Differential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages

Abstract

Background: Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue.

Methods: Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay.

Results: IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6.

Conclusion: AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.

Figure 1

Morphology and CD90 staining. MΦ visualization by Pappenheim staining (A) and electron microscopy (B). Images are representative for cell preparations from at least two different donors. C: Comparison of MΦ sizes by forward scatter as measured by flow cytometry. Light grey line: IM; filled/dark grey: AM. D: CD90 staining of AM and IM. Filled/dark grey: isotype control; light grey line: antibody staining. MFI values are given within graphs. Data show one representative out of three independent experiments with cells obtained from different donors.

Figure 2

CD14 and HLA-DR expression. AM and IM were stained and analyzed by flow cytometry. A: Data show one representative out of four independent experiments. Filled/dark grey: isotype control; light grey line: antibody staining. B: Comparison of AM and IM concerning CD14 and HLA-DR expression. Data are expressed as MFI related to AM values. Data show means ± SEM of four independent experiments with cells derived from four different donors. *P < 0.05 compared to AM values.

Figure 3

Expression of CD68, CD83 and CD1a. AM and IM as well as in vitro differentiated iDC and mDC were stained and analyzed by flow cytometry. Filled/dark grey: isotype control; light grey line: antibody staining. MFI values are given within graphs. Data show one representative out of three independent experiments with cells originating from different donors.

Figure 4

Phagocytic Activity. AM and IM were cultured with fluorescent FITC-labeled microspheres for 4 h at 37°C. As a control experiment, cells were pretreated with cytochalasin D (10 μg/ml, CytD) for 1 h. Alternatively, cells were preincubated at 4°C for 1 h and incubated with microspheres for 4 h at 4°C afterwards. Experiments were performed with cells derived from at least three different donors. A, C: representative results are shown. A: Fluoresphere-associated fluorescence (marked with black bars) was detected in AM and IM using flow cytometry. B: Average of percentage of MΦ positive for fluorosphere-associated fluorescence. Data represent mean ± SEM. *P < 0.05 as compared to cells left untreated at 37°C. C: Particle uptake in AM and IM was visualized by CLSM. F-actin was stained with rhodamin-phalloidine (red), nuclei with TOTO-3 iodide (blue). Latex beads are shown in green. Co: untreated cells.

Figure 5

Toll-like receptor expression. RNA was isolated from AM and IM and real-time RT PCR analysis for TLR1-10 was performed. Data were normalized to β-Actin values. Data show means ± SEM of independent experiments performed with cells from 3 to 4 different donors.

Figure 6

Activation of AM and IM by LPS. AM or IM were left untreated (Co) or treated with LPS (100 ng/ml) for 4 h, followed by RNA isolation and real-time PCR analysis for TNF-α (A), IL6 (B) or IL10 (C). Data are normalized to β-Actin values. D: Comparison of x-fold cytokine mRNA inductions. Data show means ± SEM of four independent experiments with cells derived from different donors. *P < 0.05.

Figure 7

Activation of AM and IM by TLR9 ligands. AM or LTM were left untreated (Co) or incubated with TLR9 ligands, followed by RNA isolation and real-time PCR analysis for TNF-α (A, B), IL10 (C, D) or IL6 (E, F). A, C, E: Cells were treated either with immunostimulatory sequences (ISS 1018 phosphorothioate-modified oligonucleotide, 20 μg/ml) or genomic DNA from M. bovis BCG (20 μg/ml) for 2 h. B, D, F: DNA isolated from virulent M. tuberculosis (H37Rv) or from the attenuated H37Ra strain (20 μg/ml) was added to AM or LTM for 2 h. Data show means ± SEM of four experiments with cells derived from two different donors. *P < 0.05.

Figure 8

LPS-induced cytokine secretion. AM or IM were left untreated (Co) or treated with LPS (100 ng/ml) for 6 h. Supernatants were removed and used for measurement of cytokine protein production. Data are normalized to total cellular protein values. Data show means ± SEM of 2-4 independent experiments performed in triplicate with cells derived from different donors. *P < 0.05.