CYTOKINES EXPRESSION AND LYE AND NO-LYE RELAXERS 113 with 25 ml phosphate-buffered saline (PBS) containing Ca++ and Mg++ and incubated at 37oC, 5% CO2, and 95% relative humidity for a period of 4, 24, or 48 h. Cytokines were extracted from the tissues into the media by shaking at 300 rpm for 15 min, and collected aliquots were kept frozen at −20°C until assayed. CYTOKINE ASSAY A panel of proinfl ammatory cytokines [PGE2, interleukin-1α (IL-1α)] and antiinfl am- matory cytokine [IL-1 receptor antagonist (IL-1ra)] in a 2-plex kit was purchased from Millipore Corporation (Billerica, MA). The ELISA kit for PGE2 was obtained from the R&D Systems (Minneapolis, MN). The concentration of mediators was determined by using a Luminex-based multiplex assay system (Luminex Corp, Austin, TX). All assays were performed by following manufacturer’s instructions. STATISTICS Each of two separate experiments using a skin model with different lot number was per- formed in triplicate, and all measurements were in duplicate. The student’s t-test was used to determine signifi cant difference where p ≤0.05 was considered signifi cant. Results were calculated as a difference between the normalized control (100%) and observed value at each time period and are given as mean ± SE. RESULTS LYE AND NO-LYE RELAXERS DIFFERENTIALLY EXPRESS PGE2 Prostaglandins are produced as by-products of arachidonic acid metabolism and are known to induce sensory discomfort in humans and animals (4,6). To explore the possible involvement of the cytokine in the differential sensory perception of discomfort between lye and no-lye relaxers, its expression was examined. As shown in Figure 2, both relaxers induce a statistically signifi cant higher level of PGE2 compared to the control at each Figure 1. Histology of EpiSkin™ showing epidermal structures. A cross section of reconstructed human epidermis stained with hematoxylin and eosin. The model consists of human-derived epidermal keratinocytes that have been grown on bovine collagen coated with collagens 1 and 5. Cultures are raised in air interface to form a multilayered structure of stratum corneum (SC), stratum granulosum (SG), stratum spinosum (SS), and basal layer (SB).

JOURNAL OF COSMETIC SCIENCE 114 time point. However, the increase is statistically higher for the lye relaxer at each time period after exposure when compared with no-lye (p 0.001). IL-1α INDUCTION LEVELS ARE INFLUENCED BY RELAXER TYPE As the main initiator of epidermal response to injury, IL-1α induction was determined for up to 48 h following application of lye and or no-lye relaxer on EpiSkin™. As with PGE2, the expression of the cytokine was increased versus control with both relaxer types at each time interval. However, in this case, it is the no-lye relaxer that elicits a higher response that is statistically different at the latter two time points (p 0.01) (Figure 3). Expression of the cytokine after exposure to lye relaxer was 368%, 256%, and 201% over 4, 24, and 48 h, respectively, over untreated control. Comparatively, no-lye induced ex- pression levels of 436%, 468%, and 338% under the same conditions and over the same period, respectively (Figure 3). EXPRESSION OF IL-1ra BY RELAXERS Excessive production of IL-1α due to injury causes signifi cant side effects (10–13) there- fore, the epidermis has developed an exquisite mechanism to counteract activities of the cytokine to maintain homeostasis and protect itself by the expression of IL-1ra (8). Figure 4A shows how the two types of relaxers affect induction of IL-1ra. No-lye relaxer was better able to cause the induction of IL-1ra than lye by over 140% in the early phase of EpiSkin response, and at 24 h postapplication, this difference has signifi cantly increased to over fourfold. The ratio of IL-1ra to IL-1α expression has been used in other reported studies to determine the control of an infl ammatory response (8). We therefore examined if the ratio of IL-1ra to IL-1α, as a result of exposure to the two relaxers correlated with differences in perceived level of discomfort. As Figure 4B shows, both relaxers have similar Figure 2. Hair relaxers differentially induce expression of PGE2. Lye or no-lye relaxer was topically applied on EpiSkin™ model for exactly 15 min and washed thoroughly with PBS and incubated in fresh media as described in the section Materials and Methods. PGE2 was extracted, and concentration was determined by ELISA. Each data point represents the mean of six tissues run in duplicate ± SE showing signifi cant differen- tial expression of the cytokine (*p 0.05).