Differential expression of Toll-like receptors on human alveolar macrophages and autologous peripheral monocytes

Abstract

Background: Toll-like receptors (TLRs) are critical components in the regulation of pulmonary immune responses and the recognition of respiratory pathogens such as Mycobacterium Tuberculosis (M.tb). Through examination of human alveolar macrophages this study attempts to better define the expression profiles of TLR2, TLR4 and TLR9 in the human lung compartment which are as yet still poorly defined.

Methods: Sixteen healthy subjects underwent venipuncture, and eleven subjects underwent additional bronchoalveolar lavage to obtain peripheral blood mononuclear and bronchoalveolar cells, respectively. Surface and intracellular expression of TLRs was assessed by fluorescence-activated cell sorting and qRT-PCR. Cells were stimulated with TLR-specific ligands and cytokine production assessed by ELISA and cytokine bead array.

Results: Surface expression of TLR2 was significantly lower on alveolar macrophages than on blood monocytes (1.2 +/- 0.4% vs. 57 +/- 11.1%, relative mean fluorescence intensity [rMFI]: 0.9 +/- 0.1 vs. 3.2 +/- 0.1, p < 0.05). The proportion of TLR4 and TLR9-expressing cells and the rMFIs of TLR4 were comparable between alveolar macrophages and monocytes. The surface expression of TLR9 however, was higher on alveolar macrophages than on monocytes (rMFI, 218.4 +/- 187.3 vs. 4.4 +/- 1.4, p < 0.05) while the intracellular expression of the receptor and the proportion of TLR9 positive cells were similar in both cell types. TLR2, TLR4 and TLR9 mRNA expression was lower in bronchoalveolar cells than in monocytes.Pam3Cys, LPS, and M.tb DNA upregulated TLR2, TLR4 and TLR9 mRNA in both, bronchoalveolar cells and monocytes. Corresponding with the reduced surface and mRNA expression of TLR2, Pam3Cys induced lower production of TNF-alpha, IL-1beta and IL-6 in bronchoalveolar cells than in monocytes. Despite comparable expression of TLR4 on both cell types, LPS induced higher levels of IL-10 in monocytes than in alveolar macrophages. M.tb DNA, the ligand for TLR9, induced similar levels of cytokines in both cell types.

Conclusion: The TLR expression profile of autologous human alveolar macrophages and monocytes is not identical, therefore perhaps contributing to compartmentalized immune responses in the lungs and systemically. These dissimilarities may have important implications for the design and efficacy evaluation of vaccines with TLR-stimulating adjuvants that target the respiratory tract.

Figure 1

Differential constitutive surface expression of TLR2, TLR4 and TLR9 on human alveolar macrophages and monocytes. Alveolar macrophages and monocytes from healthy donors were analyzed by flow cytometry using phycoerythrin (PE)-coupled mouse anti-TLR2, TLR4 and TLR9 antibodies and their corresponding isotype controls (gray thin lines). Histograms are representative of eight independent experiments.

Figure 2

Modulation of TLR2, TLR4, and TLR9 expression during macrophage maturation. Monocyte-derived macrophages (MDM) were obtained from monocytes during a 7-day culture period in plastic dishes. Surface TLR expression was assessed by flow cytometry on freshly isolated monocytes (D0) and on cultured monocytes after 1 day (D1), 4 days (D4) and 7 days (D7) of differentiation. Histograms are representative of five independent experiments.

Figure 3

Bronchoalveolar cell mRNA expression of TLR2, TLR4 and TLR9 is lower than that of monocytes. TLR gene expression in unstimulated cells was assessed by qRT-PCR and relative quantification determined using the ΔΔCT method. Gene expression was normalized to 18 S rRNA. TLR expression of bronchoalveolar cells (BAC, panel A) and monocyte-derived macrophages (MDM, panel B) is reported relative to monocytes (MN). TLR expression on monocytes was set at 1. Depicted are mean ± SE of five individuals.

Figure 4

TLR expression upon ligand recognition. Alveolar macrophages and monocytes were cultured for 30 minutes in presence of 1 ng/mL Pam3Cys (TLR2, panel A). Alveolar macrophages and monocytes were cultured for 10 minutes in presence of 100 ng/mL LPS (TLR4, panel B). TLR expression after ligand stimulation was determined by flow cytometry. Histograms are representative of six independent experiments.

Figure 5

Regulation of TLR mRNA expression after ligand exposure. Bronchoalveolar cells (BAC) and monocytes (MN) were incubated in presence of 1 ng/ml of Pam3Cys and 100 ng/ml of LPS during 1 h or 24 h. Total RNA from cell lysates was reverse transcribed and qRT-PCR performed to quantify mRNA expression. Ligand-induced TLR2, TLR4 and TLR9 expression is reported relative to that of the unstimulated autologous cells. Mean ± SE of five independent experiments are depicted.

Figure 6

TLR-ligands induce differential cytokine production. Bronchoalveolar cells (BAC) and monocytes (MN) were stimulated with 5 mg/mL DNA from M.tb H37 Rv, 100 ng/mL LPS and 1 ng/mL Pam3Cys for 20 hours. Cytokines were determined in culture supernatants by ELISA for TNF-α and IL-6 (A, C) and Cytokine Bead Array for IL-1β IL-10 and IL-12 (B, D and E). Culture medium and human DNA (5 mg/mL) were used as negative stimulation controls. Mean cytokine pg/mL ± SE of seven independent experiments are depicted. Statistically significant differences (p < 0.05): (*) comparing stimulated vs. controls (medium) within respective cell groups, (++) comparing bronchoalveolar cells with monocytes.