C-reactive protein increases C3 deposition on Leishmania donovani promastigotes in human serum.

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C-reactive protein (CRP) is the major acute phase serum protein of man, with concentrations increasing upto one thousand fold during inflammation, however, little is known about the role of CRP in defence against infection. CRP binds bacteria [ 1.21 and damaged host cells and tissues [3] and acts as an opsonin, increasing uptake into both macrophages and neutrophils. Some researchers have found that the presence of complement is necessary for CRP mediated opsonisation 14.51. whereas others have reported direct CRP dependent uptake [6]. CRP can activate the classical pathway of complement by binding to Clq [7]. CRP mediated complement activation stimulates C3 deposition but is p”r at triggering formation of the membrane attack complex and causing cell lysis[8]. Leishmania donovuni are unicellular parasites which are the causative agent of viceral leishmaniasis. These parasites are transmitted in the promastigote form from the sandfly vector. Once within the mammalian host, the parasties transform into amastigotes which perpetuate the infection within host macrophages. Promastigotes activate the complement system in human serum. However, rather than being deliterious to parasite survival, promastigotes are believed to use the complement deposited on their surface to gain entry to macrophages via macrophage complement receptors [9]. Although macrophage derived complement is capable of causing opsonisation of promastigotes in v i m , uptake is enhanced in the presence of serum [lo]. We have recently shown that CRP will bind to the surface of L.donovani promastigotes, through recognition of the surface glycocalyx of the parasites which is formed by the molecule lipophosphoglycan [I l l . One of the consequences of CRP binding to the surface of these parasites was found to be an increased uptake into human macrophages. Here, we have studied CRP binding to promastigotes in human serum and its influence on complement deposition on the parasite surface. Human serum was obtained from a healthy volunteer and assayed for CRP using a sandwich ELISA method. The,serum used was found to have a low concentration of CRP (0.12 pg/ml). The average concentration of CRP in sera from a Leishmaniasis endemic area has been shown to be 3.95 f 1.9pg/ml [12]. Therefore, serum supplemented with an additional 5.0 pg/ml CRP was also used. In addition, CRP depleted serum was prepared by incubating with phosphorylcholine-sepharose overnight at 4°C. Following exbaction, the serum concentration of CRP was <25 ng/ml. 2mM phosphorylcholine was also included in CRP-depleted serum, to bind to any remaining CRP. L.donovuni promastigotes were grown to late stationary phase as previously described [ I I] and were fixed with 4% formaldehyde before use. lo6 parasites were used for each assay and all dilutions were in 1% bovine serum albumin, 0.5mM Ca’: Tris-buffered saline. To demonstrate CRP binding to promastigotes, parasites were incubated in sera, followed by ami ty purified goat anti-CRP [ I I] and anti-goat IgG-FITC conjugate (Sigma) each for I hour at 37T , washing twice between each stage. Binding was analysed in a Becton Dickinson FACscan using lysis I1 software.