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LILRA2 selectively modulates LPS-mediated cytokine production and inhibits phagocytosis by monocytes.

Lu HK, Mitchell A, Endoh Y, Hampartzoumian T, Huynh O, Borges L, Geczy C, Bryant K, Tedla N - PLoS ONE (2012)

Bottom Line: However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown.Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS.Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6.

View Article: PubMed Central - PubMed

Affiliation: Inflammation and Infection Research Centre, School of Medical Sciences, Department of Pathology, University of New South Wales, Australia. [corrected].

ABSTRACT
The activating immunoglobulin-like receptor, subfamily A, member 2 (LILRA2) is primarily expressed on the surface of cells of the innate immunity including monocytes, macrophages, neutrophils, basophils and eosinophils but not on lymphocytes and NK cells. LILRA2 cross-linking on monocytes induces pro-inflammatory cytokines while inhibiting dendritic cell differentiation and antigen presentation. A similar activating receptor, LILRA4, has been shown to modulate functions of TLR7/9 in dendritic cells. These suggest a selective immune regulatory role for LILRAs during innate immune responses. However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown. Moreover, the effects of LILRA2 on TLR4 and FcγRI-mediated monocyte functions are not elucidated. Here, we show activation of monocytes via LILRA2 cross-linking selectively increased GM-CSF production but failed to induce IL-12 and MCP-1 production that were strongly up-regulated by LPS, suggesting functions distinct from TLR4. Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS. Furthermore, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads and serum opsonized Escherichia coli but had limited effect on phagocytosis of non-opsonized bacteria. Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6. Interestingly, LILRA2 cross-linking on monocytes caused significant inhibition of TLR4 mRNA and protein, suggesting LILRA2-mediated suppression of LPS responses might be partly via regulation of this receptor. Taken together, we provide evidence that LILRA2-mediated activation of monocytes is significantly different to LPS and that LILRA2 selectively modulates LPS-mediated monocyte activation and FcγRI-dependent phagocytosis.

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Cross-linking of LILRA2 on monocytes caused time dependent down-regulation of TLR4 mRNA and surface protein expression.A. Quantitative RT-PCR on mRNA extracted from purified monocytes that were activated through LILRA2 cross-linking caused down-regulation of TLR4 mRNA expression with significant changes observed at 12, 18 and 24 h but not at 6 h time point. B. Representative flow cytometry for the expression of TLR4 protein on the surface of monocytes activated via LILRA2 cross-linking for 6–48 hr. Dotted histograms on the left of each plot are cells stained with isotype matched negative control mAb. C. Summary of the mean fluorescence intensity (MFI) showing decreased expression of surface TLR4 on cells activated via LILRA2 cross-linking as compared to cells treated with IgG1 control with statistically significant difference seen at 12 h time point. Error bars represent SEM of 5 independent experiments, *p<0.05.
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pone-0033478-g005: Cross-linking of LILRA2 on monocytes caused time dependent down-regulation of TLR4 mRNA and surface protein expression.A. Quantitative RT-PCR on mRNA extracted from purified monocytes that were activated through LILRA2 cross-linking caused down-regulation of TLR4 mRNA expression with significant changes observed at 12, 18 and 24 h but not at 6 h time point. B. Representative flow cytometry for the expression of TLR4 protein on the surface of monocytes activated via LILRA2 cross-linking for 6–48 hr. Dotted histograms on the left of each plot are cells stained with isotype matched negative control mAb. C. Summary of the mean fluorescence intensity (MFI) showing decreased expression of surface TLR4 on cells activated via LILRA2 cross-linking as compared to cells treated with IgG1 control with statistically significant difference seen at 12 h time point. Error bars represent SEM of 5 independent experiments, *p<0.05.

Mentions: To determine whether the inhibitory effect of LILRA2 cross-linking was due to down-regulation of LPS-recognition receptors mainly TLR4, mRNA level of these receptor on monocytes was assessed. LILRA2 cross-linking consistently down-regulated TLR4 mRNA expression by up to 3fold at 12, 18 and 24 hour time points when compared to those treated with control IgG1 (Fig. 5A). Similarly, TLR4 protein expression on the surface of LILRA2 cross-linked cells was down-regulated at 6, 12 and 18 h time points with the highest and statistically significant effect observed 12 h after LILRA2 cross-linking (Fig. 5B, C). Interestingly, prolonged LILRA2 cross-linking (24–48 h) does not seem to show significant difference in TLR4 expression to control cells (Fig. 5C).


LILRA2 selectively modulates LPS-mediated cytokine production and inhibits phagocytosis by monocytes.

Lu HK, Mitchell A, Endoh Y, Hampartzoumian T, Huynh O, Borges L, Geczy C, Bryant K, Tedla N - PLoS ONE (2012)

Cross-linking of LILRA2 on monocytes caused time dependent down-regulation of TLR4 mRNA and surface protein expression.A. Quantitative RT-PCR on mRNA extracted from purified monocytes that were activated through LILRA2 cross-linking caused down-regulation of TLR4 mRNA expression with significant changes observed at 12, 18 and 24 h but not at 6 h time point. B. Representative flow cytometry for the expression of TLR4 protein on the surface of monocytes activated via LILRA2 cross-linking for 6–48 hr. Dotted histograms on the left of each plot are cells stained with isotype matched negative control mAb. C. Summary of the mean fluorescence intensity (MFI) showing decreased expression of surface TLR4 on cells activated via LILRA2 cross-linking as compared to cells treated with IgG1 control with statistically significant difference seen at 12 h time point. Error bars represent SEM of 5 independent experiments, *p<0.05.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316576&req=5

pone-0033478-g005: Cross-linking of LILRA2 on monocytes caused time dependent down-regulation of TLR4 mRNA and surface protein expression.A. Quantitative RT-PCR on mRNA extracted from purified monocytes that were activated through LILRA2 cross-linking caused down-regulation of TLR4 mRNA expression with significant changes observed at 12, 18 and 24 h but not at 6 h time point. B. Representative flow cytometry for the expression of TLR4 protein on the surface of monocytes activated via LILRA2 cross-linking for 6–48 hr. Dotted histograms on the left of each plot are cells stained with isotype matched negative control mAb. C. Summary of the mean fluorescence intensity (MFI) showing decreased expression of surface TLR4 on cells activated via LILRA2 cross-linking as compared to cells treated with IgG1 control with statistically significant difference seen at 12 h time point. Error bars represent SEM of 5 independent experiments, *p<0.05.
Mentions: To determine whether the inhibitory effect of LILRA2 cross-linking was due to down-regulation of LPS-recognition receptors mainly TLR4, mRNA level of these receptor on monocytes was assessed. LILRA2 cross-linking consistently down-regulated TLR4 mRNA expression by up to 3fold at 12, 18 and 24 hour time points when compared to those treated with control IgG1 (Fig. 5A). Similarly, TLR4 protein expression on the surface of LILRA2 cross-linked cells was down-regulated at 6, 12 and 18 h time points with the highest and statistically significant effect observed 12 h after LILRA2 cross-linking (Fig. 5B, C). Interestingly, prolonged LILRA2 cross-linking (24–48 h) does not seem to show significant difference in TLR4 expression to control cells (Fig. 5C).

Bottom Line: However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown.Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS.Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6.

View Article: PubMed Central - PubMed

Affiliation: Inflammation and Infection Research Centre, School of Medical Sciences, Department of Pathology, University of New South Wales, Australia. [corrected].

ABSTRACT
The activating immunoglobulin-like receptor, subfamily A, member 2 (LILRA2) is primarily expressed on the surface of cells of the innate immunity including monocytes, macrophages, neutrophils, basophils and eosinophils but not on lymphocytes and NK cells. LILRA2 cross-linking on monocytes induces pro-inflammatory cytokines while inhibiting dendritic cell differentiation and antigen presentation. A similar activating receptor, LILRA4, has been shown to modulate functions of TLR7/9 in dendritic cells. These suggest a selective immune regulatory role for LILRAs during innate immune responses. However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown. Moreover, the effects of LILRA2 on TLR4 and FcγRI-mediated monocyte functions are not elucidated. Here, we show activation of monocytes via LILRA2 cross-linking selectively increased GM-CSF production but failed to induce IL-12 and MCP-1 production that were strongly up-regulated by LPS, suggesting functions distinct from TLR4. Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS. Furthermore, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads and serum opsonized Escherichia coli but had limited effect on phagocytosis of non-opsonized bacteria. Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6. Interestingly, LILRA2 cross-linking on monocytes caused significant inhibition of TLR4 mRNA and protein, suggesting LILRA2-mediated suppression of LPS responses might be partly via regulation of this receptor. Taken together, we provide evidence that LILRA2-mediated activation of monocytes is significantly different to LPS and that LILRA2 selectively modulates LPS-mediated monocyte activation and FcγRI-dependent phagocytosis.

Show MeSH
Related in: MedlinePlus