TARC and RANTES, but not CTACK, are induced in two models of allergic contact dermatitis. Effects of cilomilast and diflorasone diacetate on T-cell-attracting chemokines

BACKGROUND: Skin-infiltrating T cells play a predominant role in allergic and inflammatory skin diseases such as atopic dermatitis and allergic contact dermatitis. These T cells are attracted by chemotactic factors, e.g. RANTES (regulation on activation, normal T cell expressed and secreted; CCL5), TARC (thymus and activation regulated chemokine; CCL17) and CTACK (cutaneous T-cell attracting chemokine; CCL27). OBJECTIVES: To investigate which T-cell-attracting chemokines are involved in allergic contact dermatitis in mice. METHODS: Allergic contact dermatitis was induced by application of dinitrochlorobenzene (DNCB) or toluene-2,4-diisocyanate (TDI), and chemokine concentrations were determined by enzyme-linked immunosorbent assay. The effects on chemokine concentrations of the highly selective phosphodiesterase 4 inhibitor cilomilast and the glucocorticoid diflorasone diacetate were studied in mouse ears. RESULTS: RANTES and TARC were elevated in both models of allergic contact dermatitis 24 h after challenge, whereas CTACK remained unchanged. The increase in RANTES was diminished in mouse ears pretreated with cilomilast or diflorasone diacetate. TARC was reduced by diflorasone diacetate in the DNCB model but was highly induced in the TDI model; in contrast, TARC was not influenced by cilomilast. CONCLUSIONS: TARC and RANTES, but not CTACK, are involved in these two models of allergic contact dermatitis.     

Participation of complement 3a receptor (C3aR) in the sensitization phase of Th2 mediated allergic contact dermatitis

The complement system has emerged as a bridge between innate and adaptive immune responses. An involvement of C3aR has been described during skin inflammation. The aim of the study was to investigate the role of C3a in a mouse model of allergic skin inflammation, such as allergic contact dermatitis (ACD) which is a clinical manifestation of contact sensitivity (CS). The sensitization phase was studied using the local lymph node test: Mice were sensitized on three consecutive days by application of non-irritant concentrations of toluene-2,4-diisocyanate (TDI; 0.5%) onto the ear skin. On day 5, auricular draining lymph nodes were obtained. The elicitation phase was investigated by sensitization with TDI on the depilated and tape-stripped abdominal skin and challenge with TDI on the ear skin and measuring of ear swelling in vivo and cytokine secretion in activated splenocytes in vitro respectively. Complement 3a receptor deficient (C3aRKO) mice showed increased cytokine responses (interleukin[IL]-5, IL-6, IL-17, granulocyte macrophage-colony stimulating factor [GM-CSF]) in the sensitization phase of ACD to TDI. However, no differences in CS responses to TDI were observed in C3aR KO mice compared with WT controls in the elicitation phase of ACD as assessed by measuring of ear swelling in vivo and cytokines in skin and in activated splenocytes in vitro, namely IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, interferon-γ (IFN-γ), GM-CSF and tumor necrosis factor (TNF)-α. These findings provide a new insight into the participation of C3a in the sensitization phase of CS immune responses.     

Non-steroidal and steroidal anti-inflammatory drugs vary in their modulation of dendritic cell function in the elicitation phase of allergic contact dermatitis

The role of dendritic cells (DCs) in allergic contact dermatitis has been clearly demonstrated for the induction phase. However, the situation during the elicitation phase is very complex within a distinct inflammatory response. This study was performed to exploit DC migration in the elicitation phase in a mouse model of allergic contact dermatitis and to evaluate the effects of steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) on DC migration through skin in the elicitation phase of allergic contact dermatitis. Topically and systemically administered acetylsalicylic acid (ASA) did not reduce the inflammatory response. However, systemically administered ASA significantly reduced the DC migration to the draining lymph node. In contrast, topically administered indomethacin reduced the inflammatory response, but had only minor effects on DC migration, whereas diflorasone diacetate reduced both inflammatory reaction and DC migration. Thus, NSAIDs may differ in their inhibitory action in immunological inflammation.     

A chronic contact eczema impedes migration of antigen-presenting cells in alopecia areata

Long-lasting allergen treatment is the most efficient therapy in alopecia areata (AA). The underlying mechanism is unknown. We here asked whether treatment with a contact sensitizer influences leukocyte migration such that dendritic cell (DC) migration or the recruitment of activated T-cells towards the skin become hampered. Allergen treatment of AA mice was not accompanied by a decrease in skin-infiltrating leukocytes or draining lymph node cells (LNC). However, the distribution of leukocyte subsets was changed with a dominance of monocytes in the skin and a reduced percentage of DCs in draining nodes. Chemokine and chemokine receptor expression in skin and draining nodes was strikingly increased and LNC from untreated and allergen-treated AA mice showed high migratory activity in vitro and readily homed in draining nodes and skin after intravenous injection. However, FITC labelling of the skin and subcutaneous transfer of dye-labelled DC revealed that allergen treatment created a chemokine milieu severely hampering DC migration from the skin towards the draining node. An allergic eczema-induced reduction in DC migration and antigen transfer could well contribute to insufficient T-cell activation and the recovery of hair follicle in AA and possibly be of relevance for other skin-related autoimmune diseases.     

Pimecrolimus inhibits the elicitation phase but does not suppress the sensitization phase in murine contact hypersensitivity,in contrast to tacrolimus and cyclosporine A

Pimecrolimus (SDZ ASM 981, Elidel) is a nonsteroid inflammatory cytokine inhibitor specifically developed for the treatment of inflammatory skin diseases. Its effect on the elicitation and sensitization phases of oxazolone-induced contact hypersensitivity was compared with tacrolimus and cyclosporine A (CyA) in BALB/c mice using the ear swelling assay. The compounds were administered orally. Elicitation was dose-dependently inhibited by all three compounds. The minimal effective doses were 30 mg per kg (pimecrolimus, tacrolimus) and 90 mg per kg (CyA), respectively. There was no impairment of sensitization by pimecrolimus up to the highest dose tested (120 mg per kg), in contrast to CyA (60% inhibition at 60 mg per kg) and tacrolimus (71% inhibition at 30 mg per kg). Weight and cellularity of the draining lymph nodes in mice treated with tacrolimus or CyA during sensitization were reduced. In addition, proliferation of T cells after secondary stimulation was inhibited in cell cultures from lymph nodes of mice treated with tacrolimus or CyA. Thus, in contrast to tacrolimus and CyA, pimecrolimus exerts a more selective immunomodulatory effect. It does not impair the primary immune response (sensitization phase) but effectively inhibits the secondary phase, the elicitation phase that is the clinical manifestation of contact hypersensitivity.     

Induction of cutaneous delayed hypersensitivity reactions in mice sensitized with intragastrically administered hapten: activation of Langerhans cells in the sensitization and elicitation phases

BACKGROUND: As seen in atopic dermatitis, allergic diseases often produce lesions both in the gastrointestinal tract and the skin, suggesting the involvement of an immunological relationship between the two organs in the pathogenesis. OBJECTIVES: To study the role of gastric and epidermal Langerhans cells (LCs) in the sensitization and elicitation phases, respectively, of cutaneous delayed-type hypersensitivity (DTH) reactions to intragastrically administered hapten. METHODS: BALB/c mice, which were subjected to intragastric administration of trinitrochlorobenzene 5 days previously, received an elicitative challenge of the same hapten to the ear skin. Sections of the ear were immunostained for CD4 and CD8. Epidermal sheets of the ear and epithelial sheets of the forestomach were immunostained for I-A and observed under a confocal laser scanning microscope. RESULTS: Cutaneous DTH reactions were induced in mice, as demonstrated by an increase in ear thickness and a prominent infiltration of CD4+ and CD8+ lymphocytes at 24-36 h after the elicitative challenge. In the elicitation phase, epidermal LCs showed a significant increase in size, indicating in vivo activation, at 24 h. In the sensitization phase, gastric LCs increased in size at 2 h, became round at 6 h, and decreased in number at 24 h, possibly representing the sequential events of LC activation and migration from the epithelium. CONCLUSIONS: The present study demonstrated that gastric LCs and epidermal LCs were activated in vivo in the sensitization and elicitation phases, respectively, of cutaneous DTH reactions in orally sensitized mice.     

咪唑斯汀、氯雷他定及西替利嗪对小鼠变应性接触性皮炎作用的比较研究

目的:比较咪唑斯汀、氯雷他定及西替利嗪对小鼠变应性接触性皮炎(ACD)的抑制作用。方法:建立小鼠ACD模型,采用致敏前及诱发后两种给药方法,口服不同剂量咪唑斯汀、氯雷他定及西替利嗪,观察抑制作用。结果:致敏前开始给药,3种药物均能明显抑制ACD小鼠耳肿胀(P<0.05),但咪唑斯汀的抑制作用强于氯雷他定及西替利嗪(P<0.05);诱发后开始给药,咪唑斯汀组小鼠耳肿胀消退快于氯雷他定及西替利嗪(P<0.05)。结论:咪唑斯汀对小鼠ACD抑制作用强于氯雷他定和西替利嗪。     

Langerhans cells and lymph node dendritic cells express the tight junction component claudin-1

Claudin-1 is a critical structural component of tight junctions that have an important role in adhesive properties, barrier function, and paracellular transport of epithelia and other nonhematopoietic tissues. We found claudin-1 in murine CD207+ Langerhans cells (LC) residing in epidermis. Claudin-1 was not detected in other skin dendritic cells (DC). LC expressed claudin-1 in steady state and inflamed skin. Claudin-1 was demonstrated further in lymph node LC under steady state and inflammatory conditions, including after direct tracking with tetramethylrhodamine-isothiocyanate (TRITC). All subsets of skin draining lymph node DC defined by CD205, CD11b, CD11c, and CD8, including a presumably blood-borne lymph node resident CD8+CD207+ LC population, were claudin-1+. TRITC tracking demonstrated claudin-1 in CD207- skin migrant DC in the lymph node, suggesting upregulation of this molecule during migration or once arrived in the lymph node. Claudin-1 expression in CD207+ cells was confirmed at the protein and mRNA levels. Transforming growth factor-beta, a factor critical for the induction of LC in vitro and in vivo, stimulated the accumulation of claudin-1 mRNA and protein when added to bone marrow cells cultured with GM-CSF and IL-4. Claudin-1 may thus have an important function in adhesion and/or migration of LC.     

Pimecrolimus -- an anti-inflammatory drug targeting the skin

Pimecrolimus is the most recent member of calcineurin inhibitors available for the therapy for inflammatory skin diseases. It targets T-cells and mast cells and inhibits the production and release of cytokines and other inflammatory mediators, as well as the expression of signals essential for the activation of inflammatory T-lymphocytes. Pimecrolimus has a cell-selective mode of action. In contrast to corticosteroids, it does not affect, e.g., Langerhans'cells/dendritic cells (LC/DC), as demonstrated in vitro with human monocyte-derived DC and in vivo with epidermal LC in mice, nor human primary fibroblasts. As shown in vitro with human skin and by comparison of clinical pharmacokinetic data from patients with atopic dermatitis, pimecrolimus permeates less through skin than tacrolimus and much less than corticosteroids. It, thus, has a lower potential for transcutaneous resorption after topical administration, resulting in a lower risk of systemic effects. Pimecrolimus has high anti-inflammatory activity in animal models of skin inflammation, including a model reflecting neurogenic inflammation, but a more favourable balance of anti-inflammatory vs. immunosuppressive activity than tacrolimus. Pimecrolimus does not affect sensitization in a murine model of allergic contact dermatitis and has a lower potency in various models of immunosuppression after systemic administration, compared to tacrolimus. In conclusion, the results of preclinical studies show that pimecrolimus has a selective pharmacological profile, suited for effective and safe treatment for inflammatory skin diseases.     

Studies on human skin lymph containing Langerhans cells from sodium lauryl sulphate contact dermatitis.

Immunologic processes in diseased human skin have been extensively investigated, but little is known about the effect of skin diseases on human afferent skin lymph. Starting in the papillary dermis, the skin lymphatics drain the adjacent tissue in a one-way flow toward the regional lymph nodes. The composition of the afferent lymph, therefore, reflects the immunologic inflammatory processes in the drained tissue. To obtain afferent lymph to investigate its content, we inserted a cannula, by means of microsurgery, into a superficial peripheral lymph vessel draining a defined skin area. By manipulating the drained skin area and subsequent examination of the lymph we established an in vivo system for investigating the kinetics of lymph changes during the course of skin reactions. In lymph derived from a mild sodium lauryl sulphate (SLS)--induced contact dermatitis we could demonstrate an increase of both flow and cells. In particular, the number of Langerhans cells (LC) increased enormously during the course of the skin reaction. It, therefore, seems that a large increase in the migration of LC from the skin to the regional lymph nodes is a major feature of SLS-induced contact dermatitis, suggesting that LC may play a major role in the irritant contact dermatitis reaction.     

Junctional adhesion molecules (JAM)-B and -C contribute to leukocyte extravasation to the skin and mediate cutaneous inflammation

Leukocyte extravasation is a finely tuned process, in which transmigration is the final step. Transmigration depends on molecules located at borders of endothelial cells; e.g., junctional adhesion molecules (JAM-A, -B and -C). In vivo blockade of JAM-A lead to decreased migration of monocytes into the skin. In contrast, the role of JAM-B and -C in development of cutaneous inflammation is unknown. We therefore elicited an allergic contact dermatitis in mice using 2,4-dinitro-1-fluorobenzene. RT-PCR and immunofluorescent staining of healthy skin revealed a constitutive JAM-B (66.4%+/-6.7% of all vessels) and -C expression (88.6+/-13.2%), which remained constant after induction of contact dermatitis. Functional studies, in which either JAM-B or -C neutralizing antibodies were injected into sensitized mice prior to allergen challenge showed a concentration-dependent reduction of the contact dermatitis. Decreased ear swelling was accompanied by reduction of leukocyte infiltration as analyzed by hematoxylin and eosin (H&E) histology and enzyme activity. Combined antibody treatment at doses of 1.25 mg per kg bodyweight lead to additive inhibition of allergic contact dermatitis, indicating that JAM-B and -C may have distinct functions. In conclusion, interactions with JAM-B and -C are essential for development of cutaneous inflammation.     

Pimecrolimus and tacrolimus differ in their inhibition of lymphocyte activation during the sensitization phase of contact hypersensitivity

BACKGROUND: As reported previously, oral administration of the calcineurin inhibitors (CNI) pimecrolimus and tacrolimus resulted in equipotent inhibition of the elicitation phase of contact hypersensitivity (CHS) in mice. The sensitization phase was inhibited by tacrolimus but was unaffected by pimecrolimus, even at higher doses. OBJECTIVE: The kinetics of lymph node hyperplasia and up-regulation of T and B cell activation antigens were analyzed to obtain a better understanding of the divergent CNI profile in CHS. METHODS: Lymph node (LN) cells of CNI-untreated and treated mice were examined with flow cytometry at various time points after sensitization with oxazolone. LN hyperplasia and drug levels were also determined. RESULTS: Sensitization induced a higher portion of LN cells expressing the activation antigens CD25, CD69 and CD134 and an increase in activated B cells (B220(+)/CD40(+)) compared to na?ve mice. Up-regulation of these markers was completely or profoundly blocked with tacrolimus, whereas pimecrolimus at the three-fold higher dose caused significantly less inhibition. Tacrolimus also completely blocked the sensitization-associated increase of CD11c(+) antigen presenting cells (APC) in LN, whereas pimecrolimus showed significantly less inhibition. In contrast to tacrolimus, LN weight and cellularity were not affected by pimecrolimus at any time point after sensitization. Concentration of tacrolimus in blood and in the draining LN substantially exceeded that of pimecrolimus by factors 6.7-14 and 5.6-5.8, respectively, at the same dose levels. CONCLUSION: In contrast to tacrolimus, systemic treatment of mice with pimecrolimus only weakly interferes with lymphocyte activation and does not affect hyperplasia of the draining lymph nodes during sensitization.     

Contact sensitivity (allergic contact dermatitis) to bis(tri-n-butyltin) oxide in mice

Contact sensitivity (allergic contact dermatitis) to bis(tri-n-butyltin) oxide (TBTO) was demonstrated in the mouse. TBTO (in an acetone:olive olive oil vehicle) or acetone:olive oil alone (as a control) were applied to the shaved flank under an occlusive patch and animals were challenged on the dorsum of the ear 6 days later. Ear swelling was then measured using an engineer's micrometer, 2, 24, 48 or 72 h thereafter. Significant differences in ear swelling between control and TBTO-sensitized animals were found 24 h after challenge: thereafter the elicitation reaction declined rapidly whilst irritant swelling in control animals increased. Maximal elicitation of TBTO sensitivity could only be elicited by concentrations of TBTO that caused irritation. In a separate experiment, a single application of TBTO to the ears of naive animals provoked increase in auricular lymph node weight, cell yield and proliferation of lymph node cells during overnight culture. These data support the conclusion that TBTO is a contact sensitizer and illustrate the potential usefulness of the quantitative methods of contact sensitivity assessment which have been developed in the mouse.     

Syndecan‐1 regulates dendritic cell migration in cutaneous hypersensitivity to haptens

In human dendritic cells (DCs), we previously demonstrated in vitro that syndecan‐1 (SDC1) is downregulated during maturation correlating with enhanced motility. We investigated the effects of SDC1 on DC migration in vivo during TNCB(2,4,6‐trinitro‐1‐chlorobenzene)‐induced cutaneous hypersensitivity reaction (CHS) in mice. We show that DC in SDC1‐deficient mice migrated faster and at a higher rate to lymph nodes draining the hapten‐painted skin. Adoptive transfer of SDC1‐deficient hapten‐ and fluorochrome‐labelled DC into wild‐type (WT) mice led to increased and faster migration of DC to paracortical lymph nodes, and to a stronger CHS compared to WT DC. In SDC1?/? mice, CCR7 remains longer on the DC surface within the first 15‐minutes maturation (after LPS‐induced maturation). In addition, a time‐dependent upregulation of CCL2, CCL3, VCAM1 and talin was found during maturation in SDC1?/? DC. However, no difference in T‐cell‐stimulating capacity of SDC1‐deficient DC was found compared to WT DC. Mechanistically, SDC1‐deficient DC showed enhanced migration towards CCL21 and CCL19. This may result from functional overexpression of CCR7 in SDC1?/? DC. Increased and accelerated migration of otherwise functionally intact SDC1‐deficient DC leads to an exacerbated CHS. Based on our results, we conclude that SDC1 on DC negatively regulates DC migration.     

小鼠局部淋巴结检测结合耳肿胀程度鉴别光敏物的评估

目的 建立小鼠局部淋巴结检测结合耳肿胀程度评估的方法鉴别光敏物。方法 受试物加UVA照射(剂量10J/cm²)连续3d在BALB/c小鼠耳背部进行激发,最后一次照射结束后24h切取引流的局部淋巴结,用淋巴细胞计数和MTT比色法测定淋巴细胞增殖率反映局部淋巴结的增殖情况;同时测量小鼠耳肿度以反映皮肤的刺激反应;用双抗体夹心ELISA法进一步测定局部淋巴结细胞72h培养上清液中干扰素-γ(IFN-γ),白介素-2(IL-2)以及白介素-4(IL-4)的含量。结果 强光变应原四氯水杨酰苯胺引起局部淋巴结的增殖反应而不伴耳肿胀反应;弱光变应原6甲基香豆素既不引起局部淋巴结的增殖反应又不出现耳肿胀反应;光毒物质8-甲氧补骨脂素同时引起局部淋巴结增殖和耳肿胀反应。用四氯水杨酰苯胺预先致敏后再进行激发,局部淋巴结中IL-4的分泌量明显升高(P<0.05)。结论 小鼠局部淋巴结检测结合耳肿胀程度评价的方法可较快鉴定较强的接触性光变应原和光毒物质,但对识别较弱的光变应原敏感性不足;预先致敏后激发阶段局部淋巴结中IL-4的分泌量明显升高是光变应原区别于光毒物质的突出特点。