In vitro binding and survival assays of Leishmania parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi

Abstract

BackgroundThere are a few works considering the characterization of canine monocyte-derived macrophages as well as a standardized procedure for isolation, culture, and infection of these cells with Leishmania. We have performed several modifications in order to improve the canine monocyte-derived macrophage cultures. In addition, we have done a comparative study between monocytes and monocyte-derived macrophages from dogs naturally and experimentally infected with L. chagasi.ResultsIn the presence of exogenous serum, opsonized Leishmania promastigotes binds better to monocytes/macrophages than without serum. Otherwise, this binding occurs due to the strict correlation between the opsonized biologic particles with the third receptor of the complement (CR3-CD11b/CD18). In fact, our assays with CD11b confirmed the importance of this receptor for canine cells and the L. chagasi experimental system. Moreover, monocytes obtained from naturally infected dogs have shown a higher number of monocytes bounded to promastigotes. The experimental results regarding survival have shown that promastigote forms of opsonized L. chagasi were more infective, because we found higher numbers of promastigotes bound to the different cells. As a consequence, after forty-eight hours of binding, higher numbers of amastigotes appeared inside monocyte-macrophages.ConclusionThese studies have given support to continue comparative studies involving canine monocytes, monocyte-derived macrophages and peritoneal macrophages. Since we have standardized the canine cell culture, we are looking forward to determining the phenotypic properties of these cells before and after L. chagasi infection using flow cytometry.

A Rapid and Simple Method to Obtain Canine Peripheral Blood-Derived Macrophages

“…It is of note that nearly all of the macrophages showed active phagocytosis 5 days post stimulation. Previous reports required more than 10 days of cultivation for macrophage maturation [3,8,10,16]. Shorter cultivation period would be more convenient for investigation.…”

“…This suggests that 60 ml of peripheral blood would be required to obtain approximately 4  10 5 macrophages. A modification of this method has been made by others [16]. By adding granulocyte-macrophage colony stimulating factor (GM-CSF) and replacing FBS for dog serum, Sampaio et al [16] were able to improve cell survival.…”

“…A modification of this method has been made by others [16]. By adding granulocyte-macrophage colony stimulating factor (GM-CSF) and replacing FBS for dog serum, Sampaio et al [16] were able to improve cell survival. However, the exact difference was not described.…”

“…Since it would be difficult to collect a large volume of peripheral blood from diseased dogs, a more efficient method would be necessary to reduce the blood sampling volume. Moreover, since more than 10 days of cultivation are required in all previously reported methods [3,8,10,16], a shorter cultivation period would be more convenient for investigation. The aim of this study is to establish a simple and rapid method to obtain macrophages from small volumes of dog peripheral blood.…”

“…Macrophages can be obtained from bronchoalveolar lavage fluid [18], bone marrow [20], and peripheral blood [3,8,10,16], and cultured as adherent cells on plastic plates with or without cytokine addition. Among the above-mentioned macrophage sources, the most accessible source is peripheral blood, since it does not require anesthesia for sample collection.…”