Carcinoembryonic antigen (CEA)-related cell adhesion molecules are co-expressed in the human lung and their expression can be modulated in bronchial epithelial cells by non-typable Haemophilus influenzae, Moraxella catarrhalis, TLR3, and type I and II interferons

Klaile, Esther; Klassert, Tilman E; Scheffrahn, Inka; Müller, Mario M; Heinrich, Annina; Heyl, Kerstin A; Dienemann, Hendrik; Grünewald, Christiane; Bals, Robert; Singer, Bernhard B; Slevogt, Hortense

In order to investigate the regulation of CEACAMs by cigarette smoke or in the presence of acute inflammation in human lung, NHBE cells were stimulated as described below and the expression of CEACAMs was analyzed using RT-PCR and qPCR. Untreated confluent NHBE cells expressed CEACAM5 and CEACAM6, as well as the CEACAM1 isoforms CEACAM1-4L, CEACAM1-4S, CEACAM1-3L and CEACAM1-3S. This was shown through RT-PCR analysis with pairs of primers specific for each CEACAM and isoforms ( Table 5 ). Identity of the obtained PCR-products for each CEACAM and isoforms was confirmed by sequencing. The intensity of the resulting bands after electrophoretic separation suggests that both CEACAM1 isoforms bearing the long cytoplasmic domain were expressed to a much lesser extent than the two short CEACAM1 isoforms. To address short-term effects of smoking on epithelial CEACAM expression, NHBE cells were exposed to CSE. To assess the tie of CEACAM expression in human lung cells to innate immune responses, NHBE cells were treated with IFNα, IFNβ, IFNγ, TNFα, and the TLR agonists MALP-2 (TLR2/6), poly I:C (TLR3) and flagellin (TLR5). Then, RNA from these cells was isolated and qPCR analysis was used to check for differences in CEACAM1-4L, CEACAM1-3L, CEACAM1-4S, CEACAM1-3S, CEACAM5 and CEACAM6 mRNA levels. To ensure an accurate normalization of their expression levels, we tested two candidate housekeeping genes for their stability across the unstimulated and stimulated samples. PPIB (SD (±Ct)=0.24; CV(%Ct)=1.24) as well as HPRT1 (SD(±Ct)= 0.34; CV(%Ct)=1.42) showed a highly stable expression, allowing for normalization with the geometric means of both genes. The four transmembrane CEACAM1-isoforms CEACAM1-4L, CEACAM1-3L, CEACAM1-4S and CEACAM1-3S were basically co-regulated by all reagents that elicited a difference in transcription levels (Table 5, Figure 3B ). IFNα significantly upregulated the expression of CEACAM1-4L (3.7-fold, p < 0.05), CEACAM1-4S (4.8-fold, p < 0.05) and CEACAM1-3S (4.3-fold, p < 0.001), whereas the 3.5-fold upregulation of CEACAM1-3L failed to reach significance (p = 0.124). IFNβ and IFNγ significantly increased the expression of all four splicing variants of CEACAM1 (p < 0.05 in all cases). In the case of IFNβ we could observe a slightly higher up-regulation of the two short isoforms CEACAM1-4S (5.2-fold) and CEACAM1-3S (4.7-fold) than the long isoforms CEACAM1-4L (4.4-fold) and CEACAM1-3L (3.0-fold). On the other side, IFNγ seemed to favor the two long isoforms CEACAM1-4L (6.3-fold) and CEACAM1-3L (5.2-fold) when compared to the short variants CEACAM1-4S (4.2-fold) and CEACAM1-3S (3.5-fold). However, no significant alteration in the ratios between long and short CEACAM1 isoforms was observed. Poly I:C treatment also upregulated all four isoforms of CEACAM1 in a significant manner (p < 0.001 in all cases) with expression ratios ranging between 4.9 and 7.0-fold ( Figure 3B ). CSE, TNFα, MALP-2 and flagellin had no effect on any of the four CEACAM1 transcripts despite of a significant increase of the IL-8 mRNA levels, which served as positive control of their efficacy (data not shown).