Inflammation in the lung occurring in response to endotoxin lipopolysaccharide (LPS), viruses and foreign particles results in the generation of a variety of proinflammatory mediators from resident alveolar macrophages. Mediators released from activated macrophages include cytokines such as IL-1ß and IL-6 which are crucial for maintaining optimal immune function (Mehrad et al., 1999) and the arachidonic acid metabolite LTB₄ which is involved in host defence (Janniger et al., 1987). In leukocytes the cytosolic free Ca²⁺ plays a key role in determining cellular responsiveness (Li et al., 2002). The aim of this study was to evaluate the role of Ca²⁺ influx on mediator release from the rat alveolar macrophage cell line NR8383 using known cation channel blockers.

NR8383 cells (ATCC) were cultured in Ham's F12 medium containing 2mM L-glutamine, 15% heat inactivated fetal bovine serum, 100U/ml penicillin and 100mg/ml streptomycin. For cytokine generation 5x10⁵ cells (in 1ml) were seeded into each well of a 24 well Primaria plate and co-stimulated with LPS(100ng/ml) and thapsigargin (Tg, 50nM), for 24 hr (37ºC, 5% CO₂. For the generation of LTB₄, NR8383 cells (5x105 /ml) in HBSS buffer were stimulated with arachidonic acid (AA, 50µg/ml) in 1.5ml microcentrifuge tubes (10 min, room temperature). To evaluate the effect of inhibiting Ca²⁺ influx, cells were pre-treated (10 min, room temperature) with the ion channel blockers; LOE 908 (QuChem, Northern Ireland), econazole, SK&F 96365 or the Ca²⁺ chelator, EGTA (2mM). Cell supernatants were collected by centrifugation and assayed for LTB₄, rat IL-1ß and rat IL-6 by ELISA.

Statistical significance was assumed when *P<0.05 ANOVA, Kruskal-Wallis (Dunn's) test.

Table 1. The effect of ion channel blockers on the production of IL-1ß and IL-6 (LPS+Tg-stimulated) and LTB₄ (AA-stimulated) by NR8383 cells. Data represents the percent inhibition (%I) of vehicle control mediator release and is expressed as mean ± s.e.m. (n=3-5 experiments).

This study shows that IL-1ß, IL-6 and LTB₄ generation by the rat alveolar macrophage cell line NR8383 can be inhibited by cation channel blockers (Table 1). Although IL-1ß and IL-6 levels were measured in the same samples, IL-6 production appears to be more sensitive to inhibition by SK&F 96365, LOE 908 and econazole compared to IL-1ß (Table 1), suggesting that IL-6 generation is more Ca²⁺ influx dependent. Further evidence to support this idea is demonstrated by the lack of effect of 2mM EGTA on IL-1ß release (-14±9% inhibition, n=3) whereas IL-6 production was reduced by 93±1% (P<0.05, n=3). In addition, this concentration of EGTA inhibited LTB4 release by 79±6% (P<0.05, n=3).

These data suggest that IL-1ß, IL-6 and LTB₄ have different sensitivities to the ion channel blockers used in this study. Although these observations could be explained by the differences in the stimuli used for mediator generation and/or the signalling pathways involved, another possibility could be the involvement of different Ca²⁺ influx channels. Further studies are planned with these compounds to investigate the role of Ca²⁺ influx in the inflammatory responses of NR8383 cells.

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