PI3K signalling in inflammation.

PI3Ks regulate several key events in the inflammatory response to damage and infection. There are four Class I PI3K isoforms (PI3Kα,ß,γ,δ), three Class II PI3K isoforms (PI3KC2α, C2ß, C2γ) and a single Class III PI3K. The four Class I isoforms synthesise the phospholipid 'PIP3'. PIP3 is a 'second messenger' used by many different cell surface receptors to control cell movement, growth, survival and differentiation. These four isoforms have overlapping functions but each is adapted to receive efficient stimulation by particular receptor sub-types. PI3Kγ is highly expressed in leukocytes and plays a particularly important role in chemokine-mediated recruitment and activation of innate immune cells at sites of inflammation. PI3Kδ is also highly expressed in leukocytes and plays a key role in antigen receptor and cytokine-mediated B and T cell development, differentiation and function. Class III PI3K synthesises the phospholipid PI3P, which regulates endosome-lysosome trafficking and the induction of autophagy, pathways involved in pathogen killing, antigen processing and immune cell survival. Much less is known about the function of Class II PI3Ks, but emerging evidence indicates they can synthesise PI3P and PI34P2 and are involved in the regulation of endocytosis. The creation of genetically-modified mice with altered PI3K signalling, together with the development of isoform-selective, small-molecule PI3K inhibitors, has allowed the evaluation of the individual roles of Class I PI3K isoforms in several mouse models of chronic inflammation. Selective inhibition of PI3Kδ, γ or ß has each been shown to reduce the severity of inflammation in one or more models of autoimmune disease, respiratory disease or allergic inflammation, with dual γ/δ or ß/δ inhibition generally proving more effective. The inhibition of Class I PI3Ks may therefore offer a therapeutic opportunity to treat non-resolving inflammatory pathologies in humans. This article is part of a Special Issue entitled Phosphoinositides.

PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40(phox).

The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b(558) (gp91(phox) and p22(phox)) and three soluble factors (GTP-Rac, p47(phox) and p67(phox) (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40(phox) has subsequently been discovered as a binding partner for p67(phox) (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation but through an unknown mechanism. We show that the addition of p40(phox) to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns(3)P to the PX domain of p40(phox). These results offer new insights into the roles for PI(3)Ks and p40(phox) in ROS formation and define a cellular ligand for the orphan PX domain.

Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B.

Protein kinase B (PKB) is a proto-oncogene that is activated in signaling pathways initiated by phosphoinositide 3-kinase. Chromatographic separation of brain cytosol revealed a kinase activity that phosphorylated and activated PKB only in the presence of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. Phosphorylation occurred exclusively on threonine-308, a residue implicated in activation of PKB in vivo. PtdIns(3,4,5)P3 was determined to have a dual role: Its binding to the pleckstrin homology domain of PKB was required to allow phosphorylation by the upstream kinase and it directly activated the upstream kinase.

Translocation of PDK-1 to the plasma membrane is important in allowing PDK-1 to activate protein kinase B.

BACKGROUND: Protein kinase B (PKB) is involved in the regulation of apoptosis, protein synthesis and glycogen metabolism in mammalian cells. Phosphoinositide-dependent protein kinase (PDK-1) activates PKB in a manner dependent on phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which is also needed for the translocation of PKB to the plasma membrane. It has been proposed that the amount of PKB activated is determined exclusively as a result of its translocation, and that a constitutively active pool of membrane-associated PDK-1 simply phosphorylates all the PKB made available. Here, we have investigated the effects of membrane localisation of PDK-1 on PKB activation. RESULTS: Ectopically expressed PDK-1 translocated to the plasma membrane in response to platelet-derived growth factor (PDGF) and translocation was sensitive to wortmannin, an inhibitor of phosphoinositide 3-kinase. Translocation of PDK-1 also occurred upon its co-expression with constitutively active phosphoinositide 3-kinase, but not with an inactive form. Overexpression of PDK-1 enhanced the ability of PDGF to activate PKB. PDK-1 disrupted in the pleckstrin homology (PH) domain which did not translocate to the membrane did not increase PKB activity in response to PDGF, whereas membrane-targeted PDK-1 activated PKB to the extent that it could not be activated further by PDGF. CONCLUSIONS: In response to PDGF, binding of Ptdlns (3,4,5)P3 and/or Ptdlns(3,4)P2 to the PH domain of PDK-1 causes its translocation to the plasma membrane where it co-localises with PKB, significantly contributing to the scale of PKB activation.

Phosphatidylinositol 3-phosphate is generated in phagosomal membranes.

Phagocytic cells such as neutrophils and macrophages engulf and destroy invading microorganisms. After internalization, material captured within the phagosomal membrane is destroyed by a complex process of coordinated delivery of digestive enzymes and reactive oxygen species. Several endosomal, lysosomal, and oxidase components expected to participate in these events have recently been shown to bind PtdIns3P, suggesting that this lipid may play a role in this process. We used live, digital fluorescence imaging of RAW 264.7 cells stably expressing either a PtdIns3P binding GFP-PX domain or a GFP-FYVE domain to visualize changes in the levels and subcellular localization of PtdIns3P during phagocytic uptake of IgG-opsonized zymosan particles. Very similar results were obtained using both PtdIns3P probes. The basal distribution of each PtdIns3P probe was partially cytosolic and partially localized to EEA-1-positive endosomal structures. Within about 2-3 min of zymosan attachment and concomitant with the closure of the phagosomal membrane, GFP-positive vesicles moved toward and attached to a localized area of the phagosome. A dramatic, transient accumulation of GFP probe around the entire phagosome rapidly ensued, accompanied by a transient drop in cytosolic GFP fluorescence. The magnitude and timing of this rise in PtdIns3P clearly suggest that it is an ideal candidate for controlling the early stages of phagosomal maturation.

DAPP1 undergoes a PI 3-kinase-dependent cycle of plasma-membrane recruitment and endocytosis upon cell stimulation.

BACKGROUND: Phosphoinositide (PI) 3-kinase and its second messenger products, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), play important roles in signalling processes crucial for cell movement, differentiation and survival. Previously, we isolated a 32kDa PtdIns(3,4,5)P(3)-binding protein from porcine leukocytes. This protein contains an amino-terminal Src homology 2 (SH2) domain and a carboxy-terminal pleckstrin homology (PH) domain, and is identical to the recently described DAPP1 (also known as PHISH or Bam32) protein. Here, we characterised the subcellular distribution of DAPP1 in response to cell stimulation. RESULTS: When expressed transiently in porcine aortic endothelial (PAE) cells, DAPP1 translocated from the cytosol to the plasma membrane in response to platelet-derived growth factor (PDGF). This translocation was dependent on both PI 3-kinase activity and an intact DAPP1 PH domain. Following recruitment to the plasma membrane, DAPP1 entered the cell in vesicles. Similar responses were seen in DT40 chicken B cells following antibody treatment, and Rat-1 fibroblasts following epidermal growth factor (EGF) or PDGF treatment. Colocalisation studies in PAE cells suggested entry of DAPP1 by endocytosis in a population of early endosomes containing internalised PDGF-beta receptors. DAPP1 also underwent PI 3-kinase-dependent phosphorylation on Tyr139 in response to PDGF stimulation, and this event was involved in the vesicular response. CONCLUSIONS: This is the first report of plasma-membrane recruitment and endocytosis of a PI 3-kinase effector protein in response to cell stimulation. The results suggest a novel role for DAPP1 in endosomal trafficking or sorting.

Priming of human neutrophil superoxide generation by tumour necrosis factor-alpha is signalled by enhanced phosphatidylinositol 3,4,5-trisphosphate but not inositol 1,4,5-trisphosphate accumulation.

In human neutrophils, significant agonist-stimulated superoxide anion (O2-) release is observed only after exposure to a priming agent such as TNFalpha. We have investigated the potential for TNFalpha to modulate N-formyl-Met-Leu-Phe (fMLP)-triggered Ins(1,4,5)P3 and PtdIns(3,4,5)P3 accumulation. TNFalpha pretreatment did not affect basal or stimulated Ins(1,4,5)P3 levels but greatly upregulated fMLP-stimulated PtdIns(3,4,5)P3 accumulation, in a manner that matched, both temporally and in magnitude, the increase in O2- generation implying a possible role for PtdIns(3,4,5)P3 in signalling primed O2- release.

Insulin and ATP stimulate actin polymerization in U937 cells by a wortmannin-sensitive mechanism.

ATP and insulin stimulate increases in phosphatidylinositol (3,4,5)-trisphosphate levels in myeloid-derived U937 cells. Quantification of FITC-phalloidin binding by fluorescence-activated cell sorting reveals that both ATP and insulin stimulate actin polymerization with distinctive kinetics in U937 cells. The response to ATP is rapid and dose-dependent with an EC50 of 200 nM, and is abolished by pre-incubation with the Ca2+ chelator BAPTA-AM. At 800 nM concentration, wortmannin, a potent inhibitor of phosphoinositide 3-kinase (PI3K), blocks the late, but not the early phase of actin polymerization stimulated by 100 nM ATP. Responses elicited by 10 micrograms/ml insulin are slower, smaller and more transient than responses to ATP, and are inhibited by preincubation with 100 nM wortmannin. Actin polymerization can also be stimulated by thapsigargin, but not by phorbol ester, providing further evidence for a role for Ca2+ in actin polymerization. These data implicate distinct Ca2+ and PI3K-mediated pathways in the regulation of actin polymerization.

Changes in the levels of inositol lipids and phosphates during the differentiation of HL60 promyelocytic cells towards neutrophils or monocytes.

HL60 cells were adapted to grow in a serum-free medium containing 1 mg l-1 inositol, in which they differentiated normally towards neutrophils (in 0.9% by volume dimethylsulphoxide) and towards monocytes (in 10 nM phorbol myristate acetate). Cells that had been equilibrium-labelled with [2-3H]myo-inositol contained a complex pattern of inositol metabolites, several of which were at relatively high concentrations. These included InsP5 and InsP6, which were present at concentrations of about 25 microM and 60 microM, respectively. Striking and different changes occurred in the levels of some of the inositol polyphosphates as the cells differentiated towards either neutrophils or monocytes. Most notable were a large but gradual accumulation of Ins(1,3,4,5,6)P5 as HL60 cells decreased in size and acquired neutrophil characteristics, and much more rapid and sequential declines in InsP4, InsP5 and InsP6 as the cells started to take on monocyte character. There was a marked accumulation of free inositol and of phosphatidylinositol in the cells during neutrophil differentiation, probably caused at least in part by an increased rate of inositol uptake providing an increased intracellular inositol supply. The same accumulation of Ins(1,3,4,5,6)P5 occurred during neutrophil differentiation, whether it was induced by dimethylsulphoxide or by a combination of retinoic acid and a T-lymphocyte cell line-derived differentiation factor. Ins(1,4,5)P3, a physiological intracellular mediator of Ca2+ release from membrane stores, did not change in concentration during these differentiation processes. These observations suggest that some of the more abundant cellular inositol polyphosphates play some important, but not yet understood, role either in the processes of haemopoietic differentiation or in the expression of differentiated cell character in myeloid cells.

Relationships between days post partum, observed estrus and uterine microflora in commercial dairy cows.

This study was conducted to determine relationships between the uterine microflora and reproductive characteristics of dairy cows. Uterine lumina were swabbed during estrus immediately prior to artificial insemination (A I). Swabbings were cultured for bacteria and accessed for cytologic evidence of inflammation. The time of sampling averaged 104 d post partum. Bacteria in the uterus were cultured from 65% of the cows (n = 85). The number of cows with any given microbial genus ranged from 1 to 35 and the number of different genera per cow ranged from 0 to 8. There were on significant correlations between amount and type of uterine microflora, days post partum, numbers of observed estrus, conception rate and uterine inflammation. The number of observed estrus periods was not correlated with the presence of aerobic bacteria. No significant relationships were found between microbes and uterine inflammation or conception to the A.I. service at the time of sampling. A negative correlation between uterine inflammation and conception approached significance (r=-0.24, P > 0.10). Acquisition of uterine lumen samples by the technique utilized had no effect on conception to A I service.

Positive and negative aspects of host immune response to Haemophilus, Actinobacillus and Pasteurella.

Haemophilus somnus, Actinobacillus pleuropneumoniae and Pasteurella haemolytica are economically important bacteria with pathogenic characteristics that require us to look further than killed, whole cell bacterins for induction of a protective immune response. A strong immune response is not synonymous with protection and the extreme specificity of the immune response works to our disadvantage when broad protection is needed. Detection of animals that are susceptible or immune to infection is important for the purpose of diagnosis and epidemiological study. However serum antibody levels are rarely indicative of protection unless it is known that the antibody of a particular isotype must be directed against a specific epitope for protection to occur. Parenteral vaccination with killed, whole cells of H. somnus, A. pleuropneumoniae or P. haemolytica produces, respectively, adequate protection, partial protection and increased disease. The reasons for these differences and methods of improving protection, based on an understanding of virulence determinants, are discussed.

Humoral immunity in experimental thromboembolic meningoencephalitis in cattle caused by Haemophilus somnus.

Of 23 cattle inoculated IV with Haemophilus somnus, 16 (70%) died of thromboembolic meningoencephalitis. The inoculum was prepared from a minimally subcultured isolate of H somnus that was passaged through a calf by intracisternal inoculation immediately before use. Serum antibody titers, as measured by 7 serologic tests, were not correlated with the animal's susceptibility to infection. All cattle that died had a mean 4-fold increase in agglutination titer during the acute phase of the disease, 2 to 4 days after inoculation. Similarly, high acute-phase titers were demonstrated in 15 cattle with naturally occurring disease. Haemophilus somnus was isolated more frequently and in greater numbers from the CNS and urinary tract than from other organs of cattle that died.

Vaccination of cattle against experimentally induced thromboembolic meningoencephalitis with a Haemophilus somnus bacterin.

The capability of a commercial Haemophilus somnus bacterin to protect cattle against experimentally induced thromboembolic meningoencephalitis was examined. Eighteen cattle were vaccinated twice, 8 were vaccinated once, and 14 were nonvaccinated controls. Serum antibody responses to vaccination were measured by gel immunodiffusion, bacterial agglutination test, and complement-fixation test. Deaths occurred in 8 of the 14 controls, 3 of the cattle vaccinated once, and 3 of the cattle vaccinated twice. Two vaccinations were found to give significant protection against challenge exposure (P less than 0.05). There were no cattle which gave positive reactions in the gel immunodiffusion test, and significant changes in bacterial agglutination test titers were not seen in the cattle after vaccination. There was a significant (P less than 0.01) increase in the complement-fixation test titers of cattle vaccinated twice. Serum antibody titers were unrelated to the outcome of challenge infection, regardless of vaccination status, in any of the serotests.

Isolation of Haemophilus somnus antigens and their use as vaccines for prevention of bovine thromboembolic meningoencephalitis.

Antigens extracted from Haemophilus somnus were examined for their suitability as vaccines for prevention of thromboembolic meningoencephalitis and as antigens in immunologic tests for detection of susceptible cattle. Saline extraction of whole H somnus cells produced an outer membrane complex (OMC) that contained 2 major antigens when tested against antiserum to intact cells by immunoelectrophoresis. Anion exchange chromatography was used to separate an anionic antigen (AA) from the more cationic antigen (CA). According to chemical analysis and polyacrylamide gel electrophoresis, both AA and CA were complex mixtures--probably, outer membrane fragments. Enzyme-linked immunosorbent assays were used to measure serum levels of immunoglobulin (Ig) G and IgM against AA and CA in cattle vaccinated with whole cells, OMC, CA, or AA. Protection of vaccinated cattle was assessed after IV challenge exposure to H somnus. Moderate IgG and IgM responses occurred when cattle were given 2 vaccinal doses of 0.1 mg or 1.0 mg of whole cells, OMC, or AA. Two of 10 cattle given 2 vaccinal doses (1.0 mg) of OMC died after IV challenge exposure, whereas 8 of the 10 controls died, indicating significant protection (P less than 0.05). All of the 10 cattle given 2 vaccinal doses (1.0 mg) of AA were protected from IV challenge exposure, whereas 5 controls died (P less than 0.05). Two vaccinal doses of either 0.1 mg or 1.0 mg of CA produced high IgG and IgM responses. However, 3 of 10 cattle given 2 vaccinal doses (1.0 mg) of CA died after IV challenge exposure, as did 3 of the 10 controls, indicating that CA was not protective.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevalence and distribution of Haemophilus somnus in the male bovine reproductive tract.

"Haemophilus somnus' was isolated from 77% of 31 reproductive tracts of bulls from an Ontario slaughterhouse. Identification of H somnus was based on morphologic and cultural characteristics and on fluorescent antibody and immunodiffusion tests, using antisera prepared against a known pathogenic encephalitic isolate of H somnus. The infection rate and distribution of H somnus within the tract were as follows: preputial orifice--71% preputial cavity--71%; urinary bladder--26%; accessory sex glands--19% and ampulla of ductus deferens--10%. Isolates were not obtained from the testes or epididymides. On 2 occasions, H somnus was isolated in pure culture from the preputial cavity. A higher prevalence of infection was found in young bulls. There were no differences found in infection rates between breeds. Differences in hemolytic activity and minor antigenic variation between isolates indicated that a series of biotypes within the species H somnus may exist. The study indicates that organisms presently identified as H somnus may normally form part of the flora of the bovine prepuce and that dissemination from the male bovine reproductive tract is one possible means of infection in H somnus-associated diseases. The pathogenic significance of genital isolates of H somnus awaits further study.

Selective medium for isolation of Haemophilus somnus from cattle and sheep.

Incorporation of vancomycin (5 micrograms/ml), neomycin (5 micrograms/ml), sodium azide (50 micrograms/ml), nystatin (100 iu/ml) and cyclohexamide (100 micrograms/ml) into 5 per cent horse blood agar results in a selective medium for the primary isolation of Haemophilus somnus from cattle and sheep. Addition of thiamine monophosphate (1 microgram/ml) to the medium enhanced growth of this bacterium. Gram-positive bacteria did not grow on the medium and colonies of many Gram-negative bacteria were eliminated or reduced in numbers and size. Colonies of H somnus were larger on the selective medium than on sheep blood agar but retained typical morphology. Recovery of 18 laboratory strains was 73 to 166 per cent (mean 112) on selective medium compared to sheep blood agar. H somnus was isolated from the vagina of a total of 136 (28.6 per cent) of 476 cows surveyed, 79 (16.6 per cent) on sheep blood agar and 129 (27.1 per cent) on selective medium. The selective agents and thiamine were stable indefinitely as a freeze dried mixture while prepared plates were stable for two weeks.

Metronidazole for the treatment of bovine pyometra.

Metronidazole, an antibiotic with specific activity against anaerobic bacteria, was assessed as a treatment for bovine pyometra. A preliminary experiment with metronidazole-neomycin was followed by an experiment in which metronidazole-ampicillin was compared with povidone iodine. Each treatment was given as a single intrauterine infusion. The success of therapy was judged by clinical examination, bacteriological examination before and after treatment and, in the second experiment, by post treatment reproductive performance. Before treatment 45 of the 84 cows in both experiments were infected with a mixture of anaerobic and aerobic bacteria, 23 cows were infected with aerobes alone and significant bacteria were not isolated from 16 cows. Complete bacteriological and clinical cures were achieved in 23 of 32 cows treated with metronidazole-neomycin, 15 of 32 cows treated with metronidazole-ampicillin and 1 of 20 cows treated with iodine. When only those cows with mixed anaerobe/aerobe infections were considered, complete cures occurred in 12 of 17 cows treated with metronidazole-neomycin, and 11 of 16 cows treated with metronidazole-ampicillin, but none of the 12 cows treated with iodine. Twenty-two of 29 cows treated with metronidazole-ampicillin conceived after treatment (mean 51.4 days), while 9 of 18 cows treated with iodine conceived (mean 58.7 days). Statistically, metronidazole-ampicillin treatment produced significantly better clinical and bacteriological cure rates than iodine treatment (P less than 0.05), but the differences in reproductive parameters were not significant.

The effect polyethylene intramammary devices on the somatic cell count and milk production of dairy cows.

Polyethylene intramammary devices (IMD) were inserted into all 4 quarters of 15 multiparous dairy cows. Fifteen cows, matched for parity and production, were controls. The insertion of IMD's was easily achieved and produced no adverse effects. Throughout the 150-day test period, the mean somatic cell count of cows with IMD's in situ was 216,000 cells per ml, compared with 119,000 cells per ml in controls (P less than 0.01). Total production of milk, butterfat and protein was not significantly different between the 2 groups. The new infection rate was too low to allow assessment of the value of the IMD for mastitis prevention.

Diagnosis by ELISA of bovine abortion due to Campylobacter fetus.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antigen-specific secretory IgA antibody in bovine vaginal mucus after abortion due to Campylobacter fetus subsp venerealis. Abortions were diagnosed by isolating the organism from 8 foetuses and/or foetal membranes and by histopathology. Vaginal mucus was collected from 7 cows shortly after abortion. All showed a high level of IgA antibody in their vaginal mucus when they were compared with an uninfected control group. The new ELISA is simple and practical and provides a useful tool for diagnosis of bovine venereal campylobacteriosis.

Infestation of sheep with the stored product mite Sancassania berlesei (Acaridae).

A sheep reared outdoors in Victoria was found to be heavily infested with a stored product mite, Sancassania berlesei (Acaridae), apparently subsequent to an earlier flystrike. Laboratory observation revealed the infestation to be self-sustaining. Shearing rapidly resolved the infestation but treatment with diazinon was ineffective. The infestation could be transferred to other sheep only in the presence of moisture; once established it caused an extensive skin lesion with considerable fluid loss, apparently contributing to the death of one animal. This lesion was extremely attractive to blowflies. Animals infested with mites showed few signs of irritation.