Neopterin-induced expression of intercellular adhesion molecule-1 (ICAM-1) in type II-like alveolar epithelial cells

Abstract

Production and release of proinflammatory mediators such as tumour necrosis factor-alpha and neopterin are common events following the activation of the cellular immune system. Concerning inflammatory disorders of the lung, e.g. sepsis or sarcoidosis, high serum neopterin levels have been reported to correlate well with the severity of the disease. These situations are often associated with an increased expression of ICAM-1 reported to be induced in type II alveolar epithelial cells. In our study we investigated the potential effects of neopterin on ICAM-1 synthesis in the type II-like pneumocyte cell line L2. Detection of ICAM-1 gene expression by reverse transcriptase-polymerase chain reaction revealed a dose-dependent effect of neopterin, with maximum impact following 12-h incubations. Comparable results were obtained when ICAM-1 protein synthesis was measured via a cell-based ELISA. In a second set of experiments we were able to show that coincubation of L2 cells with pyrrolidine dithiocarbamate (PDTC) significantly suppressed neopterin-induced ICAM-1 synthesis. Since PDTC is known to be a potent inhibitor of NF-kappaB, the stimulating effects of neopterin on ICAM-1 gene expression and protein generation may be mediated by activation of this transcription factor. From these data we conclude that neopterin stimulates ICAM-1 production in L2 cells. In vivo, these effects may contribute to the prolongation of the inflammatory response, including cytotoxic cell host defence mechanisms that impair the functions of the airway epithelium.

Fig. 1

Qualitative polymerase chain reaction (PCR) analysis of ICAM-1 expression detected as ICAM-1 cDNA (fragment size 515 base pairs) following 6 h, 12 h, and 24 h incubations of L2 cells with: 1 μm neopterin (N1), 10 μm neopterin (N10), 100 μm neopterin (N100), and 1000 μm neopterin (N1000), respectively. Lane C shows the result of a control experiment in unstimulated cells, lane M indicates a size standard (100 base pair ladder). Results are representative of five different experiments. To gain a better resolution the original Figure has been inverted.

Fig. 2

ELISA showing ICAM-1 protein expression after 18 h exposure of L2 cells to: 1 μm neopterin (N1), 10 μm neopterin (N10), 100 μm neopterin (N100), and 1000 μm neopterin (N1000) with (□) or without (▪) 25 μm pyrrolidine dithiocarbamate (PDTC). Control cells (C) were exposed to media alone. Five different experiments with duplicate determination of ICAM-1 protein were performed as described in Materials and Methods. Data are given as mean ± s.d. *P < 0.05 compared with controls; **P < 0.05 compared with the respective incubation without PDTC.

Fig. 3

(A) Western blot analysis of nuclear factor-κB (NF-κB) in nuclear extracts of L2 cells following 1 h incubation with: lane 1, control experiment; lane 2, 1 μm neopterin (N1); lane 3, 10 μm neopterin (N10); lane 4, 100 μm neopterin (N100); lane 5, 1000 μm neopterin (N1000). Right lane represents a prestained molecular weight marker (kD). Results are representative of three different experiments. (B) Western blot analysis of NF-κB in nuclear extracts of L2 cells following 1 h incubation with: lane 6, 25 μm pyrrolidine dithiocarbamate (PDTC) (P); lane 7, 25 μm PDTC + 1 μm neopterin (P + N1); lane 8, 25 μm PDTC + 10 μm neopterin (P + N10); lane 9, 25 μm PDTC + 100 μm neopterin (P + N100); lane 10, 25 μm PDTC + 1000 μm neopterin (P + N1000). Right lane represents a prestained molecular weight marker (kD). Results are representative of three different experiments.

Fig. 4

Qualitative polymerase chain reaction (PCR) analysis of ICAM-1 expression detected as ICAM-1 cDNA (fragment size 515 base pairs) following 6 h, 12 h, and 24 h coincubation experiments of L2 cells with 25 μm pyrrolidine dithiocarbamate either alone (P) or with the addition of: 1 μm neopterin (N1), 10 μm neopterin (N10), 100 μm neopterin (N100), and 1000 μm neopterin (N1000). Lane M, molecular mass marker. Results are representative of five different experiments. To gain a better resolution, the original Figure has been inverted.