Figure 1.
URT cells express ALDH1A mRNA.
RNA was extracted from cells and cDNA was synthesized using oligo-dT20 primers. Serial 1∶10 dilutions of the cDNA were used for PCR amplifications. Gels were loaded from left to right with PCR products from serially diluted cDNA. The left-most columns were representative of products from ∼1×103 cells. Panel A. Results are shown for NT cells (see Materials and Methods), cervical lymph nodes (CLN) and mesenteric lymph nodes (MesLN) of naïve C57BL/6 mice, separated into CD11cHi and CD11cLo/neg populations and tested for ALDH1A2 and GAPDH mRNA. Panel B. CD11cHi and CD11cLo/neg NT populations were tested for ALDH1A1, ALDH1A2, ALDH1A3 and GAPDH mRNA. Panel C. Cells were tested for ALDH1A2 mRNA. Samples included NT cells that had been FACS-sorted for the F4/80+CD11c-CD11b+ phenotype (abbreviated ‘F4/80+CD11c-’), two macrophage lines MAC INF4.29 and LIE 13–14, NT cells or lung cells enriched for epithelium by negative selection and short-term culture (see Materials and Methods), and LET cells. On a per-cell basis, the highest ALDH1A expression levels were among CD11cLo/neg cell populations.
Figure 2.
Respiratory tract epithelial cells produce ALDH1A2.
Tissue sections were prepared from the URT and lungs of C57BL/6 mice. Sections were stained with rabbit anti-mouse ALDH1A2 or rabbit anti-goat antibody (control). Bound rabbit anti-mouse ALDH1A2 antibody was detected using Alexa Fluor 568 donkey anti-rabbit IgG. The tissues were counter stained with nuclear stain DAPI. Sections of URT and lung are shown at low (10×) and high (40×) magnification. Staining indicated that the greatest ALDH1A2 protein expression was among epithelial cells lining the respiratory tract.
Figure 3.
URT cells enhance IgA production in splenocyte co-cultures.
Splenocytes were cultured with or without LPS (1 µg/ml) in the absence (A) or presence (B) of retinol (1 µM). Culture components are indicated below each bar. Where indicated, CD11cHi and/or CD11cLo/neg NT cells were added to cultures. IgA was measured (Y axis, ng/ml) from supernatants after a 7 day culture. Comparisons of IgA in cultures with splenocytes, LPS and retinol with or without CD11cLo/neg NT cells revealed a statistically significant difference (p = 0.03). Comparisons of IgA in cultures with splenocytes, CD11cLo/neg cells, and LPS with or without retinol also revealed a statistically significant difference (p = 0.04).
Figure 4.
Respiratory tract epithelial cell line enhances IgA production in splenocyte co-cultures.
IgA was tested after culture of splenocytes, LPS, and LET cells in various combinations in the absence (A) or presence (B) of retinol (1 µM). LPS-stimulated splenocytes were also tested in the absence or presence of LET cells, with or without depletion of CD11b+/Cd11c+ splenocytes (C). Panel D shows IgA expression in cultures of splenocytes or purified T and B cells in the presence of LPS, LET cells and retinol. Culture components are indicated below each bar.
Figure 5.
Cytokine production in splenocyte co-cultures.
Spleen cells were stimulated with LPS in the presence of LET cells and/or retinol. Culture components are indicated below each bar. Cytokines that were reproducibly elevated in the presence of LET cells and retinol are shown, including GM-CSF (Panel A), MCP-1 (Panel B), and IL-6 (Panel C). IFNγ levels were reduced in the presence of LET cells and retinol (Panel D). Panel E: IgA levels are shown following splenocyte/LET co-cultures in the presence of LPS and retinol, with and without neutralization using anti-IL-6 and/or anti-GMC-SF antibodies. ‘++’ indicates that antibodies were added to cultures at levels exceeding manufacturers’ recommendations, either 5× (for anti-IL-6 antibodies) or 2× (for anti-GM-CSF antibodies). Panel F: Levels of GM-CSF, MCP-1 and IL-6 are shown in LET cell cultures (without splenocytes) in the presence or absence of LPS.
Table 1.
Upregulation of cytokines/chemokines by stimulation of respiratory tract epithelial cells*.
Figure 6.
Working hypothesis for epithelial cell-mediated induction of IgA production in the respiratory tract.
The cartoon illustrates hypothesized mechanisms of vitamin A-dependent IgA production in the respiratory tract. RA = retinoic acid, Macrophage = MΦ.