. In addition, SB-251353, when used as a single agent or in combination with G-CSF, mobilized long-term repopulating stem cells capable of hematopoietic reconstitution of lethally irradiated mice. In rhesus monkeys, a single injection of SB-251353 induced rapid increases in peripheral blood hematopoietic progenitor cells at a 50-fold lower dose than in mice, which indicates a shift in potency. These studies provide evidence that the use of SB-251353 alone or in combination with G-CSF mobilizes hematopoietic stem cells with long-term repopulating ability. In addition, this treatment may (1) reduce the number of apheresis sessions and/or amount of G-CSF required to collect adequate numbers of hematopoietic stem cells for successful peripheral blood cell transplantation and (2) improve hematopoietic recovery after transplantation.

mDYRK3 kinase is expressed selectively in late erythroid progenitor cells and attenuates colony-forming unit-erythroid development.

DYRKs are a new subfamily of dual-specificity kinases that was originally discovered on the basis of homology to Yak1, an inhibitor of cell cycle progression in yeast. At present, mDYRK-3 and mDYRK-2 have been cloned, and mDYRK-3 has been characterized with respect to kinase activity, expression among tissues and hematopoietic cells, and possible function during erythropoiesis. In sequence, mDYRK-3 diverges markedly in noncatalytic domains from mDYRK-2 and mDYRK-1a, but is 91.3% identical overall to hDYRK-3. Catalytically, mDYRK-3 readily phosphorylated myelin basic protein (but not histone 2B) and also appeared to autophosphorylate in vitro. Expression of mDYRK-1a, mDYRK-2, and mDYRK-3 was high in testes, but unlike mDYRK1a and mDYRK 2, mDYRK-3 was not expressed at appreciable levels in other tissues examined. Among hematopoietic cells, however, mDYRK-3 expression was selectively elevated in erythroid cell lines and primary pro-erythroid cells. In developmentally synchronized erythroid progenitor cells, expression peaked sharply following exposure to erythropoietin plus stem cell factor (SCF) (but not SCF alone), and in situ hybridizations of sectioned embryos revealed selective expression of mDYRK-3 in fetal liver. Interestingly, antisense oligonucleotides to mDYRK-3 were shown to significantly and specifically enhance colony-forming unit-erythroid colony formation. Thus, it is proposed that mDYRK-3 kinase functions as a lineage-restricted, stage-specific suppressor of red cell development. (Blood. 2001;97:901-910)

Role of p38 mitogen-activated protein kinase in rhinovirus-induced cytokine production by bronchial epithelial cells.

The stress-activated protein kinase p38 plays a central role in the regulation of cytokine biosynthesis by various cell types in response to a wide range of stimuli. Because the local inflammatory response and the infiltration of neutrophils is thought to contribute to the symptoms and sequelae of rhinovirus infection, we investigated the role of p38 kinase in cytokine and chemokine elaboration in airway epithelial cells infected with human rhinovirus. Rhinovirus-39 infection of BEAS-2B cells resulted in synthesis of cytokines (IL-1, IL-6, G-CSF, and GM-CSF) and CXC chemokines (IL-8, epithelial neutrophil-activating protein-78, and growth-related oncogene-alpha), evident 24-72 h postinfection. Rhinovirus infection induced a time- and dose-dependent increase in tyrosine phosphorylation of p38 kinase, which peaked 30 min postinfection and remained elevated for 1 h. Treatment of infected cells with SB 239063, a potent pyridinyl imidazole inhibitor of p38 kinase, resulted in up to 100% inhibition of mediator production and partially reduced levels of IL-8 mRNA as determined by quantitative RT-PCR. Treatment with SB 239063 had no effect on virus replication and was not cytotoxic at concentrations

Subtraction cloning and initial characterization of novel epo-immediate response genes.

Recent studies of erythropoietin (Epo) receptor signalling suggest that signals for mitogenesis, survival and differentiation are relayed efficiently by receptor forms lacking at least seven of eight cytoplasmic (phospho)tyrosine [(P)Y] sites for effector recruitment. While such receptor forms are known to activate Jak2 and a limited set of known immediate response genes (IRGs), the complex activities they exert predict the existence of additional target genes. To identify such targets, a minimal Epo receptor chimera was expressed in Epo-responsive erythroid SKT6 cells, and genes whose transcription is induced via this active receptor form were cloned by subtractive hybridization. Several known genes not previously linked to Epo signalling were discovered to be Epo IRGs including two which may further propagate Epo signals [Prl1 tyrosine phosphatase and receptor activator of of NFkappaB (Rank)], and three regulators of protein synthesis (EF1alpha, eIF3-p66 and Nat1). Several Epo IRGs were novel murine clones including FM2 and FM6 which proved to represent broadly expressed IRGs, and FM3 and FL10 which were induced primarily in haematopoietic cells. Interestingly, FL10 proved to correspond to a recently discovered regulator of yeast mating-type switching, and was induced by Epo in vivo. Thus, several new Epo signalling targets are described, which may modulate haematopoietic cell development.

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.

REDK, a novel human regulatory erythroid kinase.

We have identified a novel regulatory erythroid kinase (REDK) that is homologous to a family of dual-specificity kinases. The yeast homolog of REDK negatively regulates cell division, suggesting a similar function for REDK, which is primarily localized in the nucleus. REDK is present in hematopoietic tissues, such as bone marrow and fetal liver, but the RNA is expressed at significant levels only in erythroid or erythropoietin (EPO)-responsive cells. Two novel forms of cDNA (long and short) for REDK have been isolated that appear to be alternative splice products and imply the presence of polypeptides with differing amino termini. The ratio of short-to-long forms of REDK increases dramatically in CD34(+) cells cultured with EPO, suggesting differing regulation and function for each form. REDK is predominantly found in nuclear, rather than cytoplasmic, protein extracts, and immunoprecipitated REDK is active in phosphorylating histones H2b, H3, myelin basic protein, and other coimmunoprecipitated proteins. Antisense REDK oligonucleotides promote erythroid colony formation by human bone marrow cells, without affecting colony-forming unit (CFU)-GM, CFU-G, or CFU-GEMM numbers. Maximal numbers of CFU-E and burst-forming unit-erythroid were increased, and CFU-E displayed increased sensitivity to suboptimal EPO concentrations. The data indicate that REDK acts as a brake to retard erythropoiesis. (Blood. 2000;95:2838-2846)

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.

A direct comparison of the activities of two humanized respiratory syncytial virus monoclonal antibodies: MEDI-493 and RSHZl9.

Two humanized monoclonal antibodies, MEDI-493 and RSHZ19, were developed independently as potential improvements over RSV-IGIV for prevention of respiratory syncytial virus (RSV) infection. RSV-IGIV is a polyclonal human antibody preparation for intravenous infusion enriched for RSV neutralizing activity. A phase III clinical trial showed that MEDI-493 significantly reduced hospitalizations due to RSV infection. In a separate trial, RSHZ19 failed to show significant efficacy. In new studies, the in vitro and in vivo activities of MEDI-493 and RSHZ19 were compared to determine whether the different clinical results are related to differences in biologic activity. MEDI-493 was consistently 4- to 5-fold more potent than RSHZ19 in antigen binding, RSV neutralization, and fusion inhibition assays. Although both MEDI-493 and RSHZ19 were effective against A and B subtypes of RSV in the cotton rat model of RSV infection, 2- to 4-fold higher doses of RSHZ19 were required for similar protection. The enhanced activity of MEDI-493 compared with RSHZ19 may, in part, explain its better clinical effect.

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.

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.

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.

Evaluation of the protective efficacy of reshaped human monoclonal antibody RSHZ19 against respiratory syncytial virus in cotton rats.

Reshaped human MAb RSHZ19, which is specific for the surface fusion protein of respiratory syncytial virus (RSV) is in clinical development for the prevention and treatment of RSV-induced disease in human infants. The current studies profile lung virus clearance and evaluate lung histopathology in MAb-treated, RSV-infected cotton rats, a well characterized model of RSV infection. The highest dose of this MAb (10 mg/kg) administered parenterally 24 h before infection decreased subgroup A or B RSV lung titers to below detectable levels (> or = 2.3 log10 reduction), and significantly reduced lung virus titers (> or = 2.0 log10 reduction) when administered 96 h postinfection. Prophylactic administration of 10 mg/kg RSHZ19 was significantly more protective than 1000 mg/kg conventional human immune serum globulin (HSIg), and protective serum-neutralizing titers in MAb-treated animals (1:32, which correlated with approximately 40 micrograms/ml determined by anti-idiotype ELISA) were significantly lower than those reported previously for HSIg or for convalescent human serum (1:200-1:400). MAb concentration in lung lavages was determined by ELISA to be approximately 1% of the serum MAb concentration, but was not detectable by neutralization assay. The degree of lung histopathology in MAb-treated cotton rats was proportional to lung virus titer, and inversely proportional to the RSHZ19 dose administered. There was no evidence of exacerbated disease in the lungs of MAb-treated animals. These studies thus support the potential clinical utility of RSHZ19 MAb in the prevention and treatment of RSV-induced disease in humans.

Safety and immunogenicity of a recombinant protein influenza A vaccine in adult human volunteers and protective efficacy against wild-type H1N1 virus challenge.

A recombinant influenza A vaccine (D protein), comprising a carboxy-terminal sequence from the hemagglutinin HA2 subunit of A/Puerto Rico/8/34 virus (H1N1, A/PR/34) fused to 81 amino-terminal residues of the NS1 nonstructural protein, has previously protected mice against influenza A challenge by inducing H1N1/H2N2 cross-reactive cytotoxic T cells (CTL) without hemagglutination-inhibiting (HI) or neutralizing antibody. In our dose-escalating study, the vaccine was safe in humans and induced both IgG and T cell proliferative responses to D protein but little antibody to A/PR/34 or A/Kawasaki/8/86 (H1N1, A/KW/86) viruses. Among an additional group of A/KW/86-seronegative volunteers immunized with 500 micrograms of D protein, none had a rise in serum HI or neutralizing antibody to A/KW/86, 20% had minimal IgG responses to A/KW/86 by EIA, and a minority had any increase in A/KW/86-specific CTL activity. However, viral shedding and clinical illness score were reduced in vaccines relative to A/KW/86-seronegative unimmunized controls after intranasal challenge with wild-type A/KW/86. D protein immunization conferred significant protective immunity not currently explained by any of the immune parameters measured.

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.

Induction of protective class I MHC-restricted CTL in mice by a recombinant influenza vaccine in aluminium hydroxide adjuvant.

Induction of class I MHC-restricted cytotoxic T lymphocyte (CTL) responses by soluble proteins or peptides requires complex adjuvants or carrier systems which are not licensed for use with human vaccines. The data presented in this report show that vaccination with a highly purified recombinant influenza protein antigen in aluminium hydroxide adjuvant, the only adjuvant currently licensed for clinical use, elicited class I restricted CTL and protection from lethal challenge with H1N1 and H2N2 viruses. The antigen (D protein, SK&F 106160) is produced by expression of H1N1 influenza virus-derived cDNA (strain A/PR/8/34) in Escherichia coli, and is composed of the first 81 N-terminal amino acids (aa) of the non-structural protein 1 (NS1) fused via a nine nucleotide non-viral linker sequence to the 157 C-terminal aa of the haemagglutinin 2 subunit (HA2). Previous work by Kuwano et al demonstrated that in vitro stimulation of spleen cells from influenza virus-primed mice, with a partially purified preparation of the D protein, selected for CD8+ CTL clones which facilitated lung clearance of H1N1 and H2N2 viruses. In the current study, these results were extended by studying the responses of mice actively immunized with highly purified D protein in the presence or absence of adjuvants. Vaccination of CB6F1 (H-2dxb) mice with D protein in aluminum hydroxide or Freund's complete adjuvant generated H1N1 cross-reactive, H-2d-restricted, CD8+ CTL directed against an immunodominant HA2 epitope (aa 189-199). D protein without adjuvant did not elicit CTL, regardless of the route of injection. However, long-lived (greater than 6 months) splenic memory CTL were elicited by boosting mice intraperitoneally (i.p.) with the D protein in the absence of adjuvant. In mice injected subcutaneously with D protein in aluminium hydroxide at weeks 0 and 3, survival was increased relative to controls up to 16 weeks beyond the second vaccination, after which time additional boosting was required for protection. Studies in H-2b and H-2k mice vaccinated with the D protein showed that induction of CD4+ T-cell or antibody responses, in the absence of CD8+ CTL, did not correlate with protection. Passive transfer of immune sera from CB6F1 mice was also not protective. This prototype H1N1 recombinant subunit vaccine in aluminium adjuvant should directly address the feasibility of achieving a protective cell-mediated immune response in human influenza.

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.

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.

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.