The inhibitory receptor LILRB4 (ILT3) modulates antigen presenting cell phenotype and, along with LILRB2 (ILT4), is upregulated in response to Salmonella infection

Abstract

Background: Leukocyte Ig-like receptors (LILR) are a family of innate immune receptors with immunomodulatory functions. High-level expression of the receptors LILRB2 (ILT4) and LILRB4 (ILT3) is a feature of tolerogenic antigen presenting cells and has been observed in cancer and transplant situations. There are relatively few studies regarding these receptors in the context of infection and it is not yet clear how LILRB4 exerts its inhibitory effects.

Results: We studied the effects of LILRB4 ligation on antigen presenting cell phenotype, and the expression of LILRB2 and LILRB4 on Salmonella-infected antigen presenting cells. Ligation of LILRB4 throughout in vitro culture of dendritic cells led to an upregulation of the co-stimulatory protein CD86. Alterations in the production of IL-8 and IL-10 by LILRB4-ligated macrophages were also observed. Infection with Salmonella typhimurium or TLR stimulation with Salmonella components led to an upregulation of LILRB2 and LILRB4.

Conclusion: Our results indicate that the inhibitory effects of LILRB4 do not result from a failure to upregulate co-stimulatory proteins. In addition to the high level expression that can render antigen presenting cells tolerogenic, there may be a role for lower level expression and activity of LILRB2 and LILRB4 in response to TLR signalling during an immune response to bacterial infection.

Figure 1

Cell surface phenotype of LILRB4-ligated dendritic cells. Flow cytometric analysis of cell surface markers was performed for dendritic cells cultured in the presence of an isotype control antibody (Isotype) or LILRB4-specific antibody (LILRB4) with Protein G. LPS-matured dendritic cells cultured in the presence of isotype control antibody (Isotype+LPS) or LILRB4-specific antibody (LILRB4+LPS) were also compared. Results for individual donors are indicated by joined lines. A minimum of 5 individuals were examined for each marker. Statistical analysis of isotype control versus LILRB4-ligated cells was performed using a Wilcoxon matched pairs test on Prism Software (GraphPad, USA).

Figure 2

T cell stimulatory properties of LILRB4-ligated dendritic cells. B. Dendritic cells were cultured in the presence of isotype control or LILRB4-specific antibody with cross-linking and LPS matured as before. Naïve CD4+ T cells from an allogeneic donor were added at various stimulator:responder (S:R) ratios and cultured for 6 days. CD4+ T cell proliferation was assessed by thymidine incorporation. Triplicate wells were analysed with error bars representing the SEM. Results are shown for 2 experiments, which are representative of the 5 performed.

Figure 3

LILRB4 ligation throughout macrophage culture reduces IL-8 but increases IL-10 production. Cytokine levels in supernatants of in vitro-cultured macrophages were analysed by ELISA. Macrophages were cultured in the presence of Isotype control or LILRB4 specific antibody. Three sets of triplicate samples were analysed with error bars representing the SEM. Statistical testing by two-sided Student's t test did not show any significant differences between treatment groups.

Figure 4

Salmonella typhimurium upregulates expression of LILRB2 and LILRB4 on in vitro-cultured macrophages. Real-time PCR was used to assess the expression of LILRB2 and LILRB4 mRNA in monocyte-derived macrophages treated with either live or heat-killed GFP⁺ S. typhimurium. Salmonella infection was confirmed by flow cytometic detection of GFP (data not shown). p-values were calculated using a two-tailed Wilcoxon rank test, using a 95% confidence level on Prism Software (GraphPad, USA).

Figure 5

LPS treatment upregulates expression of LILRB2 and LILRB4 at a similar level to that observed for whole bacteria. Alterations in the mRNA expression of LILRB4 and LILRB2 in monocyte-derived macrophages following stimulation with S. typhimurium LPS, Flagellin, or whole S. typhimurium (either live or heat-killed (H.K.)).

Figure 6

LPS treatment upregulates LILRB2 and LILRB4 on in vitro cultured dendritic cells. Flow cytometric analysis was used to study surface expression of LILRB4 and LILRB2 on in vitro-cultured dendritic cells following treatment with LPS or flagellin.