LRP deficiency in MΦ's results in normal IFN-γ response but decreased IL-4.

<p><b>(A)</b> Mice were challenged with 4 µg αGC/mouse and serum assayed for IFN-γ and IL-4 by ELISA. Data points show standard error and mean. WT (n = 4 and 6 mice for 12 and 24 hour, respectively) and LRP-cKO (n = 5 and 4 for 12 and 24 hours, respectively), each point represents an individual mouse from a representative experiment of three separate experiments <b>(B)</b> WT (n = 7) and LRP-cKO mice (n = 8) challenged with 4 µg αGC/mouse or vehicle control. Intracellular cytokine stain was performed for IL-4 and antibodies for TCRβ, B220, and CD1d-αGC-tetramer to select iNKT cells. iNKT cells were defined as TCRβ^intermediate, B220^-, CD1d-αGC-tetramer⁺. Each point represents an individual mouse from a representative experiment of three separate experiments. <b>(C)</b> Chimeric mice were generated by transplanting Thy1.1 and LRP-cKO (Thy1.2) at a 1∶1 ratio into LysMCre^-/- LRP^flox/flox (Thy1.2) recipient mice. Isolation of splenic iNKT cells and intracellular cytokine stain of IL-4 was performed 4 weeks after transplantation. Dot plots on the left show percentage of Thy1.1⁺ or Thy1.2⁺ iNKT cells expressing IL-4. Graph on the right shows quantification of IL-4⁺ iNKT cells. Data presented from this experiment is the compilation of two separate experiments where each data point represents a single mouse. Bars show standard error and mean. *** denotes <i>p</i><0.0001. <i>P</i>-value determined by Student's <i>t</i>-test.</p

LRP expression in CD169⁺ MΦs.

<p><b>(A)</b> Spleen and <b>(B)</b> liver mononuclear lymphocytes from 8-to-12 week old WT mice (n = 4) were stained with fluorochrome-conjugated antibodies for MΦs (F4/80) and CD169. Left panels show dot plots for F4/80 and CD169 and middle panels show representative histograms of LRP expression (blue line) versus isotype control (black line). Graphs show quantification of mean fluorescent intensity (MFI  = LRP antibody- isotype antibody). Bars represent mean and standard error. ^**denotes p<0.001. <i>P</i>-value determined by Student's <i>t</i>-test. Histograms are from one representative experiment of three separate experiments. Scatter plots show individual animals from one representative experiment of three.</p

Specific Deletion of LDL Receptor-Related Protein on Macrophages Has Skewed <i>In Vivo</i> Effects on Cytokine Production by Invariant Natural Killer T Cells

<div><p>Expression of molecules involved in lipid homeostasis such as the low density lipoprotein receptor (LDLr) on antigen presenting cells (APCs) has been shown to enhance invariant natural killer T (iNKT) cell function. However, the contribution to iNKT cell activation by other lipoprotein receptors with shared structural and ligand binding properties to the LDLr has not been described. In this study, we investigated whether a structurally related receptor to the LDLr, known as LDL receptor-related protein (LRP), plays a role in iNKT cell activation. We found that, unlike the LDLr which is highly expressed on all immune cells, the LRP was preferentially expressed at high levels on F4/80⁺ macrophages (MΦ). We also show that CD169⁺ MΦs, known to present antigen to iNKT cells, exhibited increased expression of LRP compared to CD169^- MΦs. To test the contribution of MΦ LRP to iNKT cell activation we used a mouse model of MΦ LRP conditional knockout (LRP-cKO). LRP-cKO MΦs pulsed with glycolipid alpha-galactosylceramide (αGC) elicited normal IL-2 secretion by iNKT hybridoma and <i>in vivo</i> challenge of LRP-cKO mice led to normal IFN-γ, but blunted IL-4 response in both serum and intracellular expression by iNKT cells. Flow cytometric analyses show similar levels of MHC class-I like molecule CD1d on LRP-cKO MΦs and normal glycolipid uptake. Survey of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages and no homeostatic disruption as evidenced by the absence of programmed death-1 and Ly-49 surface receptors. Mixed bone marrow chimeras showed that the inability iNKT cells to make IL-4 is cell extrinsic and can be rescued in the presence of wild type APCs. Collectively, these data demonstrate that, although MΦ LRP may not be necessary for IFN-γ responses, it can contribute to iNKT cell activation by enhancing early IL-4 secretion.</p></div

LRP-cKO mice exhibit knockout of LRP in MΦs.

<p><b>(A)</b> Spleen and liver mononuclear cells from 8-to-12 week old WT and LRP-cKO (n = 4) were stained with fluorochrome-conjugated antibodies for T cells (TCRβ), B cells (B220), DCs (CD11c) and MΦs (F4/80). <b>(B)</b> Graphs show quantification of mean fluorescent intensity (MFI  =  LRP antibody MFI- isotype antibody MFI) of LRP. Each symbol represents an individual mouse from one representative experiment of three separate experiments. Bars represent mean and standard error. *** denotes <i>p</i><0.0001 and ** denotes <i>p</i><0.001 when comparing WT MΦs to LRP-cKO MΦs, <i>p</i> value was determined by Student's <i>t</i> test.</p

LRP expression in T cells, B cells, DCs and MΦs.

<p><b>(A)</b> Representative histograms for LRP (blue line) versus isotype control (black line) staining in spleen (top row) liver (middle row) and lymph nodes (bottom row). Mononuclear lymphocytes from from 8-to-12 week old WT mice (n = 4) were stained with fluorochrome-conjugated antibodies for T cells (TCRβ), B cells (B220), DCs (CD11c) and MΦs (F4/80). <b>(B)</b> Graphs showing quantification of mean fluorescent intensity (MFI  = LRP Antibody- isotype antibody) of LRP on lineage-specific populations. Symbols represent individual mice. Bars represent mean and standard error. *** denotes <i>p</i><0.0001 compared to T cells, B cells and DCs. ^**denotes <i>p</i><0.001 compared to T cells and B cells. <i>P</i> value was determined by a one-way ANOVA with a Bonferroni post-test. Histograms are representative of three separate experiments with 4 mice each. Scatter plots show individual mice from one representative experiment of three separate experiments.</p

Induction of IgE in LRP-cKO mice is normal when compared to WT.

<p>Both WT (n = 8) and LRP-cKO (n = 6) mice were challenged with αGC (4 µg/mouse) and vehicle (WT, n = 3, LRP-cKO n = 2). Blood was collected at 0 and 6 days and total serum IgE levels measured by ELISA. Bars represent mean and standard error. These results are from one experiment.</p

LRP deletion in pMΦs does not affect IL-2 secretion by iNKT cell hybridomas.

<p><b>(A)</b> Left panel shows representative flow cytometry dotplots of WT and LRP-cKO macrophages along with histograms showing LRP expression in macrophages (CD11b⁺) B cells (B220⁺) and lineage negative cells from the peritoneal cavity. <b>(B)</b> Quantification of LRP expression in pMΦs from WT (n = 3) and LRP-cKO (n = 3) mice. Each data point represents an individual mouse and results are from one representative experiment of three separate experiments <b>(C)</b> WT (n = 3) and LRP-cKO (n = 3) pMΦs incubated with iNKT cell hybridomas for 48 hours. Each sample well was done in triplicates and IL-2 levels were determined by ELISA. Results are from one representative experiment of three separate experiments. Shown bars represent mean and standard error.</p