We have taken advantage of the availability of splenic aspirate cells from VL patients, containing parasitized macrophages as well as IL-10 secreting cells, to directly address the possible function of IL-10 in promoting the survival and growth of L donovani in the VL spleen. Direct titration culture of an equivalent fraction of the splenic aspirate cells from the 67 patients, prior to their incubation with anti-IL-10 or control antibodies, revealed a wide range in the number of viable amastigotes present in the tissue aspirate (minimum 9; maximum 4.78 × 106; median 19683; data not shown). This variability reflects the sampling variance associated with the needle aspiration procedure (ie, the volume and cell concentration of the aspirate), as well as the stage of disease at the time the aspirates were obtained.
Following 3 days of incubation of the splenic cells, the number of viable organisms in the anti-IL-10-treated cultures showed a highly significant reduction compared with the control-treated cultures (control IgG, geometric mean 14
540; anti--IL-10, geometric mean 1182; P
< .0001; ). Of the 67 paired cultures analyzed, 49 (73%) had fewer parasites, while 12 (18%) had more and 6 (9%) did not change under IL-10--neutralizing conditions. Furthermore, the IL-10 inhibition resulted in complete clearance of organisms from 20 (30%) of the cultures. In 9 of the 12 paired cultures for which the anti-IL-10 treatment resulted in increased numbers of parasites, the end-point titration differed by only a single well, representing a 2–3-fold increase. The 12 patients from whom these aspirates were obtained did not differ significantly in any clinical parameter from the 49 patients for whom their splenic parasite numbers were reduced by the IL-10 neutralization.
Figure 1. IL-10 neutralization promotes the killing of L donovani and enhances IFNγ and TNFα secretion in splenic aspirate cells from patients with active VL. Splenic cells were cultured at 37°C, 5% CO2 in the presence of anti–IL-10 (more ...)
Determination of the concentration of cytokines released by the spleen cells during the 3 days of culture revealed consistently elevated levels of both IFNγ and TNFα in the anti-IL-10-treated cultures (). Of the supernatants analyzed from 32 paired cultures, IL-10 neutralization enhanced IFNγ and TNFα secretion in 29 and 31 of the cultures, respectively, compared with the control-treated cells. The wide range of cytokine concentrations again reflects the variability in the number of cells plated in these assays. No association was found between the amount of cytokines released and the amount of killing conferred by the IL-10 neutralization in this sampling of splenic aspirate cultures submitted for analysis.
IL-10 is secreted by multiple cell types, including macrophages, dendritic cells, natural killer (NK) cells, B cells, and various T-cell subsets. We have recently identified CD4+
T cells as the major source of elevated IL-10 mRNA in the VL spleen [12
]. Our current findings do not address the source(s) of the IL-10 that promoted the survival of amastigotes present in the splenic aspirate cells, and it is possible that other cell types, such as the parasitized macrophages themselves, are the most potent source of IL-10 in this assay. The cellular target(s) of the suppression is also not addressed, although we favor a direct action of IL-10 on the infected cells to render them refractory to activation signals. Because enhanced secretion of IFNγ and TNFα was also observed, the IL-10 may have also compromised T-cell activation directly, or via effects on antigen-presenting cell (APC) function. It seems likely that in the setting of the VL spleen, infected macrophages, APCs, and T cells are all suppressed by IL-10, such that IL-10 inhibition profoundly favors the balance of activating over deactivating cytokines. With regard to the few anti--IL-10--treated cultures that demonstrated increased numbers of parasites, it is possible that they contained extracellular amastigotes, due perhaps to lysis of infected cells during the aspiration procedure. These organisms would be unaffected by IL-10 inhibition and might begin to replicate as extracellular forms.
Finally, these findings provide the most direct support to date for IL-10 as a therapeutic target in human VL. Treatment of kala-azar remains unsatisfactory, and there is an urgent need to develop new treatment strategies to reduce the dose and duration of chemotherapies, the development of drug resistance, and the persistence of parasites following clinical cure [13
]. The dose-sparing and sterilizing effects of IL-10 receptor blockade on treatments involving pentavalent antimony or amphotericin B have been demonstrated in experimental models of VL [14
]. Direct manipulation of the immune response via IL-10 inhibition, and in combination with antiparasitic drugs, provides a novel approach to optimizing treatment regimens for human VL.