The results of this study showed that human keratinocytes constitutively express TLR2 mRNA and protein. Treatment of keratinocytes with LTA and SLO stimulated TLR2 expression, especially after 24 hrs of treatment. IFN-γ and TNF-α both strongly induced TLR2 expression in keratinocytes, regardless of the concentration used. However, there were no significant changes in the expression of TLR2 associated with calcium concentrations.
Several prior studies reported that the expression of TLRs, including TLR2, in human keratinocytes was stimulated by yeast and bacteria. Most of these prior studies investigated the changes of TLR2 and/or TLR4 expression by the LPS of gram negative bacteria20-22
; however, some investigated the effects of Staphylococcus aureus
or, the LTA of Staphylococcus aureus23
. In this study we demonstrated that both SLO, a pathogenic toxin of Streptococcus pyogenes
, and LTA stimulated the expression of TLR2 in human keratinocytes. Streptococcus pyogenes
and Staphylococcus aureus
are pathogens known to cause cutaneous infectious diseases. In addition, both are the main cause of secondary infection in atopic dermatitis, and contribute to the aggravation of atopic dermatitis. Streptococcus pyogenes
plays an important role in the development of psoriasis and SLO has been associated with guttate psoriasis. Staphylococcus aureus
has been demonstrated in 20~50% of patients with psoriasis24,25
. The results of this study showed that TLR2 expression was upregulated in keratinocytes as the concentration of LTA and SLO increased, although the increase was not directly proportional. These effects were more significant after 24 hrs of stimulation, which is consistent with the results of previous reports21-23
. The increase of TLR2 expression by LTA was previously reported to be statistically significant only at concentrations of 10 µg/ml23
; however, in the present study, a significant increase of TLR2 expression was observed starting at concentrations as low as 0.1 µg/ml.
TLRs, pathogen-recognition receptors, transduce signals leading to the activation of NF-κB that subsequently drive the immune reaction by the transcriptional induction of several genes coding for cytokines, chemokines, and adhesion molecules26-28
. Many investigators have focused mainly on the downstream pathway after activation of the TLRs, such as the induction of cytokines or chemokines; several molecules have been shown to affect the activation of TLRs16-18,29-31
. However, in the present study the goal was to identify the factors that regulate or influence the expression of TLR. In this study, it was confirmed that both IFN-γ and TNF-α stimulate TLR2 expression; the effects were strong enough to increase TLR2 at 6 hrs, a relatively short duration of treatment. The stimulation by the two cytokines appears to occur regardless of their concentration. Pivarcsi et al.21
investigated co-treatment with LPS+IFN-γ, and found that 10 ng/ml increased the expression of TLR2 and the stimulation increased as the duration of treatment increased.
Therefore, induction of TLR2 by bacterial pathogens such as LTA and SLO might require a certain time interval for stimulation and this might have some correlation to their concentration. In the case of IFN-γ and TNF-α, both the duration of treatment and the concentration appeared to have little importance.
In addition, we studied whether TLR2 expression was influenced by the calcium concentration. Prior to this study, we hypothesized that TLR2 activation would be influenced by the differentiation of epidermal keratinocytes, and expected that the expression of TLR2 would change with the calcium concentration. However, there was no significant effect of the calcium concentration on TLR2 observed in this study. It is well known that a culture medium with a high calcium concentration induces keratinocytes to differentiate and proliferate. In addition, it has been reported that keratinocytes maintain a proliferative basal cell phenotype when they are cultured in media with a low calcium concentration (0.03 mM), and they are induced to differentiate by increasing the calcium concentration to above 0.1 mM32
. A change of TLR2 expression was not detected at the low calcium concentration of 0.05 mM, in which, keratinocytes have a tendency to proliferate, or at the relatively high concentration of 0.2 mM, where keratinocytes tend to differentiate. Kawai et al.33
reported that TLR2 was expressed throughout the entire epidermis. By contrast, another study reported that TLR2 was highly expressed in proliferating basal keratinocytes19
. Although it is difficult to interpret the present results, the calcium concentration itself might have little effect on TLR2 expression.
Baker et al.19
demonstrated a difference in TLR2 expression in psoriatic epidermis compared to normal epidermis; TLR2 was more highly expressed in the upper epidermis of psoriatic skin, while, in normal skin, TLR2 was expressed throughout the epidermis, with higher staining in the basal keratinocytes. These findings might explain the different TLR2 expression patterns in psoriatic epidermis. This study confirmed that LTA and SLO induce TLR2 expression, and that proinflammatory cytokines such as IFN-γ and TNF-α stimulate the expression of TLR2. In the presence of psoriasis, epidermal differentiation is altered, and the proliferation and differentiation balance is distorted34
. In addition, IFN-γ and TNF-α are the main cytokines involved in the pathogenesis of psoriasis35
. These results suggest that TLR2 is upregulated in response to the presence of Gram positive bacteria in the keratin layer24,25
. In addition, TLR2 upregulation might not be secondary to altered keratinocyte differentiation but result from the effects of proinflammatory cytokines such as TNF-α and IFN-γ present in the psoriatic lesions.
In conclusion, TLR2 plays a crucial role in the induction of antimicrobial responses in specific immune cells; it was found to be expressed in human keratinocytes even without stimulation. The expression of TLR2 increased with the concentration and duration of treatment with bacterial pathogens, and the increase was amplified by several cytokines, from activated keratinocytes and other cells. Therefore, these results help us understand the expression of TLR2 in cutaneous infectious diseases as well as inflammatory or immune-mediated skin diseases such as atopic dermatitis and psoriasis.