Author(s) : Arjen R. Companjen, Vicent H.J. van der Velden, André Vooys, Reno Debets, Robert Benner, Errol P. Prens, Department of Immunology, Erasmus University Rotterdam (EUR), PO Box 1738, 3000 DR Rotterdam, The Netherlands..
Summary : The cytokine network in the skin is a tightly regulated system in which IL-1 isoforms, as well as their receptors and antagonists have a central role. The recently discovered IL-1 isoform IL-18 (also known as interferon gamma-inducing factor (IGIF) or IL-1g), promotes IFN-g expression by T cells in concert with IL-12. Because IFN-g plays an important role in many inflammatory skin diseases by facilitating the development of Th1 cells, it is important to elucidate the role of mediators which regulate the production of this cytokine. We demonstrate that human keratinocytes constitutively express IL-18 at the mRNA as well as at the protein level. The protein was mainly expressed intracellularly in the 24 kD unprocessed pro-form, but was also secreted. Histochemistry revealed a diffuse staining of IL-18 in the epidermis of normal skin, which is in line with our in vitro data. Furthermore, we show that the level of IL-18 expressed in freshly isolated normal human epidermal cells, whether or not containing HLA-DR+ cells, significantly exceeded the expression levels of other cell types such as monocytes and bronchial epithelial cells. Finally, our results show that stimulation of the keratinocyte cell line HaCaT with PMA LPS or IL-1b, does not significantly affect intracellular or released (pro) IL-18 levels. These experiments show for the first time that human keratinocytes relative to monocytes, PBMC or leukocytes produce a considerably larger amount of pro-IL-18, which is also readily released. High constitutive levels of IL-18 may contribute to the skewing towards a Th1-like environment, which is apparent in many human inflammatory skin diseases.
Figure 1 - IL-18 expression
in normal skin.
Acetone-fixed cryostat sections were stained with an IL-18 specific monoclonal
antibody (MAB318) as described in the materials and methods section. A:
IL-18 specific staining; B: IL-18 specific staining at a higher magnification
showing cells with dendritic morphology in the dermis and epidermis (arrow
heads) (magnification: x 630); C: isotype control antibody staining
(magnification x 400).
Figure 2 - Production
of IL-18 mRNA transcripts by different cell types.
Detection by RT-PCR. HPRT mRNA expression was included as a positive control.
PBMC: peripheral blood mononuclear cells; ECS: epidermal cell suspension;
HaCaT: human keratinocyte cell line; BEAS2B: bronchial epithelial cell line
and -: negative control (H2O).
Figure 3 - IL-18 protein expression
by HaCaT cells during culture and after stimulation by LPS, PMA or IL-1beta. A: total intracellular IL-18 expression; B: total IL-18 secretion.
IL-18 levels were measured by ELISA. Total intracellular levels represent
the IL-18 expression in 5 x 105 cells. Data of one representative
experiment out of three are shown. Readings were done in duplicate.
Figure 4 - Intracellular IL-18 expression in different
cell types.
IL-18 was detected on Western blot using a monoclonal antibody against
IL-18 (MAB318). Extracts from 5 x 104 cells were loaded in
each lane. Lane 1: processed recombinant human IL-18; lane 2: normal human
epidermal cells; lane 3: normal human epidermal cells depleted of HLA-DR+
cells; lane 4: HaCaT cells; lane 5: BEAS2B cells; lane 6: processed recombinant
human IL-18, omission of primary antibody and lane 7: normal human epidermal
cells, omission of primary antibody.
Pro-IL-18: 24 kD, processed IL-18: 18 kD.
Figure 5 - Intracellular expression of IL-18 in
total and HLA-DR+ cell-depleted normal epidermal cells compared
to leukocytes, PBMC and monocytes.
IL-18 was detected on Western blot using a monoclonal antibody
specific for IL-18 (MAB318). Extracts from 3 x 105 cells were
loaded in each lane.
Lane 1: processed recombinant human IL-18; lane 2: total
epidermal cell suspension; lane 3: epidermal cell suspension depleted
of HLA-DR+ cells; lane 4: monocytes; lane 5: PBMC; lane 6:
leukocytes; lane 7: processed recombinant human IL-18, isotype control
and lane 8: total epidermal cell suspension, isotype control.
Summary
