Comments for:

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Re: PAFR-binding of LTA does not exclude TLR2-signaling in human keratinocytes

Submitter: Jeffrey B. Travers | jtravers@iupui.edu

Indiana Unviersity Department of Dermatology

Published November 23, 2005

It was with great interest that I read the letter by Drs. Mempel and colleagues regarding our recent mansucript. They expressed several concerns, including that we used KB cells instead of human keratinocytes, and that there was not a possible mechanism by which the PAF-R could induce epidermal IL-10 production.
First, KB cells were used as they do not have functional PAF-Rs--we have created a model system with PAF-R-negative and -positive cells by use of a retroviral-mediated gene transfer of the PAF-R. This model is appropriate for defining whether an agent such as LTA has PAF-R agonistic effects. That intradermal injection of a PAF-R agonist or LTA can induce IL-10 immunoreactivity in wild-type but not PAF-R-negative mice in vivo provides reasonable evidence indicating that the PAF-R is linked to IL-10 production. How the epidermal PAF-R can induce IL-10 production has been previously reported by Waltersheid and colleagues [1], who demonstrated involvement of prostaglandins (EP receptors) as COX-2 antagonists blocked this in murine keratinocyte cell line.
The clinical significance of LTA exerting effects as a PAF-R agonist (or TLR2 agonist) are unclear at this time. However, that we find significant levels of LTA on the skin of infected atopic dermatitis lesions suggests that LTA could be an important mediator of the worsening of atopic dematitis associated with staphylococcal skin infections.
1. Walterscheid, J.P., Ullrich, S.E., and Nghiem, D.X. 2002. Platelet-activating factor, a molecular sensor for cellular damage, activates systemic immune suppression. J. Exp. Med. 195:171-179.

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PAFR-binding of LTA does not exclude TLR2-signaling in human keratinocytes

Submitter: Martin Mempel, MD | m.mempel@lrz.tum.de

Department of Dermatology and Allergy, Biederstein, TU München

Published November 15, 2005

We read with interest the paper by Zhang and colleagues on the anti- inflammatory capacity of lipoteichoic acid (LTA) on human keratinocytes being mediated through the platelet-activating factor receptor (PAFR) (1). However, we think that some of the presented conclusions need clarification. Unfortunately, the authors decided to use a keratinocyte (epidermoid) cell line in which the expression of toll-like-receptors (TLR) was not investigated. From comparative activation experiments with peptidoglycan (PGN) which is not able to induce Ca-flux in their system, they conclude that TLR2, the crucial receptor for staphylococcal components plays no role in the activation by LTA. But how conclusive is such an assumption if the authors did not screen for receptor expression and if the KB cell line obviously lacks expression of TLR2 (judged on the PGN results) ? Can their results been extended to primary human keratinocytes which have been undoubtedly found positive for TLR2 by several groups (2-4) ?
While it has been demonstrated convincingly that LTA binds to PAFR in bronchial epithelial cells and mediates proinflammatory signals to this cell type (5), we have recently shown that activation of human keratinocytes by Staphylococcus aureus mainly signals through the Toll- like receptor 2 (TLR2) (6). This activation leads to the translocation of NF-kappa B which is followed by the induction of proinflammatory NF-kappa- B- controlled genes such as IL8, iNOS and also COX-II. This process, however, does not require PAFR-signaling as the two potent inhibitors HAGPH and CV3988 are not able of inhibiting this activation in contrast to the blockade of TLR-2 which abrogates NF-kappa-B activation (6). The pro- inflammatory effect of S. aureus can be partially mimicked by purified LTA and LTA-activation of human keratinocytes strongly resembles the activation induced by S. aureus (6).
Unfortunately, this pro-inflammatory effect of LTA which is repeatedly described in the literature was not investigated in the study by Zhang et al., (1).
The finding that delayed-type hypersensitivity reactions in mice can be inhibited through LTA in a PAFR-and IL10-dependent way is of great interest for the understanding of staphylococcal infections, however, we wonder if this phenomenon also plays a role in human skin diseases. First, the authors did not extend their studies to IL10-production in human keratinocytes. Second, the vast majority of human epithelial cell types produce large amounts of pro-inflammatory cytokines when activated by LTA and only minute amounts of IL10, a finding which is also seen in primary keratinocytes in our experiments (6-9). Third, in human airway epithelial cells, the binding of LTA to both potential receptors (PAFR and TLR2) is followed by the translocation of NF kappa B (5). The promoter of IL10 in turn lacks NF kappa B binding sites (10,11) so that an activation through this pro-inflammatory pathway seems unlikely. We would have appreciated a more detailed analysis of the possibly involved signalling mechanisms leading to IL10 production.
The presence of staphylococci on the skin of atopic dermatitis patients is a well known problem of this disease. The question as to weather staphylococci by themselves are a triggering factor of or if the colonization is a consequence of skin inflammation is a matter of continuing debate (12). The presented hypothesis, however, that staphylococci use the keratinocyte PAFR to induce IL10 in chronic lesions is in our eyes very speculative. Not only that neither in acute (predominately IL4 and IL13) nor in chronic lesions (IL12, IFNg and IL5) a dominant IL10-production is found (13, 14), the authors also omit to mention that there has already been a large clinical trial using the PAF- antagonist RO-24-0238 which failed to show any clinical effect (15).
1, Zhang Q., Mousdicas N., Yi Q., Al-Hassani M., Billings S.D., Perkins S.M., Howard K.M., Ishii S., Shimizu T., Travers J.B. 2005. Staphylococcal lipoteichoic acid inhibits delayed-type hypersensitivity reactions via the platelet-activating factor receptor. J Clin Invest. 115:2855-2861.
2, Kollisch G., Kalali B.N., Voelcker V., Wallich R., Behrendt H., Ring J., Bauer S., Jakob T., Mempel M., Ollert M. 2005. Various members of the Toll-like receptor family contribute to the innate immune response of human epidermal keratinocytes. Immunology. 114:531-41.
3, Pivarcsi A., Bodai L., Rethi B., Kenderessy-Szabo A., Koreck A, Szell M., Beer Z., Bata-Csorgoo Z., Magocsi M., Rajnavolgyi E.,et al., 2003. Expression and function of Toll-like receptors 2 and 4 in human keratinocytes. Int Immunol. 15:721-30.
4, Kawai K., Shimura H., Minagawa M., Ito A., Tomiyama K., Ito M. 2002. Expression of functional Toll-like receptor 2 on human epidermal keratinocytes. J Dermatol Sci. 30:185-94.
5, Lemjabbar H., Basbaum C. 2002. Platelet-activating factor receptor and ADAM10 mediate responses to Staphylococcus aureus in epithelial cells. Nat Med. 8:41-6.
6, Mempel M., Voelcker V., Kollisch G., Plank C., Rad R., Gerhard M., Schnopp C., Fraunberger P., Walli A.K., Ring J., et al., 2003. Toll-like receptor expression in human keratinocytes: nuclear factor kappaB controlled gene activation by Staphylococcus aureus is toll-like receptor 2 but not toll-like receptor 4 or platelet activating factor receptor dependent. J Invest Dermatol. 121:1389-96.
7, Uehara A., Sugawara S., Takada H. 2002. Priming of human oral epithelial cells by interferon-gamma to secrete cytokines in response to lipopolysaccharides, lipoteichoic acids and peptidoglycans. J Med Microbiol. 51:626-34.
8, Uehara A., Sugawara S., Tamai R., Takada H. 2001. Contrasting responses of human gingival and colonic epithelial cells to lipopolysaccharides, lipoteichoic acids and peptidoglycans in the presence of soluble CD14. Med Microbiol Immunol . 189:185-92.
9, Sugiyama A, Arakaki R, Ohnishi T, Arakaki N, Daikuhara Y, Takada H. 1996. Lipoteichoic acid and interleukin 1 stimulate synergistically production of hepatocyte growth factor (scatter factor) in human gingival fibroblasts in culture. Infect Immun. 64:1426-31.
10, Grandjean-Laquerriere A., Gangloff S.C., Le Naour R., Trentesaux C., Hornebeck W., Guenounou M. 2002 Relative contribution of NF-kappaB and AP-1 in the modulation by curcumin and pyrrolidine dithiocarbamate of the UVB-induced cytokine expression by keratinocytes. Cytokine. 18:168-77.
11, Kube D., Platzer C., von Knethen A., Straub H., Bohlen H., Hafner M., Tesch H. 1995. Isolation of the human interleukin 10 promoter. Characterization of the promoter activity in Burkitt's lymphoma cell lines. Cytokine. 7:1-7.
12, Mempel, M. Staphylococcus aureus and atopic eczema. 2006. In Handbook of atopic eczema, 2nd edition. J. Ring, B. Przybilla, and T. Ruzicka, editors. Springer-Verlag, Berlin, Heidelberg, New York, Germany/ U.S.A. 406-408
13, Leung D.Y., Bieber T. 2003. Atopic dermatitis. Lancet. 361:151- 60.
14, Novak N., Bieber T., Leung D.Y. 2003. Immune mechanisms leading to atopic dermatitis. J Allergy Clin Immunol. 112:S128-39.
15, Abeck D., Andersson T., Grosshans E., Jablonska S., Kragballe K., Vahlquist A., Schmidt T., Dupuy P., Ring J. 1997. Topical application of a platelet-activating factor (PAF) antagonist in atopic dermatitis. Acta Derm Venereol. 77:449-51.