Regulation of macrophage subsets and cytokine production in leishmaniasis.

Macrophages are host cells for parasites of the genus Leishmania where they multiply inside parasitophorous vacuoles. Paradoxically, macrophages are also the cells responsible for killing or controlling parasite growth, if appropriately activated. In this review, we will cover the patterns of macrophage activation and the mechanisms used by the parasite to circumvent being killed. We will highlight the impacts of the vector bite on macrophage activation. Finally, we will discuss the ontogeny of macrophages that are infected by Leishmania spp.

The Leishmania chagasi proteasome: role in promastigotes growth and amastigotes survival within murine macrophages.

Proteasomes are multisubunit proteases that exist universally among eukaryotes. They have multiple proteolytic activities and are believed to have important roles in regulating cell cycle, selective intracellular proteolysis, and antigen presentation. Here we have partially purified Leishmania chagasi proteasome. The L. chagasi proteasome rich fraction displayed the typical features of eukaryotic 20S proteasome complexes, being active towards peptidyl substrates with hydrophobic and acidic residues, and sensitive to the proteasome-specific inhibitor lactacystin. We have shown that lactacystin, or its active form clasto-lactacystin beta-lactone, but not E-64, blocks the in vitro growth of L. chagasi promastigotes, demonstrating that the interference with parasite growth is due to the lack of proteasome activity. Furthermore, pre-treatment of L. chagasi promastigotes with lactacystin did not prevent parasite entry in host cells, but markedly restricted its intracellular survival. These results demonstrate that intact parasite proteasome function is required for replication of L. chagasi and for amastigotes survival inside the vertebrate host cell.

TSG-14 transgenic mice have improved survival to endotoxemia and to CLP-induced sepsis.

Tumor necrosis factor-stimulated gene 14 (TSG-14)/PTX3 was identified originally as a TNF-alpha and IL-1beta-stimulated gene from normal, human foreskin fibroblasts and vascular endothelial cells, respectively. TSG-14 gene encodes a 42-kDa-secreted glycoprotein with a carboxy-terminal half that shares homology with the entire sequence of C-reactive protein (CRP) and serum amyloid P component (SAP), acute-phase proteins of the pentraxin family. Some experimental evidence suggests that TSG-14 plays a role in inflammation, yet its function and mechanism of action remain unclear. We have generated transgenic mice that overexpress the murine TSG-14 gene under the control of its own promoter. From eight transgenic founders, two lineages were derived and better characterized: Tg2 and Tg4, carrying two and four copies of the transgene, respectively. TSG-14 transgenic mice were found to be more resistant to the endotoxic shock induced by LPS and to the polymicrobial sepsis caused by cecal ligation and puncture (CLP). Moreover, macrophages derived from the transgenic mice produced higher amounts of nitric oxide in response to IFN-gamma, TNF-alpha, and LPS as compared with macrophages from wild-type animals, and the augmented response appears to be the consequence of a higher responsiveness of transgenic macrophages to IFN-gamma. The data shown here are the first in vivo evidence of the involvement of TSG-14 in the inflammatory process and suggest a role for TSG-14 in the defense against bacterial infections.

Macrophage damage by Leishmania amazonensis cytolysin: evidence of pore formation on cell membrane.

We have previously shown that both promastigotes and amastigotes of Leishmania amazonensis contain a lytic protein that damages erythrocytes and nucleated cells, including macrophages (F. S. M. Noronha, F. J. Ramalho-Pinto, and M. F. Horta, Infect. Immun. 64:3975-3982, 1996). Using the patch-clamp technique, we show here that cell damage by parasite extracts is mediated by the formation of nonselective pores on the target membrane. This demonstrates that L. amazonensis cytolysin is a pore-forming protein (PFP), here named leishporin. We show that the diameters of the pores formed by parasite extracts are heterogeneous, varying from approximately 1.6 to >6.1 nm according to cytolysin concentration or time. We also show that pore formation involves the binding of the PFP to the target cell membrane, a temperature-independent event that is necessary but not sufficient to lyse cells. This is followed by a temperature-dependent step that triggers lysis, probably the insertion and the polymerization of protein subunits in the lipid bilayer. We provide evidence that suggests that polymerization of single subunits must occur for pore formation. We show, in addition, that L. amazonensis expresses molecules antigenically homologous to other PFPs.

Cytolytic activity in the genus Leishmania: involvement of a putative pore-forming protein.

We describe here that parasites of the genus Leishmania contain a cytolytic activity which acts optimally at pH 5.0 to 5.5 and at 37 degrees C in vitro. or the four species examined, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) major presented considerable hemolytic activity, whereas Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis showed little and no hemolytic activity, respectively. The cytolytic factor of L. amazonensis promastigotes was characterized as a protein with no protease-, phospholipase-, or detergent-like activity, probably localized inside membranous vesicles. The use of osmotic protectants revealed the colloid-osmotic nature of hemolysis, which is indicative of pore formation in the membranes of target cells. This putative pore-forming protein also damaged nucleated cells, including macrophages, causing an increase in their membrane permeability with leakage of cytoplasmic proteins. Both promastigotes and amastigotes express this lytic activity, suggesting that the cytolysin may have a function in both stages of this parasite. The pH and temperature required for optimal activity indicate that it might be more effective within the mammalian host, particularly inside the macrophage parasitophorous vacuole. In promastigotes of L. amazonensis, the expression of lytic activity seems to be regulated during their growth in vitro, being maximal at the early stationary phase.

Identification of a putative pore-forming hemolysin active at acid pH in Leishmania amazonensis.

Several organisms, including the protozoa Entamoeba histolytica and Trypanosoma cruzi, have been shown to contain pore-forming proteins (PFP) thought to play a role in the pathogenesis of the diseases they generate. In the present report, we show that promastigotes of Leishmania amazonensis express a hemolysin that appears to cause colloid-osmotic lysis, typical of pore formation. This hemolysin affects red blood cells of different species at 37 degrees C, but not at 0 degrees C, with maximum activity at pH 5.0. The hemolytic activity is heat-labile, but lysis is not affected by protease inhibitors. These results suggest the involvement of a protein with no proteolytic or detergent activity. Hemolysis is inhibited by polyethyleneglycol, suggesting its colloid-osmotic nature. Hemolytic extracts of the parasite contain a polypeptide that reacts with antibodies to perforin from mouse cytotoxic T lymphocytes or to C9 from human complement. In addition, genomic DNA of L. amazonensis contains a fragment that hybridizes to a perforin cDNA probe. The circumstantial evidence suggests that the L. amazonensis hemolytic activity may be mediated by a PFP homologous to perforin and C9.

Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins.

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

Mechanisms of evasion of Schistosoma mansoni schistosomula to the lethal activity of complement.

Schistosomula of Schistosoma mansoni became resistant to antibody-dependent complement damage in vitro after pre-incubation with normal human erythrocytes (NHuE) whatever the ABO or Rh blood group. Resistant parasites were shown to acquire host decay accelerating factor (DAF), a 70 kDa glycoprotein attached to the membrane of NHuE by a GPI anchor. IgG2a mAb anti-human DAF (IA10) immunoprecipitated a 70 kDa molecule from 125I-labeled schistosomula pre-incubated with NHuE and inhibited their resistance to complement-dependent killing in vitro. Incubation of schistosomula with erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNHE) or SRBC, which are DAF-deficient, did not protect the parasites from complement lesion. Supernatant of 100,000 x g collected from NHuE incubated for 24 h in defined medium was shown to contain a soluble form of DAF and to protect schistosomula from complement killing. Schistosomula treated with trypsin before incubation with NHuE ghosts did not become resistant to complement damage. On the other hand, pre-treatment with chymotrypsin did not interfere with the acquisition of resistance by the schistosomula. These results indicate that, in vitro, NHuE DAF can be transferred to schistosomula in a soluble form and that the binding of this molecule to the parasite surface is dependent upon trypsin-sensitive chymotrypsin-insensitive polypeptide(s) present on the surface of the worm.