Modulation of Cytokine and Nitric Oxide Production by Keratinocytes, Epithelial Cells, and Mononuclear Phagocytes in a Co-Culture Model of Inflammatory Acne

July 2012 | Volume 11 | Issue 7 | Original Article | 834 | Copyright © July 2012


Michelle K. Watson MD MS,a Yaping E MD,b Gina Dapul MD,a Wei-Li Lee PhD,b Alan R. Shalita MD,b and Maja Nowakowski PhDa

aCenter for Allergy and Asthma Research at SUNY Downstate Medical Center, Brooklyn, NY bDepartment of Dermatology, SUNY Downstate Medical Center, Brooklyn, NY

Abstract
Introduction: Ultraviolet B (UVB, 290 nm to 320 nm) has been reported to modulate the cytokine-mediated inflammatory process in various inflammatory skin conditions, including production of TNF-α, IL-1α, IL-6, IL-8, and IL-10. We constructed an in vitro model system involving co-culture of different cell types to study the effect of UVB on the inflammatory process using nitric oxide (NO) and tumor necrosis factor (TNF)-α as markers of inflammation.
Objective: This study was conducted to quantitatively assess the products secreted by human epithelial keratinocytes in the presence and absence of macrophages/monocytes.
Methods: Cells were exposed to UVB radiation (50 mJ to 200 mJ per cm2) or treated with bacterial lipopolysaccharide (LPS) as stimulator of inflammatory response. Nitric oxide (NO) was measured by modified Griess assay and TNF-α was measured by quantitative ELISA. For the co-culture system, SC monocytes were seeded in a 24-well Transwell tissue culture plate whereas irradiated keratinocytes were seeded in the individual baskets subsequently placed on top of the monocyte cultures, and samples of culture supernatants were collected at 1 to 6 days.
Results: When primary human epidermal keratinocytes (NHEK) were irradiated with UVB, a dose-dependent stimulation of TNF-α production was observed (33% to 200% increase). TNF-α production was not changed significantly in SC monocytes/NHEK co-culture. In contrast, when macrophages were irradiated with UVB, significant inhibition of NO production (40% suppression, P<0.001) was seen.
Conclusion: This improved model of cutaneous inflammation could use multiple cells to study their interactions and to offer convenience, reproducibility, and a closer approximation of in vivo conditions.

J Drugs Dermatol. 2012;11(7):834-836.

INTRODUCTION

Ultraviolet light is intricately linked to the functional status of the cutaneous immune system. In individuals who are susceptible, UV radiation can ignite pathogenic inflammatory pathways that contribute to allergic or autoimmune responses, including acne. Irradiation of the skin by UV light induces a variety of biologically active molecules, including proinflammatory cytokines IL-1α and TNF-α, which contribute to activation of inflammatory cells, the release of other cytokines, and the stimulation of other inflammatory mediators.1, 2 In addition, induction of cytokines IL-6, IL-8, and IL-10 has been reported.3, 4 This is mainly attributed to the effects of UVB (290 nm to 320 nm), which affects mainly epidermal cells and causes profound erythema and marked inflammation.2 It also induces direct DNA damage and promotes skin cancer development in chronically exposed individuals. 5 In contrast, UVA (320 nm to 400 nm) penetrates deeper and causes fibroblast apoptosis in the superficial dermis, connective tissue degradation with decreased collagen component, the accumulation of degenerative elastic fibers and production of extracellular degrading enzymes such as interstitial collagenase I.6
Earlier in vitro models that utilized a single cell line were a simplistic approximation of in vivo processes, where many cell types interact in the immune and inflammatory responses. An improved model of cutaneous inflammation would employ multiple cells to evaluate the interaction and to offer convenience, reproducibility, and a closer approximation of in vivo conditions.
We constructed an in vitro model system involving co-culture of different cell types to study the effect of UV radiation on the inflammatory process using nitric oxide (NO) and tumor necrosis factor (TNF-α) as markers of inflammation. This study was conducted to quantitatively assess cytokine and nitric oxide production by human epithelial cells and keratinocytes in the presence and absence of monocytes.

METHODS

Cell Culture
Normal human epidermal keratinocytes (NHEK), cultured macrophages (RAW 264.7, ATCC, Rockville, MD), epithelial cells (A549, ATCC), and human peripheral blood mononuclear cells (PBMC) were maintained in Eagle's minimal essential medium (MEM) supplemented with antibiotics, glutamine, and fetal bovine serum. RAW 264.7 cells are a macrophage-like, Abelson leukemia virus transformed cell line derived from BALB/c mice.
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