Entamoeba histolytica: induction of cyclooxygenase-2 expression during amoebic liver abscess formation in hamsters (Mesocricetus auratus)
Introduction
The complete sequence of morphologic events that conclude with liver necrosis by Entamoeba histolytica invasion has been studied in hamsters successfully infected by different inoculation routes. Inoculation of live trophozoites in liver parenchyma is coupled with an acute inflammatory process, which involves mostly polymorphonuclear leukocytes (PMNs), macrophages (MOs), and lymphocytes (Shibayama et al., 1997; Tsutsumi et al., 1984). Inflammation is a complex array of adaptive responses to tissue injury in which cells and biochemical mediators participate to remove endogenous or foreign material. Prostaglandins, mainly PGE2, are considered inflammation mediators that have important immuno-regulatory effects on both cellular and humoral responses (Phipps et al., 1991). The effects of PGE2 on TH1 and TH2 subsets of CD4+ lymphocytes-cytokine secretion patterns allow it to exert suppressive actions on effector and cooperative MOs functions (Betz and Fox, 1991; DeWitt, 1991).
Production of PGE2 is the result of the activity of the enzyme cyclooxygenase (COX; also known as prostaglandin endoperoxide synthase, EC 1.14.99.1). COX exists in at least two isoforms: COX-1, a constitutive enzyme that is differentially expressed in almost all tissues and COX-2, an inducible isoform associated with inflammation and that is also strongly expressed in kidney (Hla and Neilson, 1992; Masferrer et al., 1994).
Experimental evidences suggest that during amoebic liver abscess (ALA) formation, the production of TNF-α and the expression of class II MHC antigens induced by IFN-γ in MOs, are regulated by a PGE2-dependent mechanism (Wang and Chadee, 1995; Wang et al., 1992). We have demonstrated that development of experimental ALA in hamsters curses with an increase in both, systemic levels of PGE2 and local COX activity in liver microsomes (Sánchez-Ramı́rez et al., 1997). Recently, COX-2 induction in human colonic cells was demonstrated in a severe combined immunodeficient mouse–human intestinal xenograft model of E. histolytica infection (Stenson et al., 2001). In this work, we demonstrate by immunohistochemical analysis (IHC), that COX-2 protein is induced by E. histolytica during ALA formation on MOs and PMNs. COX-2 mRNA gene was detected by reverse transcription and PCR amplification (RT-PCR) from liver samples of infected animals at different times postinfection (p.i.) suggesting that de novo synthesis of the protein may be involved in the expression of COX-2.
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Chemicals and reagents
RPMI-1640, Hanks’ balanced salt solution, penicillin–streptomycin, l-glutamine, lipopolysaccharide (LPS; Escherichia coli 0128:B12 serotype), Tris, sucrose, phenylmethanesulfonyl fluoride (PMSF), p-tosyl-l-lysine chloro-methyl ketone (TLCK), N-ethylmaleimide (NEM), iodoacetamide (IA), ethylenediaminetetraacetic acid (EDTA), ethidium bromide, absolute methanol, diethyl pyrocarbonate, acrylamide, bisacrylamide, glycine, and hydrogen peroxide from Sigma–Aldrich (México).
Oligonucleotide primers
Entamoeba histolytica induces COX-2 expression in AMAs
To determine the isoform of COX that was responsible for PGE2 production in infected animals, protein extracts from liver of infected hamsters (from near, far, and abscess areas) were prepared. The anti-COX-1 antibody revealed a specific signal using COX-1 protein purified from ram seminal vesicles as a positive control (Fig. 1A, upper panel, lane 1); COX-1 protein was not detected in LPS-stimulated PMOs (Fig. 1A, upper panel, lane 2), or in samples from infected tissue (Fig. 1A, upper panel,
Discussion
Our results confirm that E. histolytica induces COX-2 expression in MOs at the site of infection, which is consistent with the high levels of PGE2 produced by AMAs (Wang and Chadee, 1992). Since PMOs from i.p. infected hamsters did not express COX-2 when cells were recovered 3 days postinoculation, induction in zones near to the abscess probably depends on continuous interaction between AMAs and trophozoites or excretion products. This result agrees with the fact that trophozoites inoculated by
Acknowledgments
This work was supported by a grant from National Council for Science and Technology of Mexico (CONACyT Grant No. 31943-M). The authors thank Amelia Rios for critically reading the manuscript.
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Host-lipidome as a potential target of protozoan parasites
2013, Microbes and InfectionCitation Excerpt :Prostaglandins (PG) are class of bioactive lipid mediators that are produced by arachidonic acid metabolism through the action of COX-1, COX-2 and PG synthase. It is a well-known fact that several protozoan parasites increase the amount and synthesis of prostaglandins in the host cell during infection [90–92]. Earlier studies on murine splenic mononuclear cells and peritoneal macrophages infected with L. donovani have shown increased COX and LOX activities which results in more prostaglandin E2 (PGE2) and other arachidonic acid metabolite production [93].
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2012, Nanomedicine: Nanotechnology, Biology, and MedicineCitation Excerpt :The following primers were used: for COX-2 gene, sense primer 5′-CTG TAT CCC GCC CTG CTG GTG-3′ and antisense primer 3′-ACT TGC GTT GAT GGT GGC TGT CTT-5′; for GAPDH, sense primer 5′-CCT TCA TTG ACC TCA ACT AC-3′ and antisense primer 3′-GGA AGG CCA TGC CAG TGA GC-5′.26 These primers yielded amplified products of 279 base pairs (bp) for COX-2 and 574 base pairs for GAPDH; the cycling program and reaction conditions are described elsewhere.27 PCR products were analyzed by electrophoresis in a 1% w/v agarose gel containing ethidium bromide; photographs were taken using a laboratory imaging GDS-8000 Chemi System (UVP, Inc., Upland, California) and analyzed with LabWorks 3.0 software (UVP, Upland, California).
Molecular basis for prostaglandin production in hosts and parasites
2007, Trends in ParasitologyCitation Excerpt :More conclusive evidence of a COX activity in pathogenic parasites was obtained from the intestinal protozoan parasite E. histolytica. A COX-like enzyme was isolated, characterized and cloned by reverse transcriptase PCR (RT-PCR) [18]. It possesses neither the arachidonate-binding domain, nor the heme-coordinating and catalytic sites, which are conserved in other species, and showed little homology with COX-1 and COX-2 enzymes from mammals and other species at the nucleotide and amino acid levels.