Chemical and physical stimuli trigger a cutaneous response by first inducing the main epidermal cells, keratinocytes, to produce specific mediators that are responsible for the initiation of skin inflammation. Activation modulates cell communication, namely leucocyte recruitment and blood-to-skin extravasation through the selective barrier of the vascular ECs (endothelial cells). In the present study, we describe an in vitro model which takes into account the various steps of human skin inflammation, from keratinocyte activation to the adhesion of leucocytes to dermal capillary ECs. Human adult keratinocytes were subjected to stress by exposure to UV irradiation or neuropeptides, then the conditioned culture medium was used to mimic the natural micro-environmental conditions for dermal ECs. A relevant in vitro model must include appropriate cells from the skin. This is shown in the present study by the selective reaction of dermal ECs compared with EC lines from distinct origins, in terms of leucocyte recruitment, sensitivity to stress and nature of the stress-induced secreted mediators. This simplified model is suitable for the screening of anti-inflammatory molecules whose activity requires the presence of various skin cells.

INTRODUCTION

The process by which the skin reacts to stress requires the local recruitment of cells and molecules that contribute to the recovery of its integrity. The main epidermal cells, the keratinocytes, when stimulated, initiate the inflammatory response, which is characterized by the production of mediators, such as IL-8 (interleukin 8) and other chemokines (eotaxin), or lectins, such as galectin 9 [19]. These, in turn, could act towards the ECs (endothelial cells) of the dermal vessels. It is known that ECs are highly selective in their reaction to the micro-environment and they reflect the organ they belong to, as well as its biological state. Therefore it is fundamental for any cellular model of inflammation or invasive process to be able to deal with the ECs from the proper origin. This was proven first by the development and study of EC lines [10], and this is why we used in this work the skin-derived microvascular ECs and the corresponding micro-environment that was mimicked by human keratinocyte-derived medium.

The model proposed in the present study allowed us to reproduce the various steps of the skin inflammation process. Indeed, activation by keratinocyte mediators, such as chemokines and eicosanoids, leads to leucocyte binding via selectins then integrins and other EC adhesion molecules, and their migration through the EC layer into the subendothelial matrix to reach the site of inflammation [1114]. At the molecular level, this process is regulated by spatio-temporarily expressed adhesion molecules [15], present on both circulating leucocytes and on ECs and in concert with chemokines. A multi-step model of leucocyte adhesion to vascular endothelium has been developed previously [14,16] and is broadly applicable in different tissues, although the details of the signals involved differ. Re-circulation begins with blood lymphocytes interacting transiently and reversibly with the vascular endothelium through villous-expressed adhesion receptors in a process called rolling. These receptors can be either selectins, which interact with carbohydrate epitopes of addressins, or, less commonly, members of the Ig superfamily, which bind leucocyte integrins [11]. Activating factors (often chemokines for lymphocytes) bind to specific G-protein-coupled receptors on the rolling lymphocytes [17] and trigger rapid activation of integrin receptors on the leucocyte surface. These activated integrins promote arrest and stabilize adhesion by binding their Ig family ligands on the endothelium.

In a general way, exposure to UVB irradiation induces acute skin inflammation, such as erythema (sunburn) and oedema, with concomitant modulation of adhesion molecules [1820]. The resulting inflammation, which includes the release of growth factors, pro-inflammatory cells and the induction of oxidative DNA damage, is known to play a role in the aging process and also in cancer development [18,2127].

Growing experimental evidences indicate that the nervous system can directly modulate the cutaneous inflammatory response by the release of neuropeptides, such as substance P or CGRP (calcitonin-gene-related peptide) [28,29]. The dermal ECs have been described as a target for substance P. After the direct application of substance P, dermal ECs express significant levels of both ICAM-1 (intercellular adhesion molecule 1) and VCAM (vascular cell adhesion molecule), which were accompanied by increased binding to leucocytes [30]. Consequently, the modulation of the expression of these various adhesion molecules could be a good target for anti-inflammatory strategies [3133] that must be directed only to the appropriate ECs, in our present study, the dermal microvasculature. In natural defence reactions, the contribution of ECs is enhanced by the endothelium–leucocyte-specific process of recognition [3436], which make them targets for cells and molecules (reviewed in [37]).

As mentioned above, because the cells of the vascular endothelia are organo-specific [10,37,38], they specifically control and mediate inflammation and the invasion process. Furthermore, the quiescent endothelium maintains a status quo, but undergoes a series of metabolic changes in inflammation, which is known as ECA (EC activation).

ECs respond to a variety of stimuli, including pro-inflammatory cytokines IL-1, TNF-α (tumour necrosis factor α), AGEs (advanced glycation end products), oxidized lipids and environmental conditions, such as chemical (oxidative) and physical (mainly shear) stress.

ECs that are exposed to cytokines, which mediate the inflammatory response, undergo quantitative changes in the synthesis of certain gene products (proteins) that, in turn, provide ECs with the capacity to perform new functions [38,39]. The five main changes associated with ECA are: a loss of vascular integrity, the production of EC adhesion molecules, the secretion of cytokines, pro-thrombotic changes and the increased production of lymphocyte adhesion molecule ligands. Those phenotypic changes, involved in ECA, amplify local inflammation. ECA is also critical in physiological situations such as the accumulated effects of micro-inflammatory states, which contribute to the aging process [4042], as exemplified in the skin [4046].

Thus the structural and functional integrity of the barrier formed by the endothelium is essential for the maintenance of blood-vessel wall homoeostasis.

The ECs select circulating cells for recruitment to the inflammatory site, and skin-infiltrating T-cells appear to be central in controlling the initiation and maintenance of skin inflammation [4749]. Moreover, the endothelial barrier displays distinct organ- and vessel-type characteristics, which helps to explain the biological sorting that they achieve, as shown in a previous work that established phenotype-stabilized EC lines from different tissue origins [10].

Existing models are limited, because they deal with one cell type response and/or with non-relevant origin cell cultures. These have brought interesting, but partial, information about keratinocyte-specific inflammation-induced factors and their implication on later events in this response pathway [5052]. Despite the highly informative data obtained by direct and general activation of adhesion molecules that has been described in most studies with umbilical cord-derived ECs [53], the endothelial reaction has been proven to be organo-selective and micro-environment-dependent, as shown by the skin recruitment of CLA+ (cutaneous lymphocyte antigen positive) T-lymphocytes [15,47]. This is why our organo-specificity-based model is potentially able to produce fundamental and biologically relevant new information about invasion mechanisms. Hence, knowledge of the molecular mechanisms by which leucocytes are recruited to the skin could provide promising targets for the development of new anti-inflammatory molecules that are able to modulate the process of keratinocyte-initiated inflammation.

Moreover, our model, which combines different representative skin cells and CLA+ T-lymphocytes, allows the study of their specific reactions and contributes to validate the EC organo-specificity hypothesis.

EXPERIMENTAL

Preparation of human keratinocytes and cell culture

HAKs (human adult keratinocytes) were obtained after the excision of skin during plastic surgery from non-related healthy volunteers. The study was carried out with ethical approval and the volunteers gave their informed consent to participate. Skin sample (0.5 mm thick), prepared with a dermatome, was floated on to a 0.05% solution of trypsin (Gibco, France) for 30 min at 37°C. The epidermis was separated from the remaining dermis and an epidermal cell suspension was obtained by pipetting and filtering through sterile gauze.

The resulting epidermal cells were plated at high density in 100 cm2 dishes at 37°C in a humidified incubator (5% CO2). Monolayer cultures of keratinocytes were established in the serum-free medium MCDB 153 [KBM (keratinocyte basic medium), BioWhittaker] supplemented with 0.5 μg·ml−1 hydrocortisone, 5 μg·ml−1 insulin, 10 ng·ml−1 EGF (epidermal growth factor) and 56 μg·ml−1 bovine pituitary extract [54].

Preparation and culture of immortalized human microvascular EC lines

Microvascular ECs were isolated from surgical specimens, as described by Bizouarne et al. [55,56] (for mouse ECs) and adapted for use with human EC lines, and were immortalized and stabilized, as described by Kieda et al. [10]. The cell lines used were: HSkMEC.1, human skin microvascular ECs (immortalized cells from normal skin); HPLNEC.B3, human peripheral lymph node ECs, clone B3 (from high endothelial venules of a cervical lymph node of a patient with Hodgkin's lymphoma); HBrMEC, human brain microvascular ECs (immortalized from a biopsy of normal brain tissue).

ECs were cultured in OptiMEM1 with Glutamax-I (Invitrogen) supplemented with 3% FBS (foetal bovine serum; Biochrom KG, Berlin, Germany), 40 mg/ml gentamycin (Panpharma, Fougères, France) and 0.5 mg/ml fungizone (GIBCO). ECs were passaged using 0.05% (w/v) trypsin/0.02% (w/v) EDTA solution (Biochrom AG, Berlin, Germany). The experiments were performed between passages 3 to 12.

Culture of human leucocytes

Human leukaemic cell lines were used in adhesion experiments. CEMT4, a leukaemic CD4+ T-cell line, was provided by Dr P. Olivier (Department of Virology, Institut Pasteur, Paris, France) [49].

Stress generation in HAK cultures using UVB irradiation, substance P and CGRP

Subconfluent cultures of HAKs were rinsed twice with PBS. For UVB irradiation, cells were irradiated in a film of PBS using a BioSun irradiator (Vilbert Lourmat, Marne-la-Vallée, France). They were exposed to 10 mJ·cm−2 (312 nm). This is a non-cytotoxic dose, as determined by counting the number of cells remaining. For stimulation, HAKs were incubated with 0.1 μM substance P or CGRP for 24 h, or with 100 μM H2O2 for 1 h, then washed and maintained in normal medium for 16 h. CGRP, substance P and H2O2 were obtained from Sigma. Control HAKs were not irradiated or incubated with stimulators.

Anti-inflammatory assessment

The conditioned medium from stressed keratinocytes was collected and added to cultures of ECs in order to activate them by mimicking the process of inflammation. This newly created micro-environment is used to reproduce physiologically attacked skin, in which the ECs respond by producing a range of adhesion molecules to recruit competent leucocytes. Stressed keratinocytes were incubated with the anti-inflammatory molecules: 0.1 μM α-MSH (α-melanin-stimulating hormone), 1 μM dexamethasone or 1 μM indomethacin for 48 h to modulate their response. The adhesion-inhibiting properties of the cells were then assessed by an in vitro assay (see below).

Quantitative assay of leucocyte (CEMT4 cell line) adhesion to ECs

Labelling leucocytes with PKH26-GL (Paul Karl Horan 26 general labelling)

CEMT4 cells were labelled with the red fluorochrome PKH26-GL according to the manufacturer's recommendations (Molecular Probes), as adapted by Kieda et al. [10]. Cells were incubated with the label for 2–3 min at 37°C with gentle mixing, washed with PBS, and suspended in PBS (106 cells/ml).

Adhesion of leucocytes to ECs

ECs in complete OptiMEM medium were plated out in 24-well tissue culture plates (Falcon, Becton Dickinson, France) (0.75×105 cells in 400 μl per well) 24 h before the assay was carried out. Immediately before the test, the cell monolayers were washed with PBS/BSA and maintained at 4°C. CEMT4 cells were layered over the EC monolayers at a ratio of 5:1, and the plates incubated for 20 min at 4°C. Non-adhering cells were removed by two gentle washes with PBS. ECs and adhering cells were detached from the tissue culture plate by a short incubation with PBS containing 0.02% (w/v) EDTA, washed in PBS (containing 0.5 mM CaCl2 and 1.0 mM MgCl2) and analysed by flow cytometry (FACSort, Becton Dickinson, Sunnyvale, CA, U.S.A.). Data were recorded for 5000 events, using CellQuest® software. The percentages of each cell type in the samples were calculated using the CellQuest® software (Becton Dickinson). Results are presented as the number of adherent cells per EC counted at the end of the adhesion step. Note that the final counts cannot be related to the initial 5:1 ratio, because of the differential reactivity of some ECs among the whole population and the activation subsequent to early recognition (results not shown). The presented data were analysed with the Student's t test.

Adhesion of leucocytes to keratinocytes

The direct adhesion of leucocytes to keratinocytes was assessed by the same technique as above.

Detection of mediators in conditioned medium from stressed keratinocytes

Aliquots of stressed keratinocyte-conditioned medium were stored at −70°C until used to assay the effects of IL-8, s-ICAM-1 (soluble ICAM-1) and IL-7 using ELISA (R&D, Abingdon, U.K.), according to the manufacturer's instructions.

RESULTS

Cell model of skin inflammation

The cellular interactions involved in skin inflammation can be estimated in vitro by subjecting keratinocytes to stress, incubating dermal microvascular ECs with the resulting conditioned medium, and then measuring the capacity and specificity of the adhesion properties of the ECs (Figure 1A).

Protocol for measuring leucocyte adhesion to ECs or keratinocytes

Figure 1
Protocol for measuring leucocyte adhesion to ECs or keratinocytes

(A) Successive culture treatments and cell conditioning. (B) Quantification of the adhesion process by flow cytometry. Lymphocytes are FL2-positive (upper left-hand quadrant) and ECs are non-fluorescent (lower left-hand quadrant).

Figure 1
Protocol for measuring leucocyte adhesion to ECs or keratinocytes

(A) Successive culture treatments and cell conditioning. (B) Quantification of the adhesion process by flow cytometry. Lymphocytes are FL2-positive (upper left-hand quadrant) and ECs are non-fluorescent (lower left-hand quadrant).

The results of the adhesion assays were analysed by flow cytometry using the forward- and side-scattered light differences between EC and lymphocytes. Furthermore, the lymphoid cells could be identified by their fluorescence due to PKH26-GL labelling producing a bright fluorescence (FL2). Figure 1(B) shows the increased number of lymphocytes (FL2-positive cells in the upper left-hand quadrant) able to adhere to ECs (non-fluorescent cells, lower left-hand quadrant) upon stimulation by a given stress. In the subsequent experiments, the results express the number of leucocytes adhering per EC, as calculated from the dot-plot analyses shown above, and expressed as histograms.

Inflammation induction assay: validation of the inflammation model

Keratinocytes exposed to UV light released mediators that activated ECs in the medium, in terms of increased adhesion capacity, as shown for a representative experiment performed in triplicate (Figure 2A). This response was modulated by anti-inflammatory molecules, such as α-MSH, which is known to protect the skin from UV irradiation [29,43,5759].

UV effects on leucocyte recruitment in the skin

Figure 2
UV effects on leucocyte recruitment in the skin

(A) Modulation of skin EC adhesion capacity by incubation with conditioned medium from UV-induced keratinocytes and medium from cells treated with α-MSH was measured by flow cytometry analysis of CEMT4 lymphocyte cell adhesion. (B) Modulation of keratinocyte adhesion capacity by UVB irradiation and blocking of the UVB action by α-MSH was measured by flow cytometry analysis of CEMT4 lymphocyte cell adhesion. CEMT4 cells labelled with PKH26-GL were incubated for 20 min at 37°C with ECs or a HAK monolayer, in a ratio of 5:1. The results are the numbers of CEMT4 per EC or HAK cell (means±S.D, n=4).

Figure 2
UV effects on leucocyte recruitment in the skin

(A) Modulation of skin EC adhesion capacity by incubation with conditioned medium from UV-induced keratinocytes and medium from cells treated with α-MSH was measured by flow cytometry analysis of CEMT4 lymphocyte cell adhesion. (B) Modulation of keratinocyte adhesion capacity by UVB irradiation and blocking of the UVB action by α-MSH was measured by flow cytometry analysis of CEMT4 lymphocyte cell adhesion. CEMT4 cells labelled with PKH26-GL were incubated for 20 min at 37°C with ECs or a HAK monolayer, in a ratio of 5:1. The results are the numbers of CEMT4 per EC or HAK cell (means±S.D, n=4).

Moreover, the keratinocytes responded to stress by also increasing their capacity to bind leucocytes (Figure 2B). In this type of experiment, α-MSH prevented the UV-induced increase of the adhesion capacity towards CEMT4 lymphocytes when added to the keratinocytes. This decrease due to α-MSH is almost complete for the effects towards ECs stimulated with UV-induced-keratinocyte-derived conditioned medium. This is quite significant, because α-MSH by itself did not affect the adhesion properties of either keratinocytes or ECs (results not shown). This is particularly important in terms of anti-UV-induced inflammation, since it was shown by Scholzen et al. [43] that the termination of the inflammatory process is due to dermal ECs proteolytic enzymes, such as ACE (angiotensin-converting enzyme).

This indicates that the model mimics the physiological situation in which inflamed tissues are infiltrated by leucocytes. Conversely, α-MSH reduced the response of both ECs and keratinocytes, confirming the validity of the proposed assay.

Organ-selectivity of ECs: distinct adhesion/activation patterns

The studies on the adhesion process were carried out to refine the in vitro model in the light of knowledge of EC selectivity. They have shown that it is a function of the EC type and tissue origin [10]; our results, for one representative experiment (Figure 3), indicate that it is also a function of the biological stimulus encountered by the ECs. Therefore, this confirms that the ECs are reacting differently according to the organ they belong to and reflect the biological and the micro-environmental state.

Selective binding of CEMT4 leucocytes to EC lines

Figure 3
Selective binding of CEMT4 leucocytes to EC lines

Modulation of the adhesion by keratinocyte-conditioned medium (treated with UVB, substance P or CGRP). Results are expressed as means±S.D., n=4.

Figure 3
Selective binding of CEMT4 leucocytes to EC lines

Modulation of the adhesion by keratinocyte-conditioned medium (treated with UVB, substance P or CGRP). Results are expressed as means±S.D., n=4.

Under resting conditions, the CEMT4 cells adhered preferentially to ECs derived from the skin and, particularly, from brain compared with those from the peripheral lymph nodes. However, on activation by keratinocyte-derived conditioned medium with substance P and, to a lesser extent, by UVB, CEMT4 cells adhered up to 3-fold more to skin-derived ECs as compared with a 2-fold increase for the brain-derived ECs. Although the basic adhesion value due to brain-derived ECs was higher than skin-derived ECs, activation enhanced the differential behaviour. Treatment of ECs with medium from keratinocytes subjected to UV irradiation increased the adhesion ability of both HSkMECs and HBrMECs to a lesser extent. The medium from keratinocytes activated with CGRP did not increase the adhesion capacity of the ECs.

The organo-specific reactivity of the ECs was highlighted by the results from this experiment, because lymph-node-derived ECs (HPLNEC.B3) were not sensitized by keratinocyte-conditioned medium when the stress was induced by UV irradiation or substance P. They responded to medium from CGRP-treated keratinocytes differently, with a very small increase in their adhesion properties.

Modulation of dermal microvascular EC activity under inflammatory conditions

Inflammatory conditions produced by stressed keratinocytes

We tested the validity of our model to reproduce the inflammatory cascade induced by UVB irradiation, substance P or CGRP, as described previously [25,29], and its modulation by potent anti-inflammatory molecules.

Exposure to UVB irradiation induces acute skin inflammation, such as erythema (sunburn) and oedema. Inflammation, which includes the release of growth factors and pro-inflammatory cells, and the induction of oxidative DNA damage, is known to play a role in the aging processes and cancer development [2527]. The neurologic system directly modulates the inflammatory cutaneous response by the release of neuropeptides, such as substance P or CGRP [28,29]. Consequently, keratinocytes were stressed by exposure to UVB irradiation or by incubation with substance P or CGRP.

ECs are proposed to be a target; by direct application of substance P, they express significant levels of both ICAM-1 and VCAM, which was accompanied by increased binding to leucocytes [30].

The capacity of the conditioned medium from these stressed keratinocytes to modulate the properties of dermal EC was tested by assessing leucocyte adhesion as a result of inflammatory cellular recruitment in an organ-restricted manner [10,15,37,47,49].

Among the main mediators which are known to be released by keratinocytes, such as IL-1, IL-6, IL-7, IL-8, IL-10, RANTES (regulated upon activation, normal T-cell expressed and secreted) and TNF-α [79,52], we focused on IL-8 [60], RANTES [61], IL-7 [38] and s-ICAM-1 [62] to investigate their direct involvement in EC biology.

The results concerning the behaviour of these mediators on keratinocyte stress induced by UVB and substance P are shown in Table 1. Under the same keratinocyte stress conditions, we observed the effect of mediator release by keratinocytes on ECA. Results expressing the recruitment capacity in terms of number of leucocytes adhering per EC are shown in Table 2.

Table 1
Secreted mediators by keratinocytes after UVB or substance P stress

Normal human keratinocyte-secreted mediators in the culture medium were measured by ELISA. The results are expressed as a percentage of the control values (ANOVA; α=5%; *, statistically significant). n, number of experiments with keratinocytes from different donors (their response is expressed as an interval between minima and maxima); nd, not determined; X, D450.

UVB (10 mJ/cm2)Substance P (0.1 μM)
X/control (%)X/control (%)
MediatorMinimumMaximumnMinimumMaximumn
IL-8 50* 408* 38* 386* 
s-ICAM-1 362* 1218* nd nd  
RANTES 28 482* nd nd  
IL-7 303* nd nd  
UVB (10 mJ/cm2)Substance P (0.1 μM)
X/control (%)X/control (%)
MediatorMinimumMaximumnMinimumMaximumn
IL-8 50* 408* 38* 386* 
s-ICAM-1 362* 1218* nd nd  
RANTES 28 482* nd nd  
IL-7 303* nd nd  
Table 2
Stimulation of CEMT4 lymphocyte adhesion to skin ECs by stressed keratinocyte-conditionned media

The results are expressed as percentages of induction as compared with control values. After EC contact with the keratinocyte-conditioned medium (24 h), PKH26-GL-labelled human leucocytes (CEMT4) were applied on to a human skin EC monolayer (HSkMEC) in a 5:1 ratio for 20 min static adhesion at 37°C. Quantification was carried out by flow cytometry assessment of the numbers of both cells. Statistical analysis was the comparison of proportion following normal distribution [α=5%; *, statistically significant; n, number of experiments with different donors (their response is translated as an interval between minima and maxima)].

Modulation of CEMT4 adhesion to HSkMEC (%)
Keratinocyte treatmentMinimumMaximumn
UVB (10 mJ·cm−216.4* 78.8* 
Substance P (0.6 μM) 26.0* 96.0* 
Modulation of CEMT4 adhesion to HSkMEC (%)
Keratinocyte treatmentMinimumMaximumn
UVB (10 mJ·cm−216.4* 78.8* 
Substance P (0.6 μM) 26.0* 96.0* 

Taking into account the study by Middleton et al. [63] on ICAM-1 expression on keratinocytes, which shows an important donor variability, we performed the studies on several individuals. In the present study, despite the observed inter-donor differences, the applied stress caused a repetitive and significant increase in the amount of released mediators, which was accompanied by an increase in the recruitment of lymphoid cells. Futhermore, IL-8 and s-ICAM-1 appeared to be the most sensitive of the mediators studied.

This demonstrates that limiting the evaluation of inflammation to the measurement of the keratinocyte responses is not sufficient, the influence of the micro-environment on dermal microcapillaries must also be assessed. This was carried out in the present study by the use of organ-specific ECs by which method it was possible to evaluate the potential of anti-inflammatory molecules.

Effect of anti-inflammatory molecules on the activation of dermal microvascular ECs

Potent anti-inflammatory molecules, such as α-MSH, indomethacin and dexamethasone, were used to check the EC model of inflammation and to validate its potential for estimating the anti-inflammatory (or pro-inflammatory) properties of molecules. Keratinocytes were irradiated with UV light or incubated with substance P, and then incubated with the test molecule for 2 days. The resulting medium was then tested for its ability to modulate the properties of dermal ECs (leucocytes adhesion and/or adhesion molecule production).

The results indicate that the conditioned medium from keratinocytes incubated with stimulant plus anti-inflammatory agent decreased the adhesion capacity of dermal ECs as much as the medium from keratinocytes treated with anti-inflammatory agent alone (Figure 4). Dexamethasone clearly inhibited the UV-light-induced production of keratinocyte factors, but did not block activation by substance P.

Assessment of anti-inflammatory molecules by inhibition of the stress-induced adhesion of leucocytes to dermal ECs via keratinocyte-conditioned medium
Figure 4
Assessment of anti-inflammatory molecules by inhibition of the stress-induced adhesion of leucocytes to dermal ECs via keratinocyte-conditioned medium

Keratinocytes were stressed by UV irradiation or substance P, and the stresses were modulated by anti-inflammatory molecules (α-MSH, indomethacin and dexamethasone). The anti-inflammatory effects are expressed as percentages of adhesion inhibition (means±S.D., n=4).

Figure 4
Assessment of anti-inflammatory molecules by inhibition of the stress-induced adhesion of leucocytes to dermal ECs via keratinocyte-conditioned medium

Keratinocytes were stressed by UV irradiation or substance P, and the stresses were modulated by anti-inflammatory molecules (α-MSH, indomethacin and dexamethasone). The anti-inflammatory effects are expressed as percentages of adhesion inhibition (means±S.D., n=4).

The endothelial model is an effective method for assessing inflammation and the effects of anti-inflammatory molecules; it is selective and reflects biologically significant effects. We therefore believe it can be used to screen potentially therapeutic molecules and drugs.

DISCUSSION

The specific immunologically active products generated by keratinocytes in response to environmental stress, including physical stimuli, such as UV light, and endogenous signals, such as neuropeptides, initiate the amplification of cutaneous inflammation [3,6,43,55]. Our results in vitro demonstrated that keratinocytes can interact with infiltrating immune cells by releasing pro-inflammatory cytokines (Table 1) or via ICAMs, leading to modified adhesion properties (Table 2). Keratinocytes function as transducers of environmental signals, converting exogenous stimuli into the signal molecules involved in the activation of dermal ECs, and hence the recruitment and sequestration of specific leucocytes.

Their highly specific reactions indicated that the significance of such an in vitro model requires the use of appropriate cells from the skin [10,37].

This model is suitable to study EC-mediated leucocyte recruitment and modulation of the inflammatory process by keratinocyte activity. It shows the early steps of leucocyte movement from the blood into the peripheral tissues. Quantitative measurements of adhesion made it possible to assess the biological effects of immunomodulatory molecules. A growing body of experimental evidence suggests that UV-light-induced skin inflammation is influenced by the sensory nervous system acting via the neuroendocrine system, involving a complex network of cytokines, chemokines, neuropeptides and neuropeptide-degrading enzymes, including neuroendocrine hormones, such POMC (pro-opiomelanocortic) peptides and, particularly, α-MSH. α-MSH is released by stimulated epidermal cells, including keratinocytes, Langerhans cells and melanocytes, as well as immunocompetent cells. It has been recognized as a potent immunomodulator that inhibits the production and activity of immunoregulatory and pro-inflammatory cytokines. Since α-MSH modulates the NF-κB (nuclear factor κB) transcription factor, it may well affect the very early steps of the inflammation cascade [57,64,65], as confirmed in Figure 2 and by preliminary results showing that it reduces the expression of E-selectin on dermal ECs (results not shown).

This model, based on HAK, HSkMEC.1 and CEMT4 human cells (Figure 1A), can be used as a tool to study skin biology. It is suitable for evaluating the anti-inflammatory properties of potential topically active molecules and should help in the search for new anti-inflammatory therapies (Figure 4). Hence, this could lead to a better understanding of pro-inflammatory mechanisms. This cell model demonstrates that limiting the evaluation of inflammatory responses to that of keratinocytes is not sufficient, since a major effect is the resulting recruitment of competent leucocytes by adhesion to dermal microcapillaries. Any estimation of the amplitude of an inflammatory process therefore requires a study of the micro-environmental conditions that influence dermal microcapillaries. This is exactly what our organ-specific EC model provides.

The modulation of inflammation by the release of soluble factors (cytokines and soluble adhesion molecules), such as IL-7 and s-ICAM-1, appears to be quite significant, as indicated by the influence of the keratinocyte-conditioned medium on ECs. It increased the adhesion of leucocytes to ECs (Table 2). Treatment of keratinocytes with anti-inflammatory molecules reduced the capacity of the conditioned medium to induce adhesion. Consequently, the model described in the present study is valid for studying inflammatory reactions by assessing the adhesion/recruitment of leucocytes to ECs. Our results also emphasize the need to deal with appropriate organ-selective ECs (i.e. dermal), the importance of micro-environmental cross-talk between keratinocytes and microcapillary ECs (Figure 3) and the importance of the interaction between appropriately selected ECs and responsive leucocytes for studying tissue invasion [1].

In particular, one has to take into account that, in the presence of the appropriate migratory signals, the leucocytes migrate across the endothelium into tissue, where tissue-associated chemokine gradients also direct the final localization.

Consequently, the longer-term goals of this work are to understand the skin reactions that occur in response to damage and aging, and to design new active specific molecules, taking into account the organ-specific production and presentation of chemokines [66], which means, for skin-related studies, fractalkine [6769] and the skin-derived CCL27 [46,7073].

Abbreviations

     
  • CEMT4

    leukaemic CD4+ T-cell line

  •  
  • CGRP

    calcitonin-gene-related peptide

  •  
  • CLA+

    cutaneous lymphocyte antigen positive

  •  
  • EC

    endothelial cell

  •  
  • ECA

    EC activation

  •  
  • HAK

    human adult keratinocyte

  •  
  • HBrMEC

    human brain microvascular ECs

  •  
  • HSkMEC.1

    human skin microvascular ECs

  •  
  • HPLNEC.B3

    human peripheral lymph node ECs, clone B3

  •  
  • (s-)ICAM-1

    (soluble) intercellular adhesion molecule 1

  •  
  • IL

    interleukin

  •  
  • α-MSH

    α-melanin-stimulating hormone

  •  
  • PKH26-GL

    Paul Karl Horan 26 general labelling

  •  
  • RANTES

    regulated upon activation, normal T-cell expressed and secreted

  •  
  • TNF-α

    tumour necrosis factor α

  •  
  • VCAM

    vascular cell adhesion molecule

This work was supported by grants from INSERM (Progress) (C.K.), the Jerôme Lejeune foundation (C.K.) and LVMH. C.K. is Research Director at CNRS (Centre National de la Recherche Scientifique), C.C. is an ANRT-CIFRE (Agence National pour la Recherche et la Technologie-Convention Industrielle pour la Formation par la Recherche et l'Education) fellow, M.M. is an AI (Assitant Ingénieur) at CNRS.

REFERENCES

REFERENCES
1
Nickoloff
 
B. J.
 
Keratinocytes regain momentum as instigators of cutaneous inflammation
Trends Mol. Med.
2006
, vol. 
12
 (pg. 
102
-
106
)
2
Igawa
 
K.
Satoh
 
T.
Hirashima
 
M.
Yokozeki
 
H.
 
Regulatory mechanisms of galectin-9 and eotaxin-3 synthesis in epidermal keratinocytes: possible involvement of galectin-9 in dermal eosinophilia of Th1-polarized skin inflammation
Allergy
2006
, vol. 
61
 (pg. 
1385
-
1391
)
3
Purwar
 
R.
Kraus
 
M.
Werfel
 
T.
Wittmann
 
M.
 
Modulation of keratinocyte-derived MMP-9 by IL-13: a possible role for the pathogenesis of epidermal inflammation
J. Invest. Dermatol.
2008
, vol. 
128
 (pg. 
59
-
66
)
4
Frink
 
M.
Hsieh
 
Y. C.
Hsieh
 
C. H.
Pape
 
H. C.
Choudhry
 
M. A.
Schwacha
 
M. G.
Chaudry
 
I. H.
 
Keratinocyte-derived chemokine plays a critical role in the induction of systemic inflammation and tissue damage after trauma–hemorrhage
Shock
2007
, vol. 
28
 (pg. 
576
-
581
)
5
Akiba
 
H.
Kehren
 
J.
Ducluzeau
 
M. T.
Krasteva
 
M.
Horand
 
F.
Kaiserlian
 
D.
Kaneko
 
F.
Nicolas
 
J. F.
 
Skin inflammation during contact hypersensitivity is mediated by early recruitment of CD8+ T cytotoxic 1 cells inducing keratinocyte apoptosis
J. Immunol.
2002
, vol. 
168
 (pg. 
3079
-
3087
)
6
Barker
 
J. N.
Mitra
 
R. S.
Griffiths
 
C. E.
Dixit
 
V. M.
Nickoloff
 
B. J.
 
Keratinocytes as initiators of inflammation
Lancet
1991
, vol. 
337
 (pg. 
211
-
214
)
7
Wagner
 
L. A.
Brown
 
T.
Gil
 
S.
Frank
 
I.
Carter
 
W.
Tamura
 
R.
Wayner
 
E. A.
 
The keratinocyte-derived cytokine IL-7 increases adhesion of the epidermal T cell subset to the skin basement membrane protein laminin-5
Eur. J. Immunol.
1999
, vol. 
29
 (pg. 
2530
-
2538
)
8
Grone
 
A.
 
Keratinocytes and cytokines
Vet. Immunol. Immunopathol.
2002
, vol. 
88
 (pg. 
1
-
12
)
9
Li
 
J.
Ireland
 
G. W.
Farthing
 
P. M.
Thornhill
 
M. H.
 
Epidermal and oral keratinocytes are induced to produce RANTES and IL-8 by cytokine stimulation
J. Invest. Dermatol.
1996
, vol. 
106
 (pg. 
661
-
666
)
10
Kieda
 
C.
Paprocka
 
M.
Krawczenko
 
A.
Zalecki
 
P.
Dupuis
 
P.
Monsigny
 
M.
Radzikowski
 
C.
Dus
 
D.
 
New human microvascular endothelial cell lines with specific adhesion molecules phenotypes
Endothelium
2002
, vol. 
9
 (pg. 
247
-
261
)
11
Fagerholm
 
S. C.
Varis
 
M.
Stefanidakis
 
M.
Hilden
 
T. J.
Gahmberg
 
C. G.
 
α-Chain phosphorylation of the human leukocyte CD11b/CD18 (Mac-1) integrin is pivotal for integrin activation to bind ICAMs and leukocyte extravasation
Blood
2006
, vol. 
108
 (pg. 
3379
-
3386
)
12
Farkas
 
S.
Hornung
 
M.
Sattler
 
C.
Edtinger
 
K.
Steinbauer
 
M.
Anthuber
 
M.
Schlitt
 
H. J.
Herfarth
 
H.
Geissler
 
E. K.
 
Blocking MAdCAM-1 in vivo reduces leukocyte extravasation and reverses chronic inflammation in experimental colitis
Int. J. Colorectal Dis.
2006
, vol. 
21
 (pg. 
71
-
78
)
13
Granger
 
D. N.
Kubes
 
P.
 
The microcirculation and inflammation: modulation of leukocyte-endothelial cell adhesion
J. Leukoc. Biol.
1994
, vol. 
55
 (pg. 
662
-
675
)
14
Springer
 
T. A.
 
Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm
Cell
1994
, vol. 
76
 (pg. 
301
-
314
)
15
Berg
 
E. L.
Yoshino
 
T.
Rott
 
L. S.
Robinson
 
M. K.
Warnock
 
R. A.
Kishimoto
 
T. K.
Picker
 
L. J.
Butcher
 
E. C.
 
The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1
J. Exp. Med.
1991
, vol. 
174
 (pg. 
1461
-
1466
)
16
Butcher
 
E. C.
Picker
 
L. J.
 
Lymphocyte homing and homeostasis
Science
1996
, vol. 
272
 (pg. 
60
-
66
)
17
Luster
 
A. D.
 
Chemokines – chemotactic cytokines that mediate inflammation
N. Engl. J. Med.
1998
, vol. 
338
 (pg. 
436
-
445
)
18
Viac
 
J.
Goujon
 
C.
Misery
 
L.
Staniek
 
V.
Faure
 
M.
Schmitt
 
D.
Claudy
 
A.
 
Effect of UVB 311 nm irradiation on normal human skin
Photodermatol. Photoimmunol. Photomed.
1997
, vol. 
13
 (pg. 
103
-
108
)
19
Park
 
L. J.
Ju
 
S. M.
Song
 
H. Y.
Lee
 
J. A.
Yang
 
M. Y.
Kang
 
Y. H.
Kwon
 
H. J.
Kim
 
T. Y.
Choi
 
S. Y.
Park
 
J.
 
The enhanced monocyte adhesiveness after UVB exposure requires ROS and NF-κB signaling in human keratinocyte
J. Biochem. Mol. Biol.
2006
, vol. 
39
 (pg. 
618
-
625
)
20
Chung
 
K. Y.
Chang
 
N. S.
Park
 
Y. K.
Lee
 
K. H.
 
Effect of ultraviolet light on the expression of adhesion molecules and T lymphocyte adhesion to human dermal microvascular endothelial cells
Yonsei Med. J.
2002
, vol. 
43
 (pg. 
165
-
174
)
21
Heck
 
D. E.
Gerecke
 
D. R.
Vetrano
 
A. M.
Laskin
 
J. D.
 
Solar ultraviolet radiation as a trigger of cell signal transduction
Toxicol. Appl. Pharmacol.
2004
, vol. 
195
 (pg. 
288
-
297
)
22
Wang
 
L.
Eng
 
W.
Cockerell
 
C. J.
 
Effects of ultraviolet irradiation on inflammation in the skin
Adv. Dermatol.
2002
, vol. 
18
 (pg. 
247
-
286
)
23
Il'yasova
 
D.
Colbert
 
L. H.
Harris
 
T. B.
Newman
 
A. B.
Bauer
 
D. C.
Satterfield
 
S.
Kritchevsky
 
S. B.
 
Circulating levels of inflammatory markers and cancer risk in the health aging and body composition cohort
Cancer Epidemiol. Biomarkers Prev.
2005
, vol. 
14
 (pg. 
2413
-
2418
)
24
Schmuth
 
M.
Watson
 
R. E.
Deplewski
 
D.
Dubrac
 
S.
Zouboulis
 
C. C.
Griffiths
 
C. E.
 
Nuclear hormone receptors in human skin
Horm. Metab. Res.
2007
, vol. 
39
 (pg. 
96
-
105
)
25
Soter
 
N. A.
 
Acute effects of ultraviolet radiation on the skin
Semin. Dermatol.
1990
, vol. 
9
 (pg. 
11
-
15
)
26
Nishigori
 
C.
Hattori
 
Y.
Arima
 
Y.
Miyachi
 
Y.
 
Photoaging and oxidative stress
Exp. Dermatol.
2003
, vol. 
12
 
suppl. 2
(pg. 
18
-
21
)
27
Wilgus
 
T. A.
Koki
 
A. T.
Zweifel
 
B. S.
Kusewitt
 
D. F.
Rubal
 
P. A.
Oberyszyn
 
T. M.
 
Inhibition of cutaneous ultraviolet light B-mediated inflammation and tumor formation with topical celecoxib treatment
Mol. Carcinog.
2003
, vol. 
38
 (pg. 
49
-
58
)
28
Ansel
 
J. C.
Armstrong
 
C. A.
Song
 
I.
Quinlan
 
K. L.
Olerud
 
J. E.
Caughman
 
S. W.
Bunnett
 
N. W.
 
Interactions of the skin and nervous system
J. Investig. Dermatol. Symp. Proc.
1997
, vol. 
2
 (pg. 
23
-
26
)
29
Scholzen
 
T. E.
Brzoska
 
T.
Kalden
 
D. H.
O'Reilly
 
F.
Armstrong
 
C. A.
Luger
 
T. A.
Ansel
 
J. C.
 
Effect of ultraviolet light on the release of neuropeptides and neuroendocrine hormones in the skin: mediators of photodermatitis and cutaneous inflammation
J. Investig. Dermatol. Symp. Proc.
1999
, vol. 
4
 (pg. 
55
-
60
)
30
Lindsey
 
K. Q.
Caughman
 
S. W.
Olerud
 
J. E.
Bunnett
 
N. W.
Armstrong
 
C. A.
Ansel
 
J. C.
 
Neural regulation of endothelial cell-mediated inflammation
J. Investig. Dermatol. Symp. Proc.
2000
, vol. 
5
 (pg. 
74
-
78
)
31
Pitzalis
 
C.
Pipitone
 
N.
Bajocchi
 
G.
Hall
 
M.
Goulding
 
N.
Lee
 
A.
Kingsley
 
G.
Lanchbury
 
J.
Panayi
 
G.
 
Corticosteroids inhibit lymphocyte binding to endothelium and intercellular adhesion: an additional mechanism for their anti-inflammatory and immunosuppressive effect
J. Immunol.
1997
, vol. 
158
 (pg. 
5007
-
5016
)
32
Daxecker
 
H.
Raab
 
M.
Markovic
 
S.
Karimi
 
A.
Griesmacher
 
A.
Mueller
 
M. M.
 
Endothelial adhesion molecule expression in an in vitro model of inflammation
Clin. Chim. Acta
2002
, vol. 
325
 (pg. 
171
-
175
)
33
Yuan
 
H.
Goetz
 
D. J.
Gaber
 
M. W.
Issekutz
 
A. C.
Merchant
 
T. E.
Kiani
 
M. F.
 
Radiation-induced up-regulation of adhesion molecules in brain microvasculature and their modulation by dexamethasone
Radiat. Res.
2005
, vol. 
163
 (pg. 
544
-
551
)
34
Kuijpers
 
T. W.
Roos
 
D.
 
Leukocyte extravasation: mechanisms and consequences
Behring Inst. Mitt.
1993
, vol. 
92
 (pg. 
107
-
137
)
35
Carlos
 
T. M.
Harlan
 
J. M.
 
Leukocyte–endothelial adhesion molecules
Blood
1994
, vol. 
84
 (pg. 
2068
-
2101
)
36
Schon
 
M. P.
Zollner
 
T. M.
Boehncke
 
W. H.
 
The molecular basis of lymphocyte recruitment to the skin: clues for pathogenesis and selective therapies of inflammatory disorders
J. Invest. Dermatol.
2003
, vol. 
121
 (pg. 
951
-
962
)
37
Kieda
 
C.
 
How endothelial cell organo-specificity mediates circulating cell homing
Arch. Immunol. Ther. Exp. (Warsz.)
2003
, vol. 
51
 (pg. 
81
-
89
)
38
Dus
 
D.
Krawczenko
 
A.
Zalecki
 
P.
Paprocka
 
M.
Wiedlocha
 
A.
Goupille
 
C.
Kieda
 
C.
 
IL-7 receptor is present on human microvascular endothelial cells
Immunol. Lett.
2003
, vol. 
86
 (pg. 
163
-
168
)
39
Kieda
 
C.
Dus
 
D.
 
Endothelial cell glycosylation: regulation and modulation of biological processes
Adv. Exp. Med. Biol.
2003
, vol. 
535
 (pg. 
79
-
94
)
40
Hase
 
T.
Shinta
 
K.
Murase
 
T.
Tokimitsu
 
I.
Hattori
 
M.
Takimoto
 
R.
Tsuboi
 
R.
Ogawa
 
H.
 
Histological increase in inflammatory infiltrate in sun-exposed skin of female subjects: the possible involvement of matrix metalloproteinase-1 produced by inflammatory infiltrate on collagen degradation
Br. J. Dermatol.
2000
, vol. 
142
 (pg. 
267
-
273
)
41
Giacomoni
 
P. U.
Rein
 
G.
 
Factors of skin ageing share common mechanisms
Biogerontology
2001
, vol. 
2
 (pg. 
219
-
229
)
42
Bosset
 
S.
Bonnet-Duquennoy
 
M.
Barre
 
P.
Chalon
 
A.
Kurfurst
 
R.
Bonte
 
F.
Schnebert
 
S.
Le Varlet
 
B.
Nicolas
 
J. F.
 
Photoageing shows histological features of chronic skin inflammation without clinical and molecular abnormalities
Br. J. Dermatol.
2003
, vol. 
149
 (pg. 
826
-
835
)
43
Scholzen
 
T. E.
Konig
 
S.
Fastrich
 
M.
Bohm
 
M.
Luger
 
T. A.
 
Terminating the stress: peripheral peptidolysis of proopiomelanocortin-derived regulatory hormones by the dermal microvascular endothelial cell extracellular peptidases neprilysin and angiotensin-converting enzyme
Endocrinology
2007
, vol. 
148
 (pg. 
2793
-
2805
)
44
Conway
 
K. P.
Price
 
P.
Harding
 
K. G.
Jiang
 
W. G.
 
The role of vascular endothelial growth inhibitor in wound healing
Int. Wound J.
2007
, vol. 
4
 (pg. 
55
-
64
)
45
Le
 
A. D.
Zhang
 
Q.
Wu
 
Y.
Messadi
 
D. V.
Akhondzadeh
 
A.
Nguyen
 
A. L.
Aghaloo
 
T. L.
Kelly
 
A. P.
Bertolami
 
C. N.
 
Elevated vascular endothelial growth factor in keloids: relevance to tissue fibrosis
Cells Tissues Organs
2004
, vol. 
176
 (pg. 
87
-
94
)
46
Homey
 
B.
Alenius
 
H.
Muller
 
A.
Soto
 
H.
Bowman
 
E. P.
Yuan
 
W.
McEvoy
 
L.
Lauerma
 
A. I.
Assmann
 
T.
Bunemann
 
E.
, et al 
CCL27–CCR10 interactions regulate T cell-mediated skin inflammation
Nat. Med.
2002
, vol. 
8
 (pg. 
157
-
165
)
47
Hunger
 
R. E.
Yawalkar
 
N.
Braathen
 
L. R.
Brand
 
C. U.
 
The HECA-452 epitope is highly expressed on lymph cells derived from human skin
Br. J. Dermatol.
1999
, vol. 
141
 (pg. 
565
-
569
)
48
von Andrian
 
U. H.
Mackay
 
C. R.
 
T-cell function and migration. Two sides of the same coin
N. Engl. J. Med.
2000
, vol. 
343
 (pg. 
1020
-
1034
)
49
Campbell
 
D. J.
Butcher
 
E. C.
 
Rapid acquisition of tissue-specific homing phenotypes by CD4+ T cells activated in cutaneous or mucosal lymphoid tissues
J. Exp. Med.
2002
, vol. 
195
 (pg. 
135
-
141
)
50
Bruynzeel
 
I.
van der Raaij
 
L. M.
Willemze
 
R.
Stoof
 
T. J.
 
Pentoxifylline inhibits human T-cell adhesion to dermal endothelial cells
Arch. Dermatol. Res.
1997
, vol. 
289
 (pg. 
189
-
193
)
51
Choi
 
J. S.
Choi
 
Y. J.
Park
 
S. H.
Kang
 
J. S.
Kang
 
Y. H.
 
Flavones mitigate tumor necrosis factor-α-induced adhesion molecule upregulation in cultured human endothelial cells: role of nuclear factor-κB
J. Nutr.
2004
, vol. 
134
 (pg. 
1013
-
1019
)
52
Grandjean-Laquerriere
 
A.
Le Naour
 
R.
Gangloff
 
S. C.
Guenounou
 
M.
 
Differential regulation of TNF-α, IL-6 and IL-10 in UVB-irradiated human keratinocytes via cyclic AMP/protein kinase A pathway
Cytokine
2003
, vol. 
23
 (pg. 
138
-
149
)
53
Bevilacqua
 
M. P.
 
Endothelial–leukocyte adhesion molecules
Annu. Rev. Immunol.
1993
, vol. 
11
 (pg. 
767
-
804
)
54
Boyce
 
S. T.
Ham
 
R. G.
 
Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture
J. Invest. Dermatol.
1983
, vol. 
81
 (pg. 
33s
-
40s
)
55
Bizouarne
 
N.
Mitterrand
 
M.
Monsigny
 
M.
Kieda
 
C.
 
Characterization of membrane sugar-specific receptors in cultured high endothelial cells from mouse peripheral lymph nodes
Biol. Cell
1993
, vol. 
79
 (pg. 
27
-
35
)
56
Bizouarne
 
N.
Denis
 
V.
Legrand
 
A.
Monsigny
 
M.
Kieda
 
C.
 
A SV-40 immortalized murine endothelial cell line from peripheral lymph node high endothelium expresses a new α-L-fucose binding protein
Biol. Cell
1993
, vol. 
79
 (pg. 
209
-
218
)
57
Luger
 
T. A.
Scholzen
 
T.
Grabbe
 
S.
 
The role of α-melanocyte-stimulating hormone in cutaneous biology
J. Investig. Dermatol. Symp. Proc.
1997
, vol. 
2
 (pg. 
87
-
93
)
58
Oktar
 
B. K.
Alican
 
I.
 
Modulation of the peripheral and central inflammatory responses by α-melanocyte stimulating hormone
Curr. Protein. Pept. Sci.
2002
, vol. 
3
 (pg. 
623
-
628
)
59
Eves
 
P. C.
MacNeil
 
S.
Haycock
 
J. W.
 
α-Melanocyte stimulating hormone, inflammation and human melanoma
Peptides
2006
, vol. 
27
 (pg. 
444
-
452
)
60
Zhang
 
W.
Chen
 
H.
 
The study on the interleukin-8 (IL-8)
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi
2002
, vol. 
19
 (pg. 
697
-
702
)
61
Ley
 
K.
 
Arrest chemokines
Microcirculation
2003
, vol. 
10
 (pg. 
289
-
295
)
62
Witkowska
 
A. M.
Borawska
 
M. H.
 
Soluble intercellular adhesion molecule-1 (sICAM-1): an overview
Eur. Cytokine Netw.
2004
, vol. 
15
 (pg. 
91
-
98
)
63
Middleton
 
M. H.
Norris
 
D. A.
 
Cytokine-induced ICAM-1 expression in human keratinocytes is highly variable in keratinocyte strains from different donors
J. Invest. Dermatol.
1995
, vol. 
104
 (pg. 
489
-
496
)
64
Luger
 
T. A.
Scholzen
 
T.
Brzoska
 
T.
Becher
 
E.
Slominski
 
A.
Paus
 
R.
 
Cutaneous immunomodulation and coordination of skin stress responses by α-melanocyte-stimulating hormone
Ann. N.Y. Acad. Sci.
1998
, vol. 
840
 (pg. 
381
-
394
)
65
Luger
 
T. A.
Scholzen
 
T. E.
Brzoska
 
T.
Bohm
 
M.
 
New insights into the functions of α-MSH and related peptides in the immune system
Ann. N.Y. Acad. Sci.
2003
, vol. 
994
 (pg. 
133
-
140
)
66
Kunkel
 
E.
Butcher
 
E.
 
Homeostatic chemokines and the targeting of regional immunity
Adv. Exp. Med. Biol.
2002
, vol. 
512
 (pg. 
65
-
72
)
67
Hasegawa
 
M.
Sato
 
S.
Echigo
 
T.
Hamaguchi
 
Y.
Yasui
 
M.
Takehara
 
K.
 
Upregulated expression of fractalkine/CX3CL1 and CX3CR1 in patients with systemic sclerosis
Ann. Rheum. Dis.
2005
, vol. 
64
 (pg. 
21
-
28
)
68
Echigo
 
T.
Hasegawa
 
M.
Shimada
 
Y.
Takehara
 
K.
Sato
 
S.
 
Expression of fractalkine and its receptor, CX3CR1, in atopic dermatitis: possible contribution to skin inflammation
J. Allergy Clin. Immunol.
2004
, vol. 
113
 (pg. 
940
-
948
)
69
Sugaya
 
M.
Nakamura
 
K.
Mitsui
 
H.
Takekoshi
 
T.
Saeki
 
H.
Tamaki
 
K.
 
Human keratinocytes express fractalkine/CX3CL1
J. Dermatol. Sci.
2003
, vol. 
31
 (pg. 
179
-
187
)
70
Chen
 
L.
Lin
 
S. X.
Agha-Majzoub
 
R.
Overbergh
 
L.
Mathieu
 
C.
Chan
 
L. S.
 
CCL27 is a critical factor for the development of atopic dermatitis in the keratin-14 IL-4 transgenic mouse model
Int. Immunol.
2006
, vol. 
18
 (pg. 
1233
-
1242
)
71
Hayakawa
 
I.
Hasegawa
 
M.
Matsushita
 
T.
Yanaba
 
K.
Kodera
 
M.
Komura
 
K.
Takehara
 
K.
Sato
 
S.
 
Increased cutaneous T-cell-attracting chemokine levels in sera from patients with systemic sclerosis
Rheumatology
2005
, vol. 
44
 (pg. 
873
-
878
)
72
Homey
 
B.
Meller
 
S.
Savinko
 
T.
Alenius
 
H.
Lauerma
 
A.
 
Modulation of chemokines by staphylococcal superantigen in atopic dermatitis
Chem. Immunol. Allergy
2007
, vol. 
93
 (pg. 
181
-
194
)
73
Moed
 
H.
Boorsma
 
D. M.
Tensen
 
C. P.
Flier
 
J.
Jonker
 
M. J.
Stoof
 
T. J.
von Blomberg
 
B. M.
Bruynzeel
 
D. P.
Scheper
 
R. J.
Rustemeyer
 
T.
Gibbs
 
S.
 
Increased CCL27–CCR10 expression in allergic contact dermatitis: implications for local skin memory
J. Pathol.
2004
, vol. 
204
 (pg. 
39
-
46
)