Table 1.
Rat primer sequences.
Fig 1.
Rat AMφ and NR8383 cells express mRNA for AM, IMD, CGRP, CRLR, and RAMP1-3.
A) Agarose gel electrophoresis of RT-qPCR products from cells obtained by bronchoalveolar lavage (BAL) and NR8383 cells. As a negative control, RT was omitted during the cDNA synthesis step (Ø RT). β-Actin served as positive control. Shown is a representative experiment out of 3. B) ΔCt values from RT-qPCR on resting NR8383 cells. Reference gene: β-actin; low values represent high expression. Bar represents median. N = 4.
Fig 2.
Messenger RNA content of peptides and their receptor complex is regulated by LPS.
NR8383 cells were treated with LPS (1 μg/ml) or left untreated. At indicated time points, gene expression of A) AM, B) IMD, C) CGRP, D) CRLR, E) RAMP1, F) RAMP2, and G) RAMP3 was analyzed by RT-qPCR. Data is presented in arbitrary units (AU) of mRNA expression with untreated cells set as 1. Bar represents median. N = 4 (N = 24 for untreated) *, p≤0.05 (Mann-Whitney test). P value for IMD at 72 h = 0.2 compared to untreated; p value for RAMP3 at 72 h = 0.057 compared to untreated.
Fig 3.
LPS enhances secretion of AM, IMD, and CGRP by NR8383 cells.
NR8383 cells were incubated in fresh culture medium with or without LPS (1 μg/ml). At indicated time points, the concentration of A) AM, B) IMD, and C) CGRP in the media was measured using colorimetric competitive ELISA. Data is presented as mean±SEM (N = 5). **, p≤0.01, compared to untreated; §§, p≤0.01, compared to untreated at 1 h; ¶¶, p≤0.01, compared to LPS at 1 h (Mann-Whitney test). Raw data are available as S1 File.
Fig 4.
AM, IMD, and CGRP do not have direct effect on [Ca2+]i, but AM inhibits SOCE.
Ratiometric measurements of [Ca2+]i. A) N = 190 (HEPES buffer), 106 (AM), 124 (IMD), 118 (CGRP), and 98 cells (acetonitrile, vehicle control). B) To analyse SOCE, cells were kept in Ca2+-free HEPES buffer, then thapsigargin (TG) (100 μM) was applied, followed by peptides. N = 153 (HEPES buffer), 84 (AM), 42 (IMD), and 46 cells (CGRP). Data is presented as ratio of fluorescence after excitation at 340/380 nm normalized to the 1st s. ***, p≤0.001 (AM versus HEPES buffer; Mann-Whitney test). Raw data are available as S2 File.
Fig 5.
AM, IMD, and CGRP inhibit phagocytosis.
A) NR8383 cells, treated with AM (1 μM), IMD (1 μM), CGRP (1 μM), PGE2 (1 μM), or ATP (100 μM) were exposed to opsonized Texas Red-conjugated zymosan beads for 30 min. Nuclei were stained with DAPI (blue), cell membranes were stained with FITC-labeled IB4 (green). ATP is a control of positive regulation; PGE2 is a control of negative regulation. Shown are representative pictures from 6 experiments. Bar, 50 μm. B) Phagocytosis activity is presented as phagocytosis index, normalized to untreated. Bar represents median. N = 5 experiments (N = 4 for AM and IMD). *, p≤0.05, **, p≤0.01 (Mann-Whitney test).
Fig 6.
AM, IMD, and CGRP attenuate production of TNF-α.
A-C) NR8383 cells were treated with LPS (100 ng/ml) with or without AM, IMD, or CGRP at indicated concentrations for 24 h. TNF-α mRNA expression was analyzed by RT-qPCR. Data is presented as relative mRNA expression, normalized to LPS-treated cells. Bar represents median. N = 4. *, p≤0.05 (Mann-Whitney test). D) NR8383 cells were treated with LPS (100 ng/ml) with or without AM, IMD, or CGRP (each at 100 nM) for 24 h and TNF-α levels in the culture media were measured by ELISA. Bar represents median. N = 5. *, p≤0.05; **, p≤0.01 (Mann-Whitney test).
Fig 7.
Effects of AM, IMD, and CGRP on TNF-α mRNA are receptor-mediated.
NR8383 cells were treated for 24 h with LPS (100 ng/ml) with or without A) AM (100 nM) and indicated concentrations of AM(20–50); B) IMD (100 nM) and indicated concentrations of CGRP(8–37); C) CGRP (100 nM) and indicated concentrations of CGRP(8–37); D) IMD (100 nM) and indicated concentrations of AM(20–50). TNF-α mRNA expression was analyzed by RT-qPCR. Data is presented as relative mRNA expression, normalized to LPS-treated cells. Bar represents median. N = 5 (N = 4 for IMD). *, p≤0.05; **, p≤0.01; #, p≤0.05 (Mann-Whitney test).
Fig 8.
CGRP decreases LPS-induced IL-1β mRNA levels.
NR8383 cells were treated for 24 h with LPS (100 ng/ml) with or without AM, IMD, or CGRP (each at 100 nM). A), C), E) Messenger RNA expression of pro-IL-1β, IL-6, and IL-10 was measured by RT-qPCR. Data is presented as relative mRNA expression, normalized to LPS-treated cells. Bar represents median. N = 4 (N = 12 for untreated). *, p≤0.05; ***, p≤0.001 (Mann-Whitney test). B), D), F) Levels of IL-1β (N = 5), IL-6 (N = 4), and IL-10 (N = 5) in the culture media were measured by ELISA. Bar represents median. *, p≤0.05; **, p≤0.01 (Mann-Whitney test).