Control by sample location in pigeons'' matching to sample

Ultrastructural characteristics of 15 strains and isolates of ehrlichiae belonging to three genogroups, or clades of genetically related organisms united in the genera Ehrlichia, Cowdria, Anaplasma, Neorickettsia and a strain of Wolbachia pipientis which represents a fourth genogroup in this cluster of species, were studied in continuous cell culture or in vivo: E. canis (Oklahoma strain and VHE isolate), E. muris (AS 145), E. chaffeensis (Arkansas, 91HE17 and Sapulpa), human granulocytic ehrlichiae (HGE)(BDS, 96HE27, 96HE37, #54, #55 and #72), E. equi (MRK), E. sennetsu (Miyayama), E. risticii (HRC-IL). Wolbachia pipientis was studied in the naturally infected Aedes albopictus mosquito cell line Aa23. All organisms were similar in the normal ultrastructure of individual cells and in the ability to form abnormal, pathological ehrlichial cells of the same type irrespective of the species. Normally all ehrlichiae studied in cell culture existed in two morphological forms - reticulate and dense-cored cells, both of which could divide by binary fission. Most alterations were related to their membranes, especially the cell wall. Differences in the structure of intravacuolar microcolonies (morulae) of ehrlichiae and their inter-relations with the host cells allowed differentiation of the genogroups: the E. canis-E. chaffeensis-E. muris genogroup formed large morulae, with many ehrlichiae, often suspended in a fibrillar matrix, and the host cell mitochondria and endoplasmic reticulum usually aggregated near the morulae and were in contact with the morula membrane; the E. phagocytophila-E. equi-HGE group morulae had no fibrillar matrix, no contacts with host cell mitochodria, and they did not aggregate around the morulae; E. sennetsu-E. risticii group usually developed in small individual vacuoles that did not fuse with each other and divided along with the ehrlichiae.     

Interactions between Borrelia burgdorferi and eukaryote cells: comparative ultrastructural aspects

In the present study, mammalian VERO cells, human polymorphonuclear leukocytes (PMN) and mouse peritoneal macrophages (MPM) were coincubated with B31 Borrelia burgdorferi strain (Bb) and examined by transmission electron microscopy. The results indicate that the spirochete adheres to the mammalian cells mainly by the apical pole and less frequently by the lateral wall. In VERO cells and MPM cell penetration is accomplished especially by cytoplasmic membrane destruction, the spirochete appearing free in the cytoplasm, but also by phagocytosis. In this latter case the pathogen is seen in phagosome. In PMN the internalization takes place exclusively by phagocytosis, the spirochete undergoing destruction in phagosomes. Sometimes an alteration of the phagosomal membrane is observable, probably produced by the pathogen as a mechanism to avoid fusion with the lysosome. Other aspects indicate the contribution of the parasitized cell cytoskeleton to the spreading of Borrelia to adjacent cells. MPM does not represent a very efficient barrier against Bb, as can be seen from the cellular behaviour of the spirochetes in this study, PMN seems to a more efficient barrier, by its unique capacity to destroy the microorganism after phagocytosis.     

Opsonin requirements for phagocytosis of Enterococcus spp. by human polymorphonuclear leukocytes

BACKGROUND: Interaction of Enterococcus spp. and host defense mechanisms is not well known. Opsonic requirements of Enterococcus faecalis and Enterococcus faecium to be phagocyted by human polymorphonuclear leukocytes (PMN) were evaluated. METHODS: Twenty strains (10 E. faecalis and 10 E. faecium) were studied. Phagocytosis was determined by a radiometric assay. Bacterial cells were labelled with 3H-adenine and opsonized with: a) 10% of human pool sera (HPS); b) 10% of decomplemented HPS, and c) albumin and fibronectin. RESULTS: Phagocytosis of Enterococcus spp. by PMN in the presence of HPS was significantly higher than that in the absence of opsonins. The phagocytosis of E. faecium was higher than that of E. faecalis. A strain-dependent effect of complement in the phagocytosis of Enterococcus spp. was observed. Neither albumin nor fibronectin showed an opsonic activity on Enterococcus spp. CONCLUSIONS: A great heterogeneity in the opsonic requirements of Enterococcus spp. was observed. Serum opsonins show a critical role in the phagocytosis of E. faecalis and E. faecium by PMN, this effect being more relevant with E. faecium. A strain-dependent opsonic activity of complement was observed.     

Phagocytosis of Escherichia coli by insect hemocytes requires both activation of the Ras/mitogen-activated protein kinase signal transduction pathway for attachment and beta3 integrin for internalization

Insect hemocytes in response to lipopolysaccharide (LPS) of Gram-negative bacteria facilitate binding and internalization of either cell-associated or cell-free LPS (Charalambidis, N. D., Foukas L. C., and Marmaras V. J. (1996) Eur. J. Biochem. 236, 200-206). An early event in LPS signaling in hemocytes involves protein tyrosine phosphorylation (Charalambidis N. D., Zervas C. G., Lambropoulou M., Katsoris P. G., and Marmaras V. J.(1995) Eur. J. Cell Biol. 67, 32-41). Here we report further data of LPS-mediated signal transduction responsible for Escherichia coli phagocytosis. We demonstrate that both adhesion of hemocytes to substrata and LPS stimulation can cause activation of p44(MAPK) in Ceratitis capitata hemocytes but with distinct kinetics indicating different functions. In addition, we showed that Drk, a homolog protein to the mammalian GRB2, is implicated in the transmission of LPS signaling, indicating that the Ras/mitogen-activated protein kinase pathway is involved. Either the cell-free or the cell-associated LPS appears to attach to the hemocyte surface by the same mechanism that is based on the cross-linking of LPS to membrane-associated p47 via the intermediacy of tyrosine derivatives generated by the action of phenol oxidase. By contrast, the cell-free LPS internalization into the hemocytes differs from the cell-associated LPS internalization. For E. coli internalization integrin receptors as well as cytoskeletal rearrangements are required, as judged by inhibition of E. coli internalization in the presence of the RGD peptide, beta3-integrin antibodies, and cytochalasin D.     

Potential role of membrane internalization and vesicle fusion in adhesion of neutrophils in response to lipopolysaccharide and TNF

Human polymorphonuclear leukocytes (PMN) respond to LPS with strongly increased integrin-mediated adhesion. While the first step of this process has been identified as the interaction of LPS with CD14 on the cell surface, subsequent steps remain to be elucidated. The experiments presented here suggest that monomeric LPS is internalized in vesicles, and uptake may be required for signaling. Fluorescently labeled LPS presented as monomeric complexes with soluble CD14 appeared in the plasma membrane of PMN by 5 min and was concentrated in cytoplasmic vesicles by 20 min. Adhesion in response to LPS/soluble CD14 occurred only after a 15- to 20-min lag period, consistent with endocytosis occurring before signal generation. In contrast, there was no time lag for adhesion in response to the formyl peptide formyl-norleucyl-leucyl-phenylalanine (fNLLP). Adhesion in response to LPS, but not fNLLP, was completely blocked by lowering the temperature to 19 degrees C, a procedure that prevents vesicle fusion. These studies indicated that an event with the time and temperature dependence of endocytosis precedes signaling by LPS. Cytochalasin D, an inhibitor of phagocytosis, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase that blocks vesicle fusion and phagocytosis, both completely blocked adhesion in response to LPS but not in response to fNLLP. These results support the idea that LPS internalization and early endosomal fusion may be required for signal transduction. Parallel studies showed that the adhesion response to TNF had time, temperature, and inhibitor sensitivities nearly identical with those of LPS, suggesting that responses to TNF may also include an obligate vesicle fusion step.     

Proteins induced by recombinant equine interferon-beta 1 within equine peripheral blood mononuclear cells and polymorphonuclear neutrophilic granulocytes

Peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophilic granulocytes (PMN) as well as embryonic equine dermal fibroblasts and the equine fibroblast line E. Derm which were used as controls, were treated with recombinant equine interferon-beta 1 (rEqIFN-beta 1) in vitro which induced the expression of different proteins in these cells. A 74 kDa protein was induced in PBMC and an 82 kDa protein was additionally found in the equine fibroblast E. Derm cell line following treatment with rEqFN-beta 1. Both proteins reacted with anti-mouse and anti-human Mx protein antisera in immunoblot tests. The 74 kDa and perhaps the 82 kDa components may thus represent equine 'Mxanalogous proteins'. The 74 kDa protein was only detected in PBMC of ten out of 20 horses examined. The induction of Mx protein in the horse by Type 1 interferon may therefore resemble that in the mouse, where Mx protein is involved in selective resistance to influenza virus. The influence of rEqIFN-beta 1 on protein expression in equine PBMC and PMN was monitored by metabolic labeling and 2-D gel electrophoresis. Proteins of 82, 74, 58 and 40 kDa were induced in PBMC following exposure to rEqIFN-beta 1. A constitutively expressed 35 kDa protein, however, was no longer demonstrable upon treatment with interferon. None of the proteins induced within PBMC was found in highly purified PMN treated with interferon. PMN exposed to rEqIFN-beta 1 synthesized four proteins in the range of 25 to 27 kDa. These proteins have not been described in interferon-treated PMN of any other species.     

Stimulatory effect of gangliosides on phagocytosis, phagosome-lysosome fusion, and intracellular signal transduction system by human polymorphonuclear leukocytes

Gangliosides are known to be differentiation-inducing molecules in mammalian stem cells. We studied the interaction between the molecular structure of glycosphingolipids (GSLs) and their promoting mechanisms of the phagocytic processes in human polymorphonuclear leukocytes (PMN). The effect of various gangliosides from mammalian tissues on adhesion, phagocytosis, phagosome-lysosome (P-L) fusion and superoxide anion production was examined by human PMN using heat-killed cells of Staphylococcus aureus-coated with GSLs. Gangliosides GM3, GD1a, GD3 and GT1b showed a marked stimulatory effect on the phagocytosis and P-L fusion in a dose-dependent manner, while ganglioside GM1, asialo GM1 and neutral GSLs did not. The relative phagocytic rate of ganglioside GM3-coated S. aureus was the highest among the tested GSLs. Both P-L fusion rate and phagocytosis of S. aureus were elevated significantly when coated with ganglioside GD1a, GD3 or GT1b, and GT1b gave a five times higher rate than that of the non-coated control. These results suggest that the terminal sialic acid moiety is essential for the enhancement of phagocytosis and that the number of sialic acid molecules in the ganglioside is related to the enhancement of the P-L fusion process. On the other hand, the superoxide anion release from PMN was not affected by ganglioside GM2, GM3, GD1a or GT1b. Furthermore, to clarify the trigger or the signal transduction mechanism of phagocytic processes, we examined the effect of protein kinase inhibitors such as H-7, staurosporine (protein kinase C inhibitor), H-89 (protein kinase A inhibitor), genistein (tyrosine kinase inhibitor), ML-7 (myosin light chain kinase inhibitor), and KN-62 (Ca2+/calmodulin-dependent protein kinase II inhibitor) on ganglioside-induced phagocytosis. H-7, staurosporine and KN-62 inhibited ganglioside-induced phagocytosis in the range of concentration without cell damage, while H-89, genistein and ML-7 did not. Moreover, H-7 and KN-62 inhibited ganglioside-induced P-L fusion. These results suggest that protein kinase C and Ca2+/calmodulin-dependent protein kinase II may be involved in the induction of phagocytosis and P-L fusion stimulated by gangliosides.     

Orchestration of neutrophil movement by intestinal epithelial cells in response to Salmonella typhimurium can be uncoupled from bacterial internalization

Intestinal epithelial cells respond to Salmonella typhimurium by internalizing this pathogen and secreting, in a polarized manner, an array of chemokines which direct polymorphonuclear leukocyte (PMN) movement. Notably, interleukin-8 (IL-8) is secreted basolaterally and directs PMN through the lamina propria, whereas pathogen-elicited epithelial chemoattractant (PEEC) is secreted apically and directs PMN migration across the epithelial monolayer to the intestinal lumen. While most studies of S. typhimurium pathogenicity have focused on the mechanism by which this bacterium invades its host, the enteritis characteristically associated with salmonellosis appears to be more directly attributable to the PMN movement that occurs in response to this pathogen. Therefore, we sought to better understand the relationship between S. typhimurium invasion and epithelial promotion of PMN movement. First, we investigated whether S. typhimurium becoming intracellular was necessary or sufficient to induce epithelial promotion of PMN movement. Blocking S. typhimurium invasion by preventing, with cytochalasin D, the epithelial cytoskeletal rearrangements which mediate internalization did not reduce the epithelial promotion of PMN movement. Conversely, bacterial attainment of an intracellular position was not sufficient to induce model epithelia to direct PMN transmigration, since neither basolateral invasion by S. typhimurium nor apical internalization of an invasion-deficient mutant (achieved by inducing membrane ruffling with epidermal growth factor) induced this epithelial cell response. These results indicate that specific interactions between the apical surface of epithelial cells and S. typhimurium, rather than simply bacterial invasion, mediate the epithelial direction of PMN transmigration. To further investigate the means by which S. typhimurium induces epithelia to direct PMN movement, we investigated whether the same signaling pathways regulate secretion of IL-8 and PEEC. IL-8 secretion, but not PEEC secretion, was activated by phorbol myristate acetate and blocked by an inhibitor (mg-132) of the proteosome which mediates NF-kappabeta activation. Further, secretion of IL-8, but not PEEC, was activated by an entry-deficient (HilDelta) S. typhimurium mutant or by basolateral invasion of a wild-type strain. Together, these results indicate that distinct signaling pathways mediate S. typhimurium invasion, induction of IL-8 secretion, and induction of PEEC secretion in model intestinal epithelia.     

Liquid preservation of canine granulocytes obtained by counterflow centrifugation-elutriation

Granulocytes (PMN) were isolated from 120 ml of canine whole blood by a modification of the counterflow centrifugation-elutriation technique. Isolated cell suspensions of 96% granulocytes and 4% mononuclear leukocytes with a 21:1 PMN/RBC ratio were stored at 4 degrees C in 4:4:2 medium consisting of four parts HBSS minus Ca++ and Mg++, four parts MEM, twp parts autologous plasma, and 20 microgram/ml gentamicin for 15 days. Granulocytes were stored at concentrations of approximately 4 x 10(6) PMN/ml in polypropylene centrifuge tubes. The stored granulocyte suspensions were assayed in vitro 0, 1, 4, 8, and 15 days to monitor chemotaxis, bacterial growth inhibition, O2 consumption associated with phagocytosis, and enzyme activities. Cell volume analysis was used to evaluate cellular integrity of the liquid-stored granulocytes. Canine granulocytes isolated by the modified dilution technique of counterflow centrifugation-elutriation can be preserved for up to 15 days with 77 +/- 6% granulocyte survival with maintenance of morphological and organelle integrity, as well as retention of in vitro functions of recognition, migration, phagocytosis, and killing of gram-negative bacteria.     

Mature macrophage cell lines exhibit variable responses to LPS

Bacterial lipopolysaccharide (LPS) is a potent activator of cells of the macrophage/monocyte lineage. Two mature macrophage cell lines, P388D1 and RAW264.7, exhibit very different biological responses to LPS. Although RAW264.7 cells release arachidonic acid from phospholipid in response to LPS stimulation, P388D1 cells do not respond in this manner. However, LPS primes P388D1 cells to release arachidonic acid in response to other stimuli. The goal of this work is to contrast the biochemical events that occur in LPS-treated P388D1 and RAW264.7 macrophages. Enzyme assays indicate that LPS treatment induces the activation of cytosolic PLA2 in RAW264.7, but not in P388D1 cells. Phorbol ester (PMA), a receptor-independent stimulus, also fails to induce arachidonic acid release from P388D1 cells, suggesting that these cells may have a defect in the signal transduction machinery that is common to LPS and PMA. This hypothesis is supported by the observation that the expression of the LPS receptors CD14 and CD11b/CD18 is similar on P388D1 and RAW264.7 cells. Western blot analyses indicate that the erk kinases are activated upon LPS treatment of RAW264.7 but not P388D1 cells. LPS-induced arachidonic acid release is reduced in cells treated with the MEK inhibitor PD98059, suggesting that activated erk kinases mediate the phosphorylation and activation of cPLA2 in this system. Interestingly, the p42 isoform of erk (erk2) appears to be activated in resting P388D1 cells. This observation indicates that the MAP kinase cascade may be constitutively activated in P388D1 cells which may in turn limit their ability to respond to LPS. Together, these data provide evidence that mature macrophages from different sources can exhibit variable responses to LPS and highlight the danger of making generalizations regarding the effects of LPS on macrophages.     

Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood

Polymorphonuclear neutrophils (PMN) are important in host defence against bacterial infection in the bovine mammary gland and techniques are needed to evaluate their functional activities. A rapid and sensitive two-color flow cytometric method for simultaneous measurement of phagocytosis rate and oxidative burst activity of bovine PMN in small blood samples is described. The method utilizes the oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123 by oxidative burst products that were generated by incubating the PMNs with red fluorescent propidium iodide labeled Staphylococcus aureus. Phagocytosis and oxidative burst both increased with time of incubation and with increasing bacteria concentration. A 20 min phagocytosis time and a ratio of 25 bacteria to one cell were considered optimal for assaying bovine blood PMN activity. To further evaluate the proposed two-color flow cytometric method, blood samples were treated with factors known to interfere with phagocytosis and oxidative burst metabolism of human neutrophils. Incubation of whole bovine blood with cytochalasin B at 10 micrograms ml-1 resulted in an approximate 70% decrease in the phagocytosis rate with a concommitant decrease in oxidative burst activity. Staurosporine (2 micrograms ml-1) inhibited bovine neutrophil oxidative burst formation for 95% while the phagocytosis was unaffected. PMNs in whole blood samples of ten cows differed significantly in their ability to phagocytose Staphylococcus aureus and to produce reactive oxygen products. However, only a weak correlation was detected between the burst activity:phagocytosis ratio of ten individual cows as indicated by the ROD/PI index.     

Effect of cytochalasin B on the adhesion of mouse peritoneal macrophages

The adhesion of normal mouse macrophages to glass surfaces was reduced by nontoxic levels (1-50 mug/ml) of cytochalasin B in combination with a centrifugal force (1,000-8,000 g). Macrophages nonspecifically activated by Corynebacterium acnes were also detached by this treatment, but less effectively. The effects of cytochalasin B treatment on these cells were shown to be reversible. After detachment, the cells reattached to glass, appeared morphologically normal, and behaved like untreated cells as judged by adhesion, acid phosphatase levels, and phagocytosis. The effect of cytochalasin B on several parameters of phagocytosis by normal macrophages was also examined. The results demonstrate that cytochalasin B can be used to detach macrophages from surfaces and suggest a functional relationship between phagocytosis and macrophage adhesion to surfaces. Furthermore, the effect of cytochalasin B on adhesion of phagocytic cells provides a probe for further investigation of the adhesion of cells to surfaces.     

CD14-dependent endotoxin internalization via a macropinocytic pathway

Gram-negative bacterial endotoxin (a lipopolysaccharide (LPS)) specifically binds to CD14, a glycosylphosphatidyl inositol (GPI)-anchored surface myeloid glycoprotein. This interaction leads to cell activation, but it also promotes LPS internalization and detoxification. In this work, we investigated the route of LPS and CD14 internalization and the relevance of CD14 GPI anchor in the endocytic pathway. In promonocytic THP-1 cells transfected with a GPI or a chimeric integral form of CD14, we showed by differential buoyancy in sucrose density gradients that these two forms of CD14 were sorted to different plasma membrane subdomains. However, both forms of CD14 associated preferentially with the same surface microfilament-enriched microvilli or ruffles. Electron microscopic studies indicated that CD14 internalized via macropinocytosis, a process resembling that of phagocytosis, different from "classical" receptor-mediated endocytic pathways, such as clathrin-coated pits or caveolae. With cell warming, the CD14-enriched ruffles fused and formed large vesicles. Later, these vacuoles made stacks and condensed into phago-lysosomes. CD14 was specifically associated with all of these structures. Radiolabeled LPS internalization paralleled CD14 internalization. Confocal microscopic studies confirmed the co-localization of LPS and CD14 both at the cell surface and in endosomal compartments. The microfilament-disrupting, macropinocytosis blocking agent cytochalasin D inhibited LPS and CD14 internalization but did not prevent LPS-dependent activation, indicating that these two processes are dissociated.     

Necrosis and apoptosis of polymorphonuclear cells exposed to peritoneal dialysis fluids in vitro

Conventional peritoneal dialysis (PD) fluids are known to inhibit polymorphonuclear cells (PMN) phagocytosis, oxidative burst and enzyme release. However, the relative contributions of apoptosis and/or necrosis to this dysfunction have not been examined. We investigated the effects of osmolality, glucose concentration and heat-sterilization of PD fluids on necrosis and apoptosis of PMN. Polymorphonuclear cells were isolated from 8 healthy volunteers and exposed to different PD fluids for four hours. PMN were then double-stained with Hoechst 33342 and propidium iodide to study the proportion of viable, apoptotic and necrotic cells. Transmission electron microscopy (TEM) was performed to confirm the results obtained with flow cytometry. The fluids studied were conventionally heat-sterilized 1.5% Dianeal (1.5% D), conventionally heat-sterilized 4.25% Dianeal (4.25% D), 1.5% D in which the osmolality was increased to that of 4.25% D by adding mannitol (1.5% D + M), a filter-sterilized version of 4.25% D (4.25% D-F) and a 1.1% amino acid PD fluid (AA) (Nutrineal PD4). All PD fluids had their pH equilibrated (pH = 7.4) by the addition of sodium bicarbonate. Compared to PMN exposed to culture medium, a significantly higher proportion of necrosis was observed in PMN exposed to 1.5% D (P = 0.04). The 4.25% D induced greater necrosis than 1.5% D (P = 0.001), and the 4.25% D also induced significantly more necrosis (P = 0.002) compared to 4.25% D-F. These data suggest that the consequences of heat-sterilization, rather than high glucose concentration are responsible for the necrosis observed. Indeed, the proportion of necrotic PMN with 4.25% D-F was not significantly different from 1.5% D. The 1.5% D + M and AA induced significantly more apoptosis compared to 1.5% D (P = 0.006 and P < 0.05, respectively), suggesting that apoptosis can be induced by the high osmolality of PD fluids. However, 1.5% D +/- M also induced significantly more apoptosis (P = 0.007) compared to 4.25% D-F. This suggests that the apoptosis effect is specific for the osmolyte present in PD fluids, and that mannitol and amino acids induce more apoptosis than glucose. In summary, the different non-physiological components of conventional PD fluids evaluated in this study had a differential effect on PMN survival. Heat sterilization of high glucose-containing PD fluids was associated predominantly with necrosis of PMN, and high osmolality with apoptosis.     

Regulation of the major detoxication functions by phenobarbital and 3-methylcholanthrene in co-cultures of rat hepatocytes and liver epithelial cells

We have examined the rotational diffusion of the luteinizing hormone (LH) receptors binding human chorionic gonadotropin (hCG) or ovine luteinizing hormone (oLH) in MA-10 Leydig tumor cells using time-resolved phosphorescence anisotropy techniques. LH receptors binding erythrosin isothiocyanate (ErITC)-derivatized oLH were rotationally mobile with rotational correlation times of 62 micros, 48 micros, 38 micros, and 29 micros at 4 degrees C, 15 degrees C, 25 degrees C, and 37 degrees C, respectively. ErITC-hCG bound to the LH receptor was rotationally immobile, showing no anisotropy decay at 4 degrees C, 15 degrees C, 25 degrees C, and 37 degrees C. To determine whether cytoskeletal components influenced the rotational diffusion of LH receptors, we measured rotational diffusion of LH receptors on MA-10 cells treated with 20 microg/ml cytochalasin D and on plasma membrane preparations. Following 1 h exposure to cytochalasin D, the rotational correlation times for hCG-occupied LH receptors were typically 11 micros at 37 degrees C compared to > 1000 micros on untreated cells. Treatment of MA-10 cells with cytochalasin B or colchicine had no affect on LH receptor rotational diffusion. Rotational correlation times for LH-occupied receptors decreased from 29 micros to 12 micros at 37 degrees C following cytochalasin D treatment. The rotational diffusion of LH receptors on plasma membrane preparations was similar to that observed for LH- and hCG-occupied receptors on intact cells treated with cytochalasin D. These various results indicate that there are differential effects of LH and hCG binding on the interactions of LH receptors with plasma membrane proteins and that microfilaments anchor the hCG- and LH-occupied receptors.     

Interleukin-10 inhibits neutrophil phagocytic and bactericidal activity

Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells, which is dependent upon the coordinated expression of both pro- and anti-inflammatory cytokines. Interleukin-10 (IL-10) is a recently described cytokine with potent anti-inflammatory properties in vivo and in vitro. In this study we investigated whether IL-10 could directly regulate the ability of neutrophils (PMN) to phagocytose and kill bacteria. Initial studies demonstrated that human recombinant IL-10 (hrIL-10) inhibited the ability of PMN to phagocytose Escherichia coli in vitro. Inhibition of phagocytosis occurred in the absence of changes in CR1 (C3b) or Fc receptor expression, as treatment of PMN with IL-10 failed to induce significant changes in Fc gamma IIR, Fc gamma IIIR or CR1 cell surface expression. However, incubation of PMN with IL-10 resulted in a dose-dependent decrease in CDIIb (Mac-1) expression. In addition to effects on PMN phagocytosis, hrIL-10 significantly attenuated PMN microbicidal activity, as bactericidal assays revealed that co-incubation of PMN with hrIL-10 resulted in a marked decrease in killing of phagocytosed bacteria. Furthermore, IL-10 inhibited the production of superoxide from PMA-stimulated PMN, suggesting that the detrimental effects of IL-10 on PMN microbicidal activity were due, in part, to suppression of respiratory burst. In summary, our studies indicate that IL-10 inhibits PMN-dependent phagocytosis and killing of E. coli in vitro, and suggest that this cytokine may impair effective antibacterial host defense in vivo.     

Influence of twinline, an elemental diet, on the generation of nitric oxide and reactive-oxygen intermediates from mouse peritoneal macrophages and polymorphonuclear leukocytes

The influence of Twinline (SNN-6010), an elemental diet containing medium-chain triglycerides, on the generation of nitric oxide (NO) and superoxide (O2.-) has been examined in mouse peritoneal macrophages and polymorphonuclear leukocytes (PMN). When PMN and peritoneal macrophages obtained from untreated mice were cultured in medium containing 0.1% and 1% (v/v) Twinline for 48h and stimulated with phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine, their chemiluminescence and O2.- generation were strongly suppressed, as was NO generation from peritoneal macrophages. PMN and peritoneal macrophages obtained from mice fed Twinline for 30 days generated much smaller amounts of 02.- and NO compared with PMN and peritoneal macrophages from control mice. In conjunction with this suppressed NO generation, inducible NO synthase and its mRNA expression in peritoneal macrophages were suppressed by Twinline both in-vivo and ex-vivo. Although phagocytosis of PMN and peritoneal macrophages was not suppressed by Twinline; their candida-killing activity was markedly suppressed. These results indicate that Twinline suppresses the host-defence function of PMN and peritoneal macrophages by down-regulating their generation of reactive-oxygen intermediates and NO.     

v-SNARE-dependent secretion is required for phagocytosis

Phagosomes are generally believed to form by gradual apposition of the plasma membrane of leukocytes onto the surface of invading microorganisms. The internalization of the encapsulated particle is therefore predicted to reduce the surface area of the phagocyte. Contrary to this prediction, we observed that phagocytosis is associated with a net increase in cell surface area, suggesting the concomitant occurrence of exocytosis. Selective cleavage of components of the secretory machinery by microinjection or transfection of bacterial neurotoxins induced a pronounced inhibition of phagocytosis. These observations indicate that vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis of endomembranes is essential for optimal completion of particle internalization during phagocytosis.     

A 16S rRNA-based PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle

A PCR-based assay was developed for detecting DNA of granulocytic ehrlichiae in blood samples from dogs, horses, and cattle, Primers were designed from 16S rRNA sequence information to specifically amplify DNA from a newly identified Swedish Ehrlichia species. The 16S rRNA nucleotide sequence of this Swedish species differs in only two and three positions from the sequences of Ehrlichia phagocytophila and Ehrlichia equi, respectively, which were also amplified by this PCR system. For evaluation, PCR results were compared with microscopic examination of stained blood smears for the detection of granulocytes containing ehrlichiae (morulae). Thirty-four of 36 microscopically positive samples were also positive by PCR, and 6 microscopically negative samples were negative by PCR as well. Six samples, in which morulae-like structures had been seen, were negative by PCR, also at a lower annealing temperature and when a reamplification of the first PCR products was performed. The identities of the PCR products from some canine and equine isolates were verified by direct DNA sequencing and were found to be identical with the Ehrlichia sequence found in these animal species that had been obtained earlier. The sequences of a segment of approximately 600 nucleotides from two bovine isolates were identical to that of E. phagocytophila, whereas the sequence of another bovine isolate differed in two positions from that of E. phagocytophila and in three positions from the sequences of the canine and equine isolates. Serum samples were analyzed by indirect fluorescent-antibody testing. Seventy-three percent of the animals which were positive by microscopy and PCR also had positive antibody titers. However, it was not possible to rely on a single serological result for diagnosis of present infection. It was, therefore, concluded that PCR was the most reliable method, useful in the clinical laboratory for specific and early diagnosis of granulocytic ehrlichiosis in animals.