Quantitative RTPCR analysis of cytokine gene expression in the spleen. Plotted open circles represent the mean ± SEM in each experimental group. The dashed line represents the fitted plot of cosinor analysis utilizing linear harmonic regression. Values are represented as the number of transcript copies/1000 GAPDH transcripts. Abscissa labels indicate ZT time values every 3 hrs under LD 12:12 conditions; ZT0 = lights on; ZT12 = lights off. Open bar on the bottom of the x-axis indicates the light period, while crosshatched indicates darkness. TNFά mRNA attains maximum levels at the dark-light transition (pANOVA < .001; pcosinor = .003), IL-1β (pANOVA < .001; pcosinor < .001), and IL-6 (pANOVA < .001; pcosinor < .001) mRNA each displayed a prominent peak shortly after lights on ~ZT3, while IL-18 (pANOVA < .001; pcosinor = .003) achieved maximum values prior to midnight (ZT15). IL-2 and IL-12β mRNAs did not exhibit a >2-fold rhythm in LD, although the mRNA levels for both fluctuated during the course of the day.