Effects of UVB on the Synthesis of Complement Proteins by Keratinocytes

Abstract

UVB exposure of the skin results in increased production of several cytokines by keratinocytes and infiltration of inflammatory cells. We hypothesized that UVB may increase the expression of complement (C) components and C-regulatory proteins by keratinocytes. In vivo, UVB may upregulate these proteins by direct effects or via cytokines released by keratinocytes or infiltrating inflammatory cells. In vitro, UVB may upregulate these proteins only directly, because of dilution of released cytokines in the medium. To test this, we exposed cultured human keratinocytes to UVB (0-64 J per m2) and monitored C3 and Factor B release in the medium by enzyme-linked immunosorbent assay, and surface expression of decay accelerating factor, membrane cofactor protein, and CD59 by flow cytometry. Keratinocytes produced small amounts of C3 and Factor B, which remained unaffected by UVB. UVB (32 J per m2) caused a transient upregulation of all three C-regulatory proteins. Decay accelerating factor expression was maximal at 48 h (1.81 +/- 0.06-fold increase in mean fluorescence intensity over nonexposed cells), membrane cofactor protein at 72 h (2.13 +/- 0.09-fold increase in mean fluorescence intensity), and CD59 at 120 h (1.96 +/- 0.09-fold increase in mean fluorescence intensity), returning to baseline values within 96, 192, and 192 h, respectively. Exposure to 64 J per m2 resulted in significant cell death; cells surviving this dose up to 48 h expressed a higher level of all the three proteins than those surviving 32 J per m2. In conclusion, UVB upregulated membrane cofactor protein, decay accelerating factor, and CD59 on keratinocytes without affecting the constitutive release of C3 and Factor B. Thus, UVB can increase the resistance of keratinocytes against their own C known to be produced excessively in response to cytokines of inflammatory cells that infiltrate the skin following UVB exposure.

Synthesis of Complement Components C3 and Factor B in Human Keratinocytes is Differentially Regulated by Cytokines

“…In our laboratory, the concentration of TNF-a in culture medium of stimulated keratinocytes was found to be very low (< 1 U per ml) (Pasch et al, 1998) whereas that required for upregulation of C3 in our system in this study was much higher (Fig 2). Similarly, the enhancement of factor B synthesis in response to IL-1a and IL-6 appears not to have been caused by any other cytokine released in autocrine manner from keratinocytes.…”

“…ELISA for measurement of C3 and factor B For quantitation of C3, a previously described sandwich ELISA was used (Pasch et al, 1998). Brie¯y, wells of 96 well ¯at-bottom microtiter plates were coated with 0.7 mg polyclonal goat IgG anti-human C3 (Cappel, Boxtel, The Netherlands) per ml in 100 ml carbonate buffer (pH 9.6) overnight at 4°C.…”

“…Factor B was assayed by a previously described sandwich ELISA (Pasch et al, 1998). Brie¯y, wells were coated overnight at 4°C with 3 mg polyclonal goat-anti-human factor B IgG (ATAB, Stillwater, MN) per ml in carbonate buffer.…”

Ultraviolet B radiation exerts enhancing effects on the production of a complement component, C3, by interferon-γ-stimulated cultured human epidermal keratinocytes, in contrast to photochemotherapy and ultraviolet A radiation that show suppressive effects

“…3a): UVB enhanced the C3 production at doses from 10 to 50 mJ cm −2 , although UVB treatment without IFN‐γ stimulation did not affect C3 production, as reported by Pasch et al . 17 The enhancing effects were statistically significant ( P < 0·05 at 25 mJ cm −2 ; P < 0·001 at 50 mJ cm −2 ). On the other hand, UVB at higher doses of 75 and 100 mJ cm −2 reduced production.…”

Glycosylphosphatidylinositol-anchored Proteins Regulate Transforming Growth Factor-β Signaling in Human Keratinocytes

“…CD59, a 21-kDa GPI-anchored protein expressed on keratinocytes, was used as a marker for the cloning of GPI anchor-deficient cells. CD59 is an inhibitor of the membrane attack complex (25,26). After EMS treatment, cells were stained with fluorescein isothiocyanate-conjugated anti-CD59 antibody and negatively sorted by FACS analysis.…”