Binding of the non-typeable Haemophilus influenzae lipooligosaccharide to the PAF receptor initiates host cell signalling

Non-typeable Haemophilus influenzae (NTHi) invades host cells by binding of the platelet-activating factor (PAF) receptor via lipooligosaccharide (LOS) glycoforms containing phosphorylcholine (ChoP). The effect of NTHi infection on host cell signalling and its role in NTHi invasion was examined. The infection of human bronchial epithelial cells with NTHi 2019 increased cytosolic Ca²⁺ levels, and the invasion of bronchial cells by NTHi 2019 was inhibited by pretreatment with the cell-permeant intracellular Ca²⁺ chelator BAPTA-AM ( P  = 0.022) or thapsigargin ( P  = 0.016). Cytosolic inositol phosphate (IP) levels were also increased after infection with NTHi 2019 ( P  < 0.001), but not after infection with isogenic mutants expressing altered LOS glycoforms lacking ChoP. PAF receptor antagonist reduced NTHi 2019-stimulated IP production in a dose-dependent manner. NTHi 2019 invasion was inhibited by pertussis toxin (PTX) and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002. The less invasive strain NTHi 7502 also initiated IP production, but was unaffected by PAF receptor antagonist or PTX. These data demonstrate that the binding of the PAF receptor by NTHi initiates receptor coupling to a PTX-sensitive heterotrimeric G protein complex, resulting in a multifactorial host cell signal cascade and bacterial invasion. Moreover, the data suggest that NTHi strains initiate cell signalling and invade by different mechanisms, and that invasion mediated by PAF receptor activation is more efficient than macropinocytosis.     

The roles of epithelial cell contact,respiratory bacterial interactions and phosphorylcholine in promoting biofilm formation by Streptococcus pneumoniae and nontypeable Haemophilus influenzae

Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) often share a common niche within the nasopharynx, both associated with infections such as bronchitis and otitis media. This study investigated how the association between NTHi and S. pneumoniae and the host affects their propensity to form biofilms. We investigated a selection of bacterial strain and serotype combinations on biofilm formation, and the effect of contact with respiratory epithelial cells. Measurement of biofilm showed that co-infection with NTHi and S. pneumoniae increased biofilm formation following contact with epithelial cells compared to no contact demonstrating the role of epithelial cells in biofilm formation. Additionally, the influence of phosphorylcholine (ChoP) on biofilm production was investigated using the licD mutant strain of NTHi 2019 and found that ChoP had a role in mixed biofilm formation but was not the only requirement. The study highlights the complex interactions between microbes and the host epithelium during biofilm production, suggesting the importance of understanding why certain strains and serotypes differentially influence biofilm formation. A key contributor to increased biofilm formation was the upregulation of biofilm formation by epithelial cell factors.     

Neutrophil migration across monolayers of cytokine-prestimulated endothelial cells: a role for platelet-activating factor and IL-8

In a previous study we observed that neutrophils respond with a rapid rise in [Ca2+]i during adherence to cytokine-activated endothelial cells (EC), caused by EC membrane-associated platelet-activating factor (PAF). In the present study, we investigated whether this form of PAF was important in neutrophil adherence and migration across monolayers of rIL-1 beta- or rTNF alpha-prestimulated EC. PAF receptor antagonists prevented neutrophil migration across cytokine-pretreated EC by approximately 60% (P less than 0.005) without interfering with the process of adherence. The antagonists WEB 2086 and L-652,731 had no effect on neutrophil migration across resting EC induced by formylmethionyl-leucyl-phenylalanine (FMLP). A murine anti-IL-8 antiserum was found to also partially inhibit the neutrophil transmigration across cytokine-activated EC. When the anti-IL-8 antiserum was used in combination with a PAF receptor antagonist, neutrophil migration across cytokine-pretreated monolayers of EC was completely prevented. During transmigration, LAM-1 and CD44 on the neutrophils were down-modulated; both WEB 2086 and anti-IL-8 antiserum partially prevented this down-modulation caused by cytokine-prestimulated EC. Our results indicate that human neutrophils are activated and guided by EC-associated PAF and EC-derived IL-8 during the in vitro diapedesis in between cytokine-stimulated EC.     

Genetic and functional analysis of the phosphorylcholine moiety of commensal Neisseria lipopolysaccharide

Phosphorylcholine (ChoP) is a common surface feature of many mucosal organisms, including Neisseria spp., in which it is present exclusively on pili of pathogenic Neisseria and on the lipopolysaccharide (LPS) of commensal Neisseria (Cn). Its presence in Cn has been confirmed by nuclear magnetic resonance. It appears that choline is the main source for the production of ChoP by Cn. We have sequenced a locus, containing four genes (licA-D) with 47-73% identity to the lic1 locus of Haemophilus influenzae (Hi) and 21-40% identity to lic genes in Streptococcus pneumoniae, involved in the production and incorporation of ChoP. The arrangement of the Cn genes and the presence of CAAT repeats, responsible for phase variation of ChoP expression, resemble Hi and differ from S. pneumoniae. Cn DNA flanking the lic locus contains genes ilvE and NMA2149 with >85% identity to the pathogenic Neisseria genes. However, there are no lic genes in the corresponding location or elsewhere in pathogenic Neisseria. This suggests either the loss of the locus from pathogenic Neisseria or a horizontal transfer of genes to Cn, perhaps from H. influenzae spp. As in Hi, ChoP enhances adherence to and invasion of human epithelial cells via the receptor for platelet-activating factor. However, ChoP expression also increases susceptibility to serum killing mediated by complement and C-reactive protein. Taken together, these observations support the hypothesis that the ability of many organisms to switch off ChoP expression rapidly represents an important adaptation to different environments encountered during the colonization/infection process and that the ChoP moiety apparently synthesized by distinct means in pathogenic and commensal Neisseria represents an advantage in the colonization properties of these bacteria.     

Effect of various components of the fungal cell wall on adhesion of Candida albicans to the mouth mucosa epithelium in vitro]

An influence of mannan++, its component methyl-D-mannopyranoside+ and N-acetylglucosamine on in vitro adhesion of Candida albicans strains to buccal mucosal epithelium was studied. These substances inhibited adhesion when added to adherence test in a concentration of 10 mg/ml and 25 mg/ml despite whether were added to the test incubation medium or when preincubated with fungi or epithelial cells. Preincubation of fungal cells and epithelial cells with mannan had no influence on attachment; preincubation of epithelial cells with methyl-D-mannopyranoside+ and N-acetylglucosamine decreased adherence significantly. On the other hand preincubation of fungal calls with methyl-D-mannopyranoside+ increased their adhesive properties, having no influence on adherence after preincubation of fungi with N-acetylglucosamine.     

Adherence of Staphylococcus aureus to cell monolayers

W yatt , J.E., P oston , S.M. & N oble , W.C. 1990. Adherence of Staphylococcus aureus to cell monolayers. Journal of Applied Bacteriology 69 , 834–844.
Adherence of four strains of Staphylococcus aureus to eukaryotic cell monolayers was assayed with [³H]-thymidine labelled bacterial cells and the results were analysed by non-parametric statistical tests. Adherence to primary (human mesothelial) and semi-continuous (human embryonic lung) cell monolayers was significantly better than to continuous cell lines (HEp2, HeLa and Vero). HEp2 cell monolayers provided the most reliable assay substrate of the continuous cell lines tested. Variation occurred between bacterial culture batches but the assay measured significant differences between adhesion levels of the strains and distinguished between high level (RN92, 8325–4) and low level (Wood46, ISP458) adhering strains. Adherence to different batches of cell monolayers also varied but relative adherence values for strains were similar and the ranking of strains according to adhesion values -was unchanged.
Potential adhesion mediators have been monitored for their effect on adhesion of a highly adherent strain (RN92) to HEp2 monolayers. Fibronectin, protein A and anti-protein A did not significantly affect adhesion. Lipoteichoic acid caused a significant inhibition of adhesion. With critical statistical analysis to accommodate inherent variations, this assay provides a useful model to study factors involved in adherence of Staph. aureus to eukaryotic cells.     

The structural heterogeneity of the lipooligosaccharide (LOS) expressed by pathogenic non-typeable Haemophilus influenzae strain NTHi 9274

Nontypeable Haemophilus influenzae (NTHi) is an important pathogen responsible for otitis media in children and of pneumonitis in adults with depressed resistance. NTHi is acapsular and, therefore, capsular polysaccharide-based vaccines are ineffective for preventing infections by this pathogen. Recently it was found that a detoxified lipooligo-saccharide (LOS) conjugate from NTHi 9274 induced bactericidal antibodies effective against a large number of NTHi isolates, and conferred protection against NTHi otitis media in chinchillas (X.-X.Gu et al., 1996, Infect. Immun.,64, 4047-4053; X. -X.Gu et al., 1997., Infect. Immun.,65, 4488-4493). In this paper we report the chemical character-ization of the LOS from NTHi 9274 LOS. NTHi is capable of expressing a heterogenous population of LOS exhibited by multiple oligosaccharide (OS) epitopes. OSs released from the LOS of NTHi 9274 by mild acid hydrolysis were purified using Bio-Gel P4 gel permeation chromatography. The OSs were characterized by glycosyl composition analysis, glycosyl linkage analysis, nuclear magnetic resonance spectroscopy (NMR), fast atom bombardment mass spectro-metry (FAB-MS), matrix-assisted laser desorption time of flight mass spectro-metry (MALDITOF-MS), and tandem MS/MS. At least 17 different OS molecules were observed. These contained variable glycosyl residues, phosphate (P), and phospho-ethanolamine (PEA) substituents. These molecules contained either three, four, or five hexoses, and all contained four heptosyl residues. The four heptosyl residues consisted of one D,D-Hep and three L,D-Hep. Dephosphorylation of the OSs with aqueous 48% hydrofluoric acid (HF) reduced the number of molecules to about to seven; Hex(1)-(7)Hep(4)Kdo(1). Of these seven, Hex(2)Hep(4)Kdo(1), Hex(3)Hep(4)Kdo(1), and Hex(4)Hep(4)Kdo(1)were the major constituents. Thus, this NTHi LOS preparation is very heterogeneous, and contains structures different from those previously published for Haemophilus influenzae. The tandem MS/MS analysis and glycosyl linkage data suggest that the LOS oligosaccharides have the following structures where Hex is either a Glc or Gal residue.     

Adherence of Staphylococcus aureus to cell monolayers

Adherence of four strains of Staphylococcus aureus to eukaryotic cell monolayers was assayed with [3H]-thymidine labelled bacterial cells and the results were analysed by non-parametric statistical tests. Adherence to primary (human mesothelial) and semi-continuous (human embryonic lung) cell monolayers was significantly better than to continuous cell lines (HEp2, HeLa and Vero). HEp2 cell monolayers provided the most reliable assay substrate of the continuous cell lines tested. Variation occurred between bacterial culture batches but the assay measured significant differences between adhesion levels of the strains and distinguished between high level (RN92, 8325-4) and low level (Wood46, ISP458) adhering strains. Adherence to different batches of cell monolayers also varied but relative adherence values for strains were similar and the ranking of strains according to adhesion values was unchanged. Potential adhesion mediators have been monitored for their effect on adhesion of a highly adherent strain (RN92) to HEp2 monolayers. Fibronectin, protein A and anti-protein A did not significantly affect adhesion. Lipoteichoic acid caused a significant inhibition of adhesion. With critical statistical analysis to accommodate inherent variations, this assay provides a useful model to study factors involved in adherence of Staph. aureus to eukaryotic cells.     

Nontypeable Haemophilus influenzae: Pathogenesis and Prevention

In this paper, we describe the ability of nontypeable Haemophilus influenzae (NTHi) to coexist with the human host and the devastating results associated with disruption of the delicate state of balanced pathogenesis, resulting in both acute and chronic respiratory tract infections. It has been seen that the strains of NTHi causing disease show a marked genetic and phenotypic diversity but that changes in the lipooligosaccharide (LOS) and protein size and antigenicity in chronically infected individuals indicate that individual strains of NTHi can remain and adapt themselves to avoid expulsion from their infective niche. The lack of reliance of NTHi on a single mechanism of attachment and its ability to interact with the host with rapid responses to its environment confirmed the success of this organism as both a colonizer and a pathogen. In vitro experiments on cell and organ cultures, combined with otitis media and pulmonary models in chinchillas, rats, and mice, have allowed investigations into individual interactions between NTHi and the mammalian host. The host-organism interaction appears to be a two-way process, with NTHi using cell surface structures to directly interact with the mammalian host and using secreted proteins and LOS to change the mammalian host in order to pave the way for colonization and invasion. Many experiments have also noted that immune system evasion through antigenic variation, secretion of enzymes and epithelial cell invasion allowed NTHi to survive for longer periods despite a specific immune response being mounted to infection. Several outer membrane proteins and LOS derivatives are discussed in relation to their efficacy in preventing pulmonary infections and otitis media in animals. General host responses with respect to age, genetic makeup, and vaccine delivery routes are considered, and a mucosal vaccine strategy is suggested.     

Mass spectral characterization of lipooligosaccharides from Haemophilus influenzae 2019

Lipooligosaccharide (LOS) glycoforms from Haemophilus influenzae 2019 were profiled using the high-resolution and accurate mass capabilities of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Sequence and linkage for two previously unknown LOS glycoforms were subsequently obtained through MSn analyses on FT-ICR and quadrupole ion trap (qIT) instruments. MSn analysis of negative ion precursors confirmed structural details within the lipid moiety, while CID spectra of sodiated precursor ions provided monosaccharide sequence and linkage for the oligosaccharide portion of the molecule. Results obtained in this study indicate that extensive heterogeneity exists within the oligosaccharide moieties in LOS from H. influenzae 2019. More importantly, the data suggest that additional hexose moieties, which are added onto the LOS, are not simple extensions of one particular core structure but rather that structural isomers with different connectivities are present within the heterogeneous mixture.     

Regulation of adhesion of mouse bone marrow-derived mast cells to laminin

We have reported that mast cells adhere to laminin after activation with PMA. In this study, we demonstrate that the cross-linking of cell surface high-affinity IgE-R on mast cells derived from mouse bone marrow cultured for 3 wk in the presence of WEHI-3-conditioned media acts as a highly sensitive physiologic stimulus for this attachment and that receptor activation is also induced by calcium ionophore A23187. Adherence occurred at threefold log concentrations less of A23187 and Ag than required for histamine release in a selective subpopulation comprising 20 to 30% of the total cells. At higher concentrations of agonist that permitted histamine release, the time course for degranulation was shown to be more rapid than that of adherence. Adherence was inhibited by antibodies to laminin and laminin receptor and was calcium ion and temperature dependent. Treatment of cells with dibutyryl cAMP, which activates protein kinase A, inhibited both adherence and histamine release induced by Ag or calcium ionophore. Treatment of cells with staurosporin, which inhibits protein kinase C, also inhibited adherence and histamine release induced by calcium ionophore, but was not significantly active against either adherence or histamine release induced by Ag. It thus appears that agents which modulate intracellular signaling mechanisms are equally effective toward histamine release and adherence, suggesting these two events are intimately linked in stimulus secretion coupling. Specific cytokines stimulating mast cell adhesion to laminin could not be found; however, culture of mast cells with TGF-beta 1 was determined to enhance IgE-mediated adherence to laminin. Hence, the high-affinity IgE-R on the mast cell functions not only in exocytosis but also facilitates the process of mast cell adherence to laminin.     

Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways

Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5_NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.     

A lectin-like receptor on mammalian macrophages

Rat Kupffer cells in vitro strongly bind neuraminidase-treated rat erythrocytes but not untreated erythrocytes. Binding between cells is inhibited by preincubation of macrophages with D-galactose and related sugars, but not with unrelated saccharides. We therefore suggest that cell adherence is mediated by a galactose-specific receptor on the Kupffer cell membrane.     

Development of peptide mimotopes of lipooligosaccharide from nontypeable Haemophilus influenzae as vaccine candidates

Nontypeable Haemophilus influenzae (NTHi) is a common cause of otitis media in children and lower respiratory tract diseases in adults. So far there is no effective vaccine against NTHi. A major surface-exposed component of NTHi, lipooligosaccharide (LOS), is a virulence factor as well as a potential protective Ag. LOS is too toxic to be administered in humans. However, detoxified LOS is a T cell-independent small molecule and is poorly immunogenic in vivo, so we converted LOS into a nontoxic T cell-dependent Ag through the use of peptides that mimic the LOS by screening a phage-display peptide library with a rabbit Ab specific for NTHi LOS. Fifty-six phage clones were found to share LOS mimicry molecules. Among them, 22 clones were subjected to DNA sequencing, and four consensus sequences were identified as NMMRFTSQPPNN, NMMNYIMDPRTH, NMMKYISPPIFL, and NMMRFTELSTPS. Three of the four synthetic peptides showed strong binding reactivity to the rabbit anti-LOS Ab and also a mouse bactericidal monoclonal anti-LOS Ab in vitro, and elicited specific serum anti-LOS Abs in rabbits (27- to 81-fold) after conjugation with keyhole limpet hemocyanin. Passive immunization with the rabbit antisera resulted in a significantly enhanced pulmonary bacterial clearance in a mouse model. The enhanced bacterial clearance was eliminated if the rabbit serum was preabsorbed with NTHi LOS. These data indicate that the peptide mimotopes of LOS that we have identified might be potential components of peptide vaccines against NTHi.     

Role of endothelial leukocyte adhesion molecule-1 and platelet-activating factor in neutrophil adherence to IL-1-prestimulated endothelial cells. Endothelial leukocyte adhesion molecule-1-mediated CD18 activation.

Adherence of neutrophils to endothelium is a key event in the sequence of inflammatory leukocyte responses. Double-color FACS analysis was used to determine the extent and kinetics of neutrophil adherence to rIL-1 beta-pretreated endothelial cells (EC). Neutrophils bound very avidly when the EC were prestimulated for 4 to 6 h with rIL-1 beta. Anti-ELAM-1 F(ab)2 fragments inhibited this adherence for more than 80%. On the other hand, anti-CD18 F(ab)2 fragments also inhibited the neutrophil adherence (40 to 50%). Combined use of anti-ELAM-1 and anti-CD18 F(ab)2 fragments completely prevented adherence. Neutrophils became activated as soon as they made contact with the rIL-1 beta-pretreated EC. First, neutrophils depleted of intracellular ATP showed a clearly decreased adherence completely dependent on ELAM-1-mediated binding, i.e., without additional effects of CD18 adhesion proteins. Thus, CD18 is activated during neutrophil adherence and then participates in the binding process. Secondly, the neutrophils responded with a transient rise in [Ca2+]i upon binding to rIL-1 beta-pretreated EC, which was demonstrated to be caused by endothelial cell-associated platelet-activating factor (PAF). However, the extent of neutrophil adherence to rIL-1 beta-pretreated EC was not affected by the use of the PAF-receptor antagonist WEB 2086, or removal of the EC-bound PAF. The only effect was a complete dependency of the neutrophil adherence on ELAM-1-mediated binding, although anti-CD18 mAb still induced 40 to 50% inhibition under these conditions. We therefore conclude that ELAM-1-mediated binding is the major mechanism for CD18 activation during neutrophil adherence to rIL-1 beta-pretreated EC.     

Leukocyte inhibitory factor stimulates neutrophil-endothelial cell adhesion

We investigated the ability of the human lymphokine leukocyte inhibitory factor (LIF) to modulate neutrophil-endothelial cell (EC) adherence. EC were cultured from collagenase-treated human umbilical cord veins and grown in complete medium supplemented with EC growth factor. Adherence was measured as the percent of 51Cr-labeled neutrophils remaining adherent to the EC after gentle lavage. Polymorphonuclear neutrophils (PMN) were pretreated with LIF (0.5 to 8 U/ml), extensively washed, and allowed to interact with the EC monolayers. LIF was demonstrated to induce an increase in the capacity of PMN to bind EC in a dose-dependent fashion (from 30.9 +/- 2.1% adherence with control-treated PMN to 68.6 +/- 3.0% at 4 U LIF; p less than 0.001). In subsequent experiments we demonstrated that 10 min was a sufficient preincubation time for LIF to modulate the capacity of the PMN to adhere to EC. LIF has previously been observed to up-regulate expression of C receptor type 3 on PMN, a receptor which has been shown to be involved in PMN-EC binding. Exposure of PMN to anti-C receptor type 3 antibody before their incubation with LIF abrogated its effect as did inactivation of LIF by an esterase inhibitor. We also investigated the ability of LIF to stimulate EC to bind untreated PMN. EC were pretreated with LIF (0.25 to 4 U/ml), extensively washed, and adherence measured as before. LIF was shown to induce a dose-dependent increase in the capacity of the EC to bind PMN (from 28.8 +/- 3.1% for untreated EC to 91.1 +/- 4.0% at 4 U LIF; p less than 0.001). Modulation of EC function required a minimum of 30 min and was inhibited in the presence of cycloheximide or actinomycin D. Neither anti-TNF-alpha or -beta antibodies nor polymixin B abrogated the augmentation by LIF. However, anti-IL-1 antibody partially inhibited the stimulation of EC adhesiveness by LIF, suggesting the possible involvement of this cytokine. These studies provide further evidence that LIF may mediate an important pro-inflammatory role in vivo.     

Human endothelial cells modulate granulocyte adherence and chemotaxis

Although human endothelial cells (EC) and granulocytes interact in several ways, the factors that regulate such interactions are not well defined. In this study we found that EC and their products directly altered granulocyte adherence (GA) and chemotactic activity. The spontaneous adherence of granulocytes to human umbilical vein EC monolayers was significantly reduced at 5, 15 and 30 min if the granulocytes were preincubated with EC that were stimulated by rocking. At 30 min the spontaneous adherence of EC-preincubated granulocytes was 36% of that of control granulocytes (P = 0.004). The augmented adherence stimulated by FMLP was also decreased (54% of control) by preincubation of the granulocytes with rocked EC. The ability of stimulated EC to inhibit GA was attenuated when the EC monolayers that were used for preincubation with the granulocytes were pretreated with the cyclooxygenase inhibitor indomethacin. GA to unstimulated EC was not significantly altered by indomethacin or aspirin, suggesting that cyclooxygenase products do not influence GA under resting conditions. Preincubation of granulocytes with rocked EC monolayers or supernatant media from rocked EC monolayers diminished their chemotactic response to FMLP by 45 to 65. This inhibition was also attenuated by pretreatment of the EC with indomethacin. EC supernatant medium caused a rapid increase in granulocyte intracellular cyclic AMP, with a maximum increase to 200% of control at 1 min. These data indicate that stimulated EC release one or more arachidonic acid products that alter spontaneous and inflammatory mediator-stimulated granulocyte activity. Prostacyclin, a major cyclooxygenase product of EC arachidonate, depressed inflammatory mediator-augmented GA to EC monolayers and chemotaxis when present in nanomolar concentrations. We conclude that EC-derived prostacyclin, alone or in combination with other EC products, alters GA and chemotaxis stimulated by inflammatory mediators. This provides a mechanism by which EC may modulate granulocyte distribution as well as granulocyte responses that are influenced by adherence, such as the release of toxic oxygen metabolites and granular enzymes.     

Blockades of phospholipase A(2) and platelet-activating factor receptors reduce the hemocyte phagocytosis in Rhodnius prolixus: in vitro experiments

The hemocytes phagocytosis in response to microorganisms may play an important role in the cellular immune responses of insects. Here, we have evaluated the effects of the platelet-activating factor (PAF) and eicosanoids in the phagocytosis of hemocyte monolayers of Rhodnius prolixus to the yeast Saccharomyces cerevisiae. Experiments showed that the phagocytosis of yeast cells by Rhodnius hemocytes is very efficient in both controls and cells treated with PAF and arachidonic acid. Phagocytosis of yeast particles is significantly blocked when the specific phopholipase A(2) inhibitor, dexamethasone, is applied on the hemocytes. By contrast, dexamethasone-pretreated hemocyte monolayers exhibit a drastic increase in the quantity of yeast cell-hemocyte internalization when the cells are treated by arachidonic acid. In addition, phagocytosis presents significant reduction in hemocyte monolayers treated with a specific PAF receptor antagonist, WEB 2086. Nevertheless, inhibition of phagocytosis with WEB 2086 is counteracted by the treatment of the hemocyte monolayers with PAF. In conclusion, phagocytosis of yeast cells by hemocytes is related to the activation of PAF receptors and eicosanoid pathways in the bloodsucking bug, R. prolixus.     

Role of Platelet-Actlvatlng-Factor (PAF) on Cellular Responses after Stimulation with Leptospire Lipopolysaccharide

Leptospire lipopolysaccharide (LPS) stimulated the adherence of polymorphonuclear neutrophils (PMNs) to human umbilical vein endothelial cells (HUVEC). Enhanced PMN adherence in response to leptospire LPS can be mediated by platelet-activator-factor (PAF), because a PAF antagonist reduced adherence. Leptospire LPS also induced the adherence platelets or U937. The second experiment involved leptospire LPS elicited platelet aggregation in a PMN-platelet mixture, because leptospire LPS stimulated human PMN but not the human platelets. The platelet response was observed only in the mixture system and was inhibited by a PAF antagonist. PAF could be an important pathogenic factor in human leptospirosis.