Phenotypical and functional characterization of alveolar macrophage subpopulations in the lungs of NO2-exposed rats

Abstract

Background: Alveolar macrophages (AM) are known to play an important role in the regulation of inflammatory reactions in the lung, e.g. during the development of chronic lung diseases. Exposure of rats to NO2 has recently been shown to induce a shift in the activation type of AM that is characterized by reduced TNF-alpha and increased IL-10 production. So far it is unclear, whether a functional shift in the already present AM population or the occurrence of a new, phenotypically different AM population is responsible for these observations.

Methods: AM from rat and mice were analyzed by flow cytometry for surface marker expression and in vivo staining with PKH26 was applied to characterize newly recruited macrophages. Following magnetic bead separation, AM subpopulations were further analyzed for cytokine, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP) mRNA expression using quantitative RT-PCR. Following in vitro stimulation, cytokines were quantitated in the culture supernatants by ELISA.

Results: In untreated rats the majority of AM showed a low expression of the surface antigen ED7 (CD11b) and a high ED9 (CD172) expression (ED7-/ED9high). In contrast, NO2 exposure induced the occurrence of a subpopulation characterized by the marker combination ED7+/ED9low. Comparable changes were observed in mice and by in vivo labeling of resident AM using the dye PKH26 we could demonstrate that CD11b positive cells mainly comprise newly recruited AM. Subsequent functional analyses of separated AM subpopulations of the rat revealed that ED7+ cells showed an increased expression and production of the antiinflammatory cytokine IL-10 whereas TNF-alpha production was lower compared to ED7- AM. However, iNOS and IL-12 expression were also increased in the ED7+ subpopulation. In addition, these cells showed a significantly higher mRNA expression for the matrix metalloproteinases MMP-7, -8, -9, and -12.

Conclusion: NO2 exposure induces the infiltration of an AM subpopulation that, on the one hand may exert antiinflammatory functions by the production of high amounts of IL-10 but on the other hand may contribute to the pathology of NO2-induced lung damage by selective expression of certain matrix metalloproteinases.

Figure 1

Flow cytometric analysis of AM from NO₂-exposed and control rats. Rats were exposed to NO₂ for the indicated times and BAL cells were stained with antibodies to ED7, ED9, RM-4, and OX-6. To overcome autofluorescence signals, primary antibodies were detected using a biotin-PE/streptavidin-anti-streptavidin enhancing system and labeling of AM was analyzed by flow cytometry following gating by help of forward and sideward scatter properties. Shown are representative results of at least six animals per group.

Figure 2

Flow cytometric analysis of ED7 and ED9 expression of AM following magnetic bead separation. AM of 3 days NO₂-exposed rats were separated due to their expression of the cell surface molecule ED7 using magnetic bead separation. Susbsequently, ED7 (left) and ED9 (right) expression was analyzed in unseparated AM (A), ED7-positive AM (B), and ED7-negative AM (C). Numbers right of each histogram represent the mean fluorescence of the respective cell population. The figure clearly demonstrates that ED7-positive AM show a lower ED9 expression compared to ED7-negative AM. Shown is a representative data set of more than twenty animals.

Figure 3

FACS analysis of CD11b and PKH26 labeling of AM from NO₂-expsoed C57BL/6 mice. Mice were intravenously given PKH26 in combination with diluent C three days prior to onset of a seven days NO₂-exposure. Afterwards, AM were stained with F4/80-Alexa647 and CD11b-FITC. Isotype control (A), CD11b (B) and PKH26 (C) staining was subsequently analyzed by flow cytometry within the F4/80-positive cell population. (C) The proportion of PKH26-negative cells is shown in blue. Part (D) shows a separate analysis of CD11b-expression in PKH26-negative (blue histogram) and PKH26-positive AM (pink histogram) thereby clearly demonstrating that the CD11b-positive cell population mainly consists of PKH26-negative, newly recruited AM. Shown are representative results of eight animals per group.

Figure 4

Cytokine and iNOS mRNA expression in AM subpopulations of NO₂-exposed rats. ED7-positive and ED7-negative AM were separated from 3 days NO₂-exposed rats and total RNA was prepared immediately after cell separation. Cytokine (TNF-α, IL-10, IL-12 p40) and iNOS mRNA expression was analyzed by quantitative RT-PCR with L32 as house-keeping gene control in ED7-negative (blank bars) and ED7-positive AM (hatched bars). Data are presented as relative expression with mean expression in ED7-negative AM was 100 %. Shown are mean ± SD of six animals per group. Significance of differences was tested using the U-test according to Mann and Whitney and is indicated by * for p < 0.05 or ** for p < 0.01.

Figure 5

Differential cytokine production by LPS-stimulated AM subpopulations of NO₂-exposed rats. ED7-positive and ED7-negative AM were separated from 3 days NO₂-exposed rats and cultured in vitro for 24 hours in the presence of 100 μg LPS. Subsequently, TNF-α, IL-10, and IL-12 p70 were quantitated in the culture supernatants of ED7-negative (blank bars) and ED7-positive AM (hatched bars) by ELISA. Data are presented as mean ± SD of at least six animals per group. Significance of differences was tested using Students t-test and is indicated by ** for p < 0.01 or *** for p < 0.001.

Figure 6

mRNA expression for several MMPs in AM subpopulations of NO₂-exposed rats. ED7-positive and ED7-negative AM were separated from 3 days NO₂-exposed rats and total RNA was prepared immediately after cell separation. MMP-2, -7, -8, -9, and -12 mRNA expression was analyzed by quantitative RT-PCR with L32 as house-keeping gene control in ED7-negative (blankbars) and ED7-positive AM (hatched bars). Data are presented as relative expression with mean expression in ED7-negative AM was 100 %. Shown are mean ± SD of six animals per group. Significance of differences was tested using the U-test according to Mann and Whitney and is indicated by * for p < 0.05 or ** for p < 0.01.