Up-regulation of the chemokine CCL21 in the skin of subjects exposed to irritants

Skin derived Dendritic cells (DC) such as Langerhans cells (LC) and interstitial DC play vital roles in immune surveillance because they have efficient uptake and antigen presentation mechanisms. Antigens are captured and processed, and few hours later DC reach peripheral lymph nodes (PLN) via afferent lymphatics [1].

The molecular mechanisms that regulate DC emigration are not fully understood. It seems to depend on the selective responsiveness of DC to special sets of signals. Cytokines such as IL-1β, TNF-α, IL-18 [2], CCL19 [3], and other molecules like Osteopontin [4], MMPs [5], prostaglandins [6, 7] leukotriene C4 [8] and, Lactoferrin [9], are proved to regulate DC migration by several mechanisms. One of the most important mechanisms is the up-regulation of CCR7 in the emigrating DC, which seems to be crucial for these cells to reach PLN through lymphatics [10]. Only some subsets of DC and lymphocytes express CCR7, allowing them to be attracted by the specific ligands CCL21 and CCL19 [11].

CCL21 has been reported as the major chemokine responsible for DC emigration from peripheral tissues [1, 12]. This chemokine is expressed in murine and human high endothelial venules (HEV) within secondary lymphoid organs. Saeki et al demonstrated that mice lymphatic endothelial cells (LEC) produce CCL21 [1], and more recently a similar observation has been reported in human normal skin [13]. Since it was discovered, CCL21 was classified as a constitutive chemokine. However our group and others have clearly demonstrated up-regulation of this chemokine by LEC and blood endothelial cells (BEC) in different inflammatory skin diseases mediated by specific immune response [13–15]. The selective expression of CCR7 on immune cells, and the constitutive and inflammatory CCL21 production would suggest two different roles for this chemokine.

We conducted this study in order to investigate this chemokine in human irritant contact dermatitis (ICD). We found a weak expression of CCL21 in dermal LEC from normal skin; however after contact with several irritant agents, CCL21 was up-regulated.

The kinetic study performed with 10% Sodium Lauryl Sulphate (SLS) showed at 10 hours a small number of inflammatory cells being mostly PC in non-perivascular areas when compared with perivascular areas (Table 2). The total number of infiltrating cells in irritated skin peaked at 48 hours and decreased significantly at 96 hours and 168 hours.

In order to investigate CCL21 expression we performed immunohistochemical studies with an amplifier system to improve the sensitivity. Weak CCL21 expression was found in all normal skin biopsies, but this expression increased to moderate or intense in skin biopsies taken at 24 hours after contact with any of the irritant agents and at 48 hours in the ACD patients (Figure 1 and Table 2). In normal and damaged skin, CCL21 was found in small sub-epidermal lymphatic vessels but in ICD and ACD lesions it was also seen in lymphatic vessels next to blood vessels surrounded by inflammatory cells (data not shown). These findings were obtained in all biopsies independently of the irritant agent applied.

When we investigated levels of CCL21 expression in the skin biopsies obtained at different times from one ICD patient we observed moderate levels of this chemokine at 10 hours, which further increased at 48 hours and declined thereafter (Figure 2).

In some individuals with ICD we perform immunohistochemistry in serial sections of several biopsies using anti CCL21 and anti Prox-1 (specific marker for lymphatic vessels). Vessels expressing CCL21 were also positive for Prox-1, however we could not get good quality pictures with this antibody (data not shown).

Positive correlation was found when we compared the macroscopic response with CCL21 expression (r = 0.69; p = 0.0008; CI = 0.34–0.87) but there was no correlation between the number of infiltrating cells and CCL21 expression in all the patients studied (r = 0.29; p = 0.2155; CI = -0.19–0.66).

Irritant contact dermatitis (ICD) is a multifactor disease, which involves a combination of endogenous and exogenous factors. The exact mechanisms involved in ICD responses are not fully understood. ICD can only be distinguished from allergic contact dermatitis (ACD) by patch tests as histological examination of skin biopsies from both entities shows MC infiltration at 48 hours [16]. In this study we observed in ICD patients a predominance of PC in areas away from blood vessels and the epidermis at 24 hours after contact with several irritants, whereas in patients with ACD we previously found mostly MC in the dermis and epidermis as early as 10 h after challenge with the relevant antigen [17].

These differences are probably due to the nature of the immune responses in each entity; ICD is a non-immunological local reversible inflammatory reaction induced by contact with irritants [18] whereas ACD is a specific immune response mediated by Th1/Tc1 lymphocytes [19]. In ICD, PC are attracted to the inflammatory area at a very early stage followed by bystander lymphocytes. By contrast, specific lymphocytes attracted to the site of hapten contact in ACD and are responsible for the inflammatory response.

In 1997 Hedrick et al.[20] and Nagira et al. [21], identified a novel human CC chemokine mapped at chromosome 9p13. Using Northern blot analysis they found human CCL21 expression restricted to lymphoid organs, while murine CCL21 showed a broader tissue distribution with the highest mRNA levels found in spleen and lung. Later on, Gunn et al found CCL21 expression HEV from secondary lymphoid organs and lymphatics from several murine tissues [22].

CCL21 expression by HEV has been reported to be involved in CCR7⁺ naive lymphocytes recruitment in secondary lymphoid organs in human and mice [22–24]. Around the same time Saeki et al demonstrated that CCL21 production by LEC is crucial for the emigration of murine CCR7⁺ mature LC from the dermal compartment [1].

A couple of years ago it was demonstrated by in vitro experiments that human LEC constitutively secrete CCL21 at their basal side [25], and more recently Weninger et al. found expression of this chemokine by LEC from normal human skin using immunofluorescence staining [13]. Our previous studies and other investigations did not find CCL21 expression by LEC in normal human skin. Since these could be attributed to the low sensitivity of the assay used, in the present study, we run an immunohistochemical technique with a catalyzed amplification signal system to increase the level of detection of this chemokine. We reported here that all normal skin biopsies express low levels of CCL21 in LEC.

Recent reports have provided evidence that CCL21 plays a role during inflammatory processes. Up-regulation of this chemokine by human BEC was demonstrated by Christopherson et al using conventional immunohistochemistry on skin biopsies taken from patients with inflammatory skin diseases such as atopic dermatitis, lichen planus and graft-versus-host [26]. Weninger et al performing immunofluorescence staining found CCL21 expression by BEC from rheumatoid arthritis and ulcerative colitis biopsies, but not psoriasis where CCL21⁺ vessels were rare [13]. They suggested that this could be related to the significant number of CCR7⁺ T cells in biopsies from rheumatoid arthritis and ulcerative colitis patients, but not in psoriasis, supporting the idea that CCL21 was only recruiting naive T lymphocytes to rheumatoid arthritis and ulcerative colitis tissues. These data identify CCL21 expression by BEC as an important determinant for naive T cell migration to tertiary lymphoid tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases associated with naive T cell recruitment.

On the other hand, our group and others have demonstrated that up-regulation of CCL21 could also take place in dermal LEC from patients with other diseases. In this regard, Katou et al. have shown by RT-PCR and immunohistochemistry CCL21 expression only in LEC from chronically inflamed skin infected with Candida albicans. Moreover, the lymphocytes that infiltrate this inflamed tissue were mostly CCR7^- memory T cells [15].

Using in situ hybridization and immunohistochemistry, we have previously found low levels of CCL21 in lymphatic vessels at 2 hours, which further increased at 10 and 48 hours in positive patch tests from patients with ACD. Very low expression of this chemokine was observed at 48 hours in skin biopsies from the sites challenged with an irrelevant antigen [14]. In this study we showed that CCL21 up-regulation was not related with an increase of CCR7⁺ infiltrating lymphocytes but it was associated with the disappearance of Langerin+ cells from the epidermis. Since Langerin is a marker of immature LC [27], we speculate that under this inflammatory condition the increased production of CCL21 could facilitate the emigration of CCR7⁺ cells through lymphatic vessels. Based on these evidences we have proposed that CCL21 is probably much more than a constitutive chemokine [28].

In this paper we found that CCL21 is also up-regulated by LEC during ICD by all of the irritant agents used. This up-regulation was found as early as 10 hours and reached a maximum level at 48 hours after contact with irritant agents, and it correlated with the macroscopic response. It is worth mentioning that Cumberbatch et al [29] have shown that skin application of 10 % SLS in mice resulted in a significant elevation of DC numbers in PLN, which could also be mediated by up-regulation of this chemokine by dermal lymphatics in those animals.

In spite of the fact that cytokines and chemokines involvement in ICD and ACD responses has been broadly studied [30–32], the molecular mechanisms responsible for up-regulation of CCL21 remind to be elucidated. We are currently investigating this unanswered question.

Based upon these results, we concluded that two different types of stress signals affecting the skin, antigen specific or non-specific immune responses are capable of inducing CCL21 up-regulation by LEC. This may enhance the steady state emigration of CCR7⁺ LC.