Eicosanoid production by density-defined human peritoneal macrophages during inflammation

Density-defined macrophages isolated from fluids of patients with liver cirrhosis mainly generated the 5-lipoxygenase products leukotriene B₄ (LTB₄, 16%) and 5-hydroxy-eicosatetraenoic acid (5-HETE, 24%) and the cyclooxygenase products 12-hydroxyheptadecatrienoic acid (HHT, 22%) and thromboxane B₂ (TXB₂, 18%). The synthesis of eicosanoids was linear with the maturity of the macrophage subpopulations, suggesting that eicosanoid production is increased inin-vivo activated macrophages.

     

Eicosanoid production by density-defined human peritoneal macrophages during inflammation

Density-defined macrophages isolated from fluids of patients with liver cirrhosis mainly generated the 5-lipoxygenase products leukotriene B₄ (LTB₄, 16%) and 5-hydroxy-eicosatetraenoic acid (5-HETE, 24%) and the cyclooxygenase products 12-hydroxyheptadecatrienoic acid (HHT, 22%) and thromboxane B₂ (TXB₂, 18%). The synthesis of eicosanoids was linear with the maturity of the macrophage subpopulations, suggesting that eicosanoid production is increased inin-vivo activated macrophages.     

Mechanisms of killing of Toxoplasma gondii by rat peritoneal macrophages.

Rats are resistant to Toxoplasma infection, and macrophages are thought to mediate this resistance. We performed a series of experiments to investigate the mechanism of the anti-Toxoplasma activity of resident rat peritoneal macrophages. Resident rat peritoneal macrophages killed more than 90% of ingested Toxoplasma gondii in vitro. This capacity was reduced progressively with the prolongation of culturing of macrophages in vitro before challenge with T. gondii. Exhaustion of the respiratory burst of macrophages with phorbol myristate acetate impaired their ability to kill and limit the replication of T. gondii. Histidine and diazabicyclooctane, presumed scavengers of singlet oxygen, were the only members of a battery of scavengers of metabolites of the respiratory burst that impaired the anti-Toxoplasma activity of macrophages. Ingestion of heat-killed Candida albicans by macrophages reduced large amounts of intracellular Nitro Blue Tetrazolium dye, whereas little dye was reduced by the ingestion of T. gondii. Challenge of macrophages with T. gondii released no detectable superoxide anion, as measured by the reduction of ferricytochrome c, whereas stimulation of macrophages with phorbol myristate acetate or ingestion of heat-killed Candida by macrophages released abundant superoxide anion. These data are consistent with the contributions of oxygen-dependent and oxygen-independent mechanisms to the anti-Toxoplasma activity of rat peritoneal macrophages. In addition, neonatal rats are known to be susceptible to Toxoplasma infection in vivo. However, resident neonatal rat peritoneal macrophages ingested and killed T. gondii to the same extent as did adult macrophages. Thus, the susceptibility of neonatal rats to Toxoplasma infection probably resides in other aspects of macrophage function or the immune response.     

A Toxoplasma gondii rhoptry protein associated with host cell penetration has unusual charge asymmetry.

The monoclonal antibody Tg49 both recognizes a Toxoplasma gondii rhoptry protein (ROP1) and inhibits penetration enhancing factor. The latter is a proteinaceous factor found in Toxoplasma lysates or conditioned media that increases the efficiency with which parasites invade host cells. Tg49 was used to screen a lambda gt11 cDNA library and the clone obtained was identified as the cognate gene for ROP1 by several criteria: (1) recombinant protein reacted with the monoclonal; (2) antiserum against the recombinant reacted with the same bands on Western blots as did Tg49; and (3) antiserum against the recombinant recognized a protein in the rhoptries. The ROP1 gene is a single copy gene with a message of approximately 2.1 kb. The predicted polypeptide sequence of ROP1 shows an unusual charge and amino acid asymmetry. There is a highly acidic, proline-rich domain in the amino-terminal portion of the predicted protein, followed by a strongly basic carboxy-terminal domain. An octapeptide repeat is found almost midway through the peptide sequence toward the end of the acidic domain. The ROP1 gene was expressed in a bacterial system, and the resulting polypeptide exhibited anomalous migration on polyacrylamide gel electrophoresis. Given that Tg49 inhibits penetration enhancing factor, it seems likely that the ROP1 protein is a component of that factor, and that the unusual sequence of this protein plays some role in host cell penetration by T. gondii.     

Lymphokine activation of J774G8 cells and mouse peritoneal macrophages challenged with Toxoplasma gondii.

In vitro activation of macrophage cell line J774G8 and mouse peritoneal macrophages resulted in oxygen-dependent and oxygen-independent killing of intracellular Toxoplasma gondii. Activation was characterized by oxygen-dependent killing detectable by enhanced lysosome fusion and digestion of T. gondii. The toxoplasmacidal activity of activated J774G8 cells and peritoneal macrophages was prevented by adding the oxygen intermediate scavengers catalase or superoxide dismutase during culture. Activated J774G8 cells and peritoneal macrophages also inhibited replication of those Toxoplasma organisms which survived the initial microbicidal activity. The inhibition of Toxoplasma replication was not significantly affected by exogenous catalase or superoxide dismutase. Peritoneal macrophages from Toxoplasma-immune mice showed similar microbicidal and inhibitory responses, supporting the model that activation leads to destruction of intracellular parasites by two different mechanisms.     

Endothelial cell invasion by Toxoplasma gondii: differences between cell types and parasite strains

Toxoplasma gondii disseminates and causes congenital infection by invasion of the endothelial cells. The aim of this study was to analyze the ability of two strains to invade two endothelial cell types. Tachyzoites of the RH and ME49 strains were expanded in Balb/c and C57BL6-RAG2?/? mice, respectively. Tachyzoites were harvested from 72 h Vero cell cultures and incubated for 30 min to 4 h at 10:1 parasite/cell ratio in 24-well plates, containing monolayers of either HMEC-1 line or human umbilical cells (HUVECs). The number of infected cells and parasitic vacuoles per infected cell were counted in Wright stained slides. A slow increase in the proportion of infected cells occurred but varied according to cell type–parasite strain combination: ME49 tachyzoites invaded up to 63 % HMEC-1 cells, while RH parasites infected up to 19 % HUVECs. ME49 and RH tachyzoites invaded 49 and 46 % HUVECs and HMEC-1 cells, respectively. Reinvasion and formation of new parasitophorous vacuoles of infected cells was more frequent than invasion of noninfected cells. The results support that the factors influencing invasion, and thus dissemination and vertical transmission, are parasite type, host cell type/subtype, and activation state. Interestingly, T. gondii virulence does not seem to relay on its invasion efficiency, but probably on replication speed.

Transmission and scanning electron microscopy of host cell entry by Toxoplasma gondii.

Entry by tachyzoites of Toxoplasma gondii, the causative agent of toxoplasmosis, into peritoneal cells was investigated with transmission and scanning electron microscopy. The process of entry is initiated by the parasite contacting the host cell with its anterior end, creating a small depression in the plasmalemma of the host cell. Occasionally, a small portion of the host cell cytoplasm protrudes and contaccts the anterior end of the parasite. A cylindrical structure (35 nm in diameter) extends from the pellicle of the parasite to the host cell. Such structures appear to assist host cell entry by T. gondii. As the entry process progresses, pseudopods of the host cell surround theparasite and finally T gondii becomes intracellular, being located in a vacuole separated from the host cell cytoplasm by a unit membrane. (Am J. Pathol 87:285-296, 1977).     

Protein palmitoylation inhibition by 2-bromopalmitate alters gliding, host cell invasion and parasite morphology in Toxoplasma gondii

Protein palmitoylation is the reversible covalent attachment of palmitic acid onto proteins. This post-translational modification has been shown to play a part in diverse processes such as signal transduction, cellular localization and regulation of protein activity. Although many aspects of protein palmitoylation have been identified in mammalian and yeast cells, little is known of this modification in Toxoplasma gondii. In order to determine the functional role of protein palmitoylation in T. gondii, tachyzoites were treated with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Parasites treated with 2-BP displayed a significant increase in non-circular trails which were longer than those trails left by non-treated parasites. Furthermore, 2-BP treatment reduced the invasion process to the host cells. Long-term treatment of intracellular tachyzoites resulted in major changes in parasite morphology and shape in a dose-dependent manner. These results suggest that palmitoylation could be modifying proteins that are key players in gliding, invasion and cytoskeletal proteins in T. gondii.     

Attachment of Toxoplasma gondii to host cells is host cell cycle dependent.

The initial attachment of Toxoplasma tachyzoites to target host cells is an important event in the life cycle of the parasite and hence critical in the pathogenesis of this infection. The efficiency of Toxoplasma attachment to synchronized populations of Chinese hamster ovary cells and bovine kidney cells was investigated by using a glutaraldehyde-fixed host cell assay system. For both cell lines, parasite attachment increased as the synchronized host cells proceeded from the G1 phase to the mid-S phase and then decreased as the cells entered the G2-M boundary. Postulating that these differences in attachment reflect the upregulation of a specific receptor, polyclonal antibodies were generated against whole MDBK antigen at 0 and 4 h into the S phase. Both antisera were shown to inhibit parasite attachment to both synchronous and asynchronous host cell populations. However, the attachment blockade observed with the 4-h antiserum was significantly greater than that with the 0-h antiserum, completely abolishing the cell cycle-dependent increase in attachment found in control samples. These findings suggest that Toxoplasma tachyzoites bind specifically to a host cell receptor which is upregulated in the mid-S phase of the cell cycle.     

Eicosanoid production by peritoneal and splenic macrophages in mice depleted of bone marrow by 89Sr

Previous studies showed that the prostaglandin-forming macrophages (M phi) induced in the spleens of CBA/J mice by intraperitoneal administration of Corynebacterium parvum (CP) could not be demonstrated following the depletion of bone marrow and blood monocytes with 89Sr. The present study compares prostaglandin E2 (PGE2), leukotriene C4 (LTC4), and LTB4 release by splenic and resident peritoneal M phi in 89Sr-treated mice and 88Sr controls following in vivo CP and in vitro incubation with zymosan, calcium ionophore A23187, or phorbol ester (PMA). Intraperitoneal administration of CP resulted in the appearance of PGE2- and LTB4-releasing M phi in the spleens of control but not 89Sr mice. The incorporation and quantitative distribution of 3H-arachidonic acid into membrane lipids, however, were comparable in test and control mice. Neither zymosan nor any of the other stimulatory agents was able to effect significant release of PGE2 in vitro. No release of LTC4 by splenic M phi was detectable under experimental or control conditions. In contrast, the capacity of resident peritoneal M phi to release PGE2, LTC4, and LTB4 was apparently unaffected by 89Sr-induced bone marrow and monocyte depletion with virtually no demonstrable elicitation. Resident peritoneal M phi removed after CP in such mice showed a dramatic decrease in PGE2 release when incubated in vitro with zymosan, A23187, or PMA. These results, taken with earlier findings, demonstrate characteristically different phenotypic expression of metabolism of certain eicosanoids by splenic M phi from the spleen and the peritoneal cavity and suggest in addition that the induction of PGE2-synthesizing M phi in the spleen by CP is dependent on either an immigrant cell originating in the bone marrow or a regulatory agent derived from a bone marrow cell.     

Induction of bradyzoite-specific Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages.

By using stage-specific monoclonal antibodies, an in vitro model has been developed to analyze the kinetics of expression of stage-specific antigens during the conversion process between tachyzoites and bradyzoites of Toxoplasma gondii. Following infection of murine macrophages with bradyzoites, the expression of bradyzoite-specific antigens declined, whereas the expression of tachyzoite-specific antigens increased during the first 72 h postinfection. Conversely, in gamma interferon-treated murine macrophages infected with tachyzoites, the inhibitory effect of gamma interferon on replication of parasites was accompanied by the induction of bradyzoite-specific antigens.     

Toxoplasma gondii: redistribution of tachyzoite surface protein during host cell invasion and intracellular development

Immunoperoxidase localisation of antigen at the electron microscope level confirms that parasite surface proteins, in association with membrane, are shed from the surface of the zoite on invasion, while varying amounts are also internalised. SAG 1 is stable on intracellular zoites for up to 48h, although new protein is also synthesised. SAG1 is present on the surface of daughter zoites and is found throughout the infected cell in distinct vacuoles; these vacuoles represent either direct extensions of the parasitophorous vacuole or true export of parasite surface material.     

Eicosanoid production and phospholipase A2 secretion by peritoneal macrophages from rats with adjuvant-induced arthritis

The molecular mechanism of action of several non-immunological histamine releasers has been investigated using pertussis toxin which interfers, via ADP-ribosylation, with some G-proteins. Pertussis toxin (100 ng/ml) inhibited histamine release induced by compound 48/80, substance P, mastoparan, peptide 401, bradykinin and spermine showing that a G-protein sensitive to pertussis toxin was involved in the non-immunological histamine release. All these compounds directly activate purified G-proteins. The sensitivity to pertussis toxin of this direct stimulatory effect was demonstrated for compound 48/80, mastoparan and substance P. Altogether these results suggest that a direct activation of G-protein might be the molecular mechanism of action of histamine secretagogues acting through a pertussis toxin sensitive G-protein and in this way mimic agonist-ligand receptor interaction.     

The production of ceroid by mouse peritoneal macrophages in vitro.

Murine resident peritoneal macrophages accumulated lipid droplets and subsequently insoluble, ceroid-like material when cultured in vitro in a medium containing 33% fetal calf serum. At least some of this insoluble lipid was membrane-bound and by light microscopy it often appeared as ''rings'' with a hollow centre. It is suggested that the production of ceroid may be the consequence of the uptake of lipids from the extracellular medium and the activity of the macrophage''s membrane-bound oxidative microbicidal mechanisms. The results indicate that macrophages are capable of rendering lipids insoluble, supporting the suggestion that this might occur in the atherosclerotic plaque.     

Toxoplasma gondii infection in the peritoneal macrophages of rats treated with glucocorticoids

It is well known that toxoplasmosis can be life threatening to immunocompromised individuals such as AIDS and organ transplantation patients. Glucocorticoids (GCs) are widely used in the clinic for the treatment of autoimmune diseases and organ transplantation resulting in acute toxoplasmosis in these patients. However, the interaction and mechanism between the development of acute toxoplasmosis and GC therapy are still unknown. The aims of this study were to investigate the infection of Toxoplasma gondii in the peritoneal macrophages of rats treated with glucocorticoids. Our results showed that the growth rate of T. gondii RH strain was significantly increased in the peritoneal macrophages of rats treated with glucocorticoids in vivo. For instance, 242 (±16) tachyzoites were found in 100 macrophages from the rats treated with methylprednisolone (MP), while only 16 (±4) tachyzoites were counted in the macrophages from the non-treated control rats 24 h after infection (P?<?0.01). We also demonstrated that a significant inhibition of nitric oxide (NO) production was detected in the macrophages collected from the rats post-treated with GCs with 12.90 μM (±0.99 μM) of nitrite production from the rats treated with MP, while 30.85 μM (±1.62 μM) was found in the non-treated control rats 36 h after incubation (P?<?0.01). Furthermore, glucocorticoids could significantly inhibit the expression of inducible nitric oxide synthase mRNA and its protein in the rat peritoneal macrophages. Our results strongly indicate that the decrease of NO in the rat peritoneal macrophages is closely linked to the cause of acute toxoplasmosis in the host. Additionally, there was a significant increase in the number of cysts produced by the naturally cyst forming, T. gondii Prugniaud strain with an average of 2,795 (±422) cysts of the parasite being detected in the brains of the rats treated with dexamethasone, while only 1,356 (±490) cysts were found in the non-treated control animals (P?<?0.01). As rats and humans are both naturally resistant to T. gondii infection, these novel data could lead to a better understanding of the development of acute toxoplasmosis during glucocorticoid therapy in humans.     

Specific antibody-dependent killing of Toxoplasma gondii by normal macrophages.

The requirement for specificity of antibody-dependent inhibition or killing of intracellular Toxoplasma gondii trophozoites by normal mouse peritoneal macrophages was evaluated in vitro using light microscopy and autoradiography. Anti-toxoplasma antibody in the presence of 'accessory factor' rendered extracellular T. gondii trophozoites non-viable and non-infectious for cells, whereas exposure of extracellular trophozoites to heat-inactivated immune serum did not appear to damage the parasites. Although pretreatment of extracellular trophozoites with heat-inactivated immune serum neither diminished nor prevented infection of normal mouse peritoneal macrophages, it did confer upon macrophages the ability to inhibit or kill the organisms once they were intracellular. In contrast, pretreatment of trophozoites with either heat-inactivated normal or Besnoitia jellisoni immune serum did not enable normal macrophages to inhibit or kill T. gondii; rather, such organisms multiplied intracellularly in normal macrophages. Thus, pretreatment with specific antibody alone prepared T. gondii trophozoites for intracellular destruction by normal mouse peritoneal macrophages. These results suggest that spesific antibody acting in concert with normal macrophages may play a role in controlling infection with T. gondii.     

Regulation of tumour necrosis factor production by mouse peritoneal macrophages: the role of cellular cyclic AMP.

We have studied the role of cyclic adenosine 3':5' monophosphate (cAMP) in the regulation of lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF) production by mouse peritoneal macrophages. LPS did not alter the intracellular levels of cAMP. However, prostaglandin E2 (PGE2) and cholera toxin, both of which are known to increase intracellular levels of cAMP, consistently inhibited LPS-induced TNF production. The cAMP analogues, dibutyryl cAMP (DbcAMP) and 8-bromo cAMP (8BrcAMP), also inhibited TNF production, whereas pertussis toxin, which inactivates the inhibitory guanine nucleotide-binding protein (Gi), had no effect. These observations suggested that LPS did not itself modify macrophage adenylate cyclase activity, while agents that increased intracellular cAMP levels were able to inhibit LPS-induced macrophage TNF production.