Suppressive effects of transdermal clonidine administration on contact hypersensitivity reactions in guinea pigs

In the present study, immunopharmacological effects of clonidine-TTS on allergic contact dermatitis (ACD) to non-related, established contact sensitizers were investigated in guinea pigs. First, to evaluate the hypotensive effect of clonidine-TTS in guinea pigs, intra-arterial blood pressure was recorded. After 4 days of treatment with one (or two) TTS per animal, a reduction of arterial blood pressure from 71 +/- 1 to 51 +/- 2 mm Hg was observed. We subsequently assessed the effects of clonidine-TTS on contact hypersensitivity reactions to 2,4-dinitrochlorobenzene (DNCB) and 4-ethoxymethylene-2-phenyl-oxazolone (Ox). This study indicates that clonidine-TTS suppressed the elicitation of contact hypersensitivity reactions. The observed immunosuppressive effect of clonidine may account for the relatively weak hypersensitivity reactions to this drug in experimental animal studies. Further studies are needed to determine whether such findings are of relevance to the clinical use of clonidine in patient populations.     

Endoplasmic reticulum stress and the inflammatory response in allergic contact dermatitis

Maintaining homeostasis is central to organismal health. Deviation is detected by a variety of sensors that react to alarm signals arising from injury, infection, and other inflammatory triggers. One important element of this alarm system is the innate immune system, which recognizes pathogen-/microbe- or damage-associated molecular patterns via pattern recognition receptors localized in the cytosol or in membranes of innate immune cells such as macrophages, dendritic cells, and mast cells but also of T cells, B cells, and epithelial cells. Activation of the innate immune system results in inflammation and is a pre-requisite for activation of the adaptive immune system. Another important element is represented by the unfolded protein response (UPR), a stress response of the endoplasmic reticulum. The UPR regulates proteostasis and also contributes to the course of inflammatory diseases such as cancer, diabetes, obesity, and neurodegenerative diseases. In addition, the UPR is instrumental in allergic contact dermatitis. This inflammatory skin disease, affecting 5–10% of the population, is caused by T cells recognizing low-molecular weight organic chemicals and metal ions. In this mini-review, we discuss the orchestration of inflammatory responses by the interplay of the innate immune system with cellular stress responses in allergic contact dermatitis, with a focus on the UPR.     

The effect of some immunomodulating agents on the sensitization phase of experimental contact allergic reactions

In an experimental model which allows comparison of the macroscopic changes with the differentiated dermal inflammatory cell infiltrate of the allergic contact reaction to oxazolone in guinea pigs, the effects of single doses of the cytostatic agents cyclophosphamide, methotrexate and azathioprine and the anti-lymphocyte agent cyclosporin A administered prior to sensitization were studied. All the agents tested increased the degree of erythema and edema of reactions compared to controls. The dermal inflammatory mononuclear cell response showed little significant increase and would not seem to explain the enhancement of reactions. The number of basophils in reactions was not significantly different from controls, and there is no evidence for early arrival, degranulation and destruction of basophils prior to our first routine assessment at 24 h. The number of mast cells in the reactions was also unchanged. A role for the latter two cells involving degranulation without changes in the number of cells could provide a possible explanation for the augmentation of the vascular response. A mechanism for the effect of cyclophosphamide other than T-suppressor lymphocyte susceptibility based on selective cytostatic effects on precursor cells may need to be considered.     

Ontogeny of the allergic inflammatory response

The ability to produce allergic responses begins early in fetal life along with the development of other elements of the immune system. Among the most interesting questions related to the development of allergic disease are whether the fetus in utero commonly is exposed to sufficient allergen to induce IgE production and how much the mother's immune responses affect the developing fetal immune system. After birth, it seems that many factors, including the frequency and severity of infections and the timing and intensity of allergen and animal exposures, continue to influence immune development.     

Differential ability of occupational chemical contact and respiratory allergens to cause immediate and delayed dermal hypersensitivity reactions in mice.

Trimellitic anhydride (TMA) is known to cause occupational respiratory allergy associated with the presence of specific IgE antibody. Other chemicals, such as 2,4-dinitrochlorobenzene (DNCB), while exhibiting a clear potential for contact sensitization, apparently lack the ability to induce respiratory allergy in man. It has been shown previously that although both chemicals are immunogenic in mice, each provoking contact sensitization, exposure only to TMA results in an IgE antibody response. In the present study, to examine further the characteristics of human allergens, we have compared the ability of TMA and DNCB to elicit immediate and delayed cutaneous hypersensitivity reactions in mice. Topical exposure to both chemicals resulted in delayed (24 h) hypersensitivity. However, only TMA induced, in addition, an immediate (1 h) dermal reaction following local challenge. Serum from TMA-immune mice, but not from untreated mice or mice sensitized with DNCB, was able to transfer immediate hypersensitivity to naive recipients. The kinetics of passive sensitization with TMA-immune serum, together with the fact that immediate hypersensitivity to DNCB could be induced with monoclonal IgE anti-dinitrophenol (DNP) antibody, suggests that the immediate dermal responses caused by TMA are effected by hapten-specific IgE. These data demonstrate that different classes of occupational chemical allergen exhibit a variable potential to elicit immediate and delayed dermal hypersensitivity reactions in mice, and provide a novel approach to the classification and characterization of human allergens.     

Effects of clonidine on bilateral pain behaviors and inflammatory response in rats under the state of neuropathic pain

This study was conducted to investigate the effects of clonidine on bilateral pain behaviors and inflammatory responses in neuropathic pain induced by partial sciatic nerve ligation (PSNL), and to better understand whether the antinociception of clonidine was related to α₂-adrenoceptor mechanisms. Rats were divided randomly into five groups: sham-operation with saline, i.p.; PSNL with clonidine (0.2 mg/kg) or saline, i.p.; PSNL with yohimbine (2 mg/kg) followed by clonidine (0.2 mg/kg), i.p.; and PSNL with naloxone (0.3 mg/kg) followed by clonidine (0.2 mg/kg), i.p. On post-operative days 1, 3, 7, 14, and 21, both ipsilateral and contralateral pain behaviors were measured. In rats receiving antagonists, bilateral behavioral changes were measured on day 14. Bilateral paw pressure threshold and paw withdrawal latencies were measured, and the extent of glial activation was dertermined by measuring macrophage antigen complex-1 (Mac-1) and glial fibrillary acidic protein (GFAP). Additionally, the levels of tumor necrosis factor α (TNF-α) and interleukin (IL)-6 were determined. PSNL induced bilateral behavioral hyperalgesia, with the ipsilateral level displaying a higher extent of behavior changes than the contralateral side. In addition, the glial activation markers and cytokine production were augmented bilaterally. Clonidine caused significant attenuation of bilateral mechanical allodynia and thermal hyperalgesia, accompanied by inhibition of glial activation and the expression of cytokines. The effects of clonidine were blocked by the α₂-adrenoceptor antagonist yohimbine and partially reversed by the μ-opioid receptor antagonist naloxone. These data suggest that the bilateral antinoceptive effects of clonidine might mediate through immunomodulation by acting on α₂-adrenoceptor in rats undergoing neuropathic pain.     

Effects of salmeterol and terbutaline on IgE-mediated dermal reactions and inflammatory events in skin chambers in atopic patients

The objective of the present study was to investigate the potential of the long-acting p-aonist salmeterol as an inhibitor of various components of IgE-mediated inflammation in man. For this purpose, we measured gross skin reactivity (diameters of wheal and flare reaction [WFR] and late cutaneous reaction [LCR]) as well as inflammatory cells, mediators, and protein in cutaneous suction blister chambers in eight subjects with allergic rhinitis. Blisters were induced, two on each forearm, by gentle suction and heating, and were unroofed 12 h later, after which plastic chambers were placed over the denuded area. The chambers were challenged for 2 h with antihuman IgE (titer 1:10) in the presence and absence of salmeterol or terbutaline. Normal goat IgG served as negative control. Chamber fluids were removed hourly for the first 4 h, and this was followed by a 4-h incubation before final collection. Salmeterol (10^-6M) and terbutaline (10^-5 M) injected intradermally 30 min before, as well as together with anti-IgE (titer 1:100), inhibited the WFRs by up to 30%. The effect of salmeterol on the ensuing LCR (75% inhibition at 24 h) tended to be more pronounced than the corresponding inhibition by terbutaline. Both salmeterol and terbutaline very effectively inhibited the anti-IgE-induced extravasation of α₂-macroglobulin into skin chambers, with a significantly more sustained effect by salmeterol. Interestingly, only terbutaline reduced the histamine release evoked by anti-IgE. With the present experimental design, where both drugs were washed out from the chambers after 2 h, neither drug inhibited recruitment of leukocytes (including eosinophils). Taken together, salmeterol had a more sustained inhibitory effect than terbutaline on indices of IgE-mediated edema formation (late induration and plasma protein extravasation). On the other hand, under the present experimental conditions, salmeterol failed to reduce the histamine release (in contrast to terbutaline), and neither salmeterol nor terbutaline affected the recruitment of leukocytes.     

Effects of topically applied glucocorticosteroids on patch test responses and recruitment of inflammatory cells in allergic contact dermatitis

The effects of repeated topical application of a strong glucocorticosteroid (GCS) on patch test responses and the inflammatory infiltrate were studied in twenty nickel allergic patients. Patch test responses were strongly inhibited in 18 out of 20 patients. Immunohistochemical analysis revealed marked reductions in CD1⁺ (T6⁺) Langerhans cells, activated inflammatory T cells and of mast cells in the skin. It is concluded that GCS suppress contact allergic responses by reduction, or functional inhibition of antigen presenting cells. The reduced number of mast cells release less vasoactive mediators, inhibiting recruitment of inflammatory cells.This study was supported by The Praeventiefonds, The Netherlands.     

Effects of gliotoxin on Langerhans' cell function: contact hypersensitivity responses and skin graft survival.

Dendritic Langerhans' cells (LC), which are essential for the induction of cutaneous immunity, express high concentrations of class II major histocompatibility (MHC) glycoproteins (Ia in the mouse) on their plasma membrane. Application of gliotoxin, a member of the epipolythiodoxopiperazine (ETP) group of fungal metabolites, reduces epidermal LC density and alters their morphology from highly dendritic to a more rounded form. Here we demonstrate that gliotoxin also alters LC function, reducing contact hypersensitivity (CHS) responses due to the development of suppressor cells, and enhancing C57BL tail skin graft survival on BALB/c recipients. The reduction in LC density following gliotoxin application was shown to enhance skin graft survival, by reducing the concentration of Ia antigens within the graft, by using congenic mouse strains: B10.A(2R) x B10.A, differing only at H-2D, and B10.A(2R) x B10.A(4R), differing only at H-2 I-E. Treatment of B10.A(2R) tail skin with gliotoxin for 1 week did not affect its survival when grafted onto H-2D-disparate B10.A mice, whereas, when grafted onto H-2 I-E-disparate B10.A(4R) hosts, the grafts were not only accepted permanently, but induced specific unresponsiveness. It is concluded that gliotoxin has a marked effect on LC function, inhibiting CHS responses by the induction of suppressor cells and prolonging graft survival between H-2-disparate and congenic mouse strains.     

Contribution of CD4+ and CD8+ T-cells in contact hypersensitivity and allergic contact dermatitis

Allergic contact dermatitis, also referred to as contact hypersensitivity, is one the most frequent inflammatory skin diseases, and is characterized by redness, papule and vesicles, followed by scaling and dryness. Allergic contact dermatitis is elicited upon skin contact with nonprotein chemicals, haptens, and corresponds to a cutaneous delayed type hypersensitivity reaction, mediated by hapten-specific T-cells. During the sensitization phase, both CD4⁺ and CD8⁺ T-cell precursors are activated in the draining lymph nodes by presentation of haptenated peptides by skin dendritic cells. Subsequent hapten painting on a remote skin site induces the recruitment and activation of specific T-cells at the site of challenge. This leads to apoptosis of keratinocytes, recruitment of inflammatory cells and development of clinical symptoms. Experimental studies from the last 10 years have demonstrated that, in normal contact hypersensitivity responses to strong haptens, CD8⁺ type 1 T-cells are effector cells of contact hypersensitivity through cytotoxicity and interferon-γ production, while CD4⁺ T-cells are endowed with downregulatory functions. The latter may correspond to the recently described CD4⁺CD25⁺ regulatory T-cell population. However, in some instances, especially when there is a deficient CD8⁺ T-cell pool, CD4⁺ T-cells can be effector cells of contact hypersensitivity. Ongoing studies will have to confirm that the pathophysiology of human allergic contact dermatitis is similar to the mouse contact hypersensitivity and that the contact hypersensitivity response to common weak haptens, most frequently involved in human allergic contact dermatitis, is similar to that reported for strong haptens.     

IL-35抑制炎症反应和T细胞反应性减轻变应性鼻炎的机制研究

目的：探讨IL-35在变应性鼻炎中对炎症反应和T细胞反应性的作用及其机制。方法：选取2012年1月至2016年1月间本院收治的37例变应性鼻炎患者（观察组）及35例行变应性鼻炎过敏原检测后排除变应性鼻炎的健康志愿者（对照组）为研究对象,采集观察组和对照组的外周血液,ELISA检测患者血清中IL-35水平。建立小鼠变应性鼻炎动物模型,收集小鼠外周血液,ELISA检测小鼠血清中IL-35及IgE水平。小鼠鼻切片后组织染色检测嗜酸性粒细胞;分离小鼠脾脏细胞后加入卵清蛋白抗原,加入或不加入IL-35至培养基中,检测卵清蛋白特异性T细胞反应性;ELISA检测T细胞培养上清中细胞因子IL-2、IL-4、IL-5、IL-10、IL-13,IL-17、 IL-23、IL-27以及TNF-α含量;荧光定量PCR（Real-time PCR）检测培养T细胞中IL-2、IL-4、IL-5、IL-10、IL-13、IL-17、IL-23、IL-27以及TNF-α mRNA表达水平;Western blot 检测培养T细胞JNK、Erk1/2及p38信号途径的激活水平。结果：观察组血清IL-35水平显著低于对照组（P＜0.05）;变应性鼻炎组小鼠组织染色结果显示嗜酸性粒细胞浸润数量显著高于正常组（P＜0.05）,而血清IL-35水平则显著低于正常小鼠（P＜0.05）; 卵清蛋白特异性T细胞反应性检测显示IL-35能显著抑制其反应性;ELISA及Real-time PCR结果均显示,IL-35能够显著下调IL-4、IL-5、IL-13、IL-17、IL-23及TNF-α的表达,上调IL-2、IL-10及IL-27的表达。Western blot结果显示,IL-35能够显著抑制卵清蛋白特异性T细胞中JNK、Erk1/2及p38信号途径的激活水平。结论：IL-35能够调控炎症反应中的炎症因子表达和T细胞的反应性,从而减轻变应性鼻炎,其机制可能是通过调控JNK、Erk1/2及p38信号途径的激活水平。     

Differential and sequential expression of multiple chemokines during elicitation of allergic contact hypersensitivity

Regulation of chemokine-mediated leukocyte migration within inflammatory tissues is a complex event that cannot be mimicked and analyzed in vitro. We therefore investigated the role of macrophage- and T-lymphocyte-specific chemoattractants involved in the positioning of immune effector cells during the elicitation phase of contact hypersensitivity, a prototype of a T-lymphocyte-mediated immune reaction. Serial sections of skin biopsies obtained from sensitized individuals at distinct time intervals after epicutaneous application of allergens were hybridized with anti-sense probes of a large panel of chemokines or immunohistologically labeled with leukocyte-specific antibodies. Multifocal expression of monocyte chemoattractant protein-1 (MCP-1) was already detected after 6 hours in basal keratinocytes clearly preceding the infiltration of monocytes and T cells. Increasing basal expression of MCP-1 and, in addition, of regulated upon activation, normal T-cell expressed and secreted (RANTES) after 12 hours was accompanied by dermal expression of MCP-1, macrophage-derived chemoattractant (MDC), and RANTES and paralleled by infiltration of mononuclear cells into dermis and epidermis. Expression of the T-lymphocyte-specific chemokines IP-10 and MIG in epidermis and dermis and of MDC, pulmonary and activation-regulated chemokine (PARC), and thymus and activation-regulated chemokine (TARC) exclusively in the dermis started after 12 hours reaching maximum levels at 72 hours and was associated with infiltration of T cells into the epidermal compartment. Our data provide evidence that migrating effector cells encounter multiple chemoattractant signals in a complex spatial and temporal pattern. In particular, keratinocytes contribute to the vigorous immigration by sequential expression of MCP-1, RANTES, and interferon-inducible protein-10 (IP-10) monokine induced by gamma interferon (MIG), indicating that chemokine-mediated nonimmunological mechanisms precede and corroborate antigen-specific mechanisms during elicitation of contact hypersensitivity.     

Characteristics of the development of experimental allergic contact dermatitis]

Histological and electron microscopic examination of changes in the skin and blood revealed three different stages in the development of experimental allergic contact dermatitis (ACD). The primary contact reaction (24 hours) had features of nonspecific inflammation with some morphological signs of initial sensitization. Inflammation in the flare-up reaction (5-7 days) is the result of the immune process, and basophilic infiltration of the skin is the obligatory component of this reaction. ACD (patch test at 15 days) was shown to have morphological manifestations typical of the delayed type hypersensitivity and some signs of the immediate type reactions.     

Effects of contact sensitization and delayed hypersensitivity reactions on immune responses to non-related antigens. Modulation of Immune responses.

Guinea pigs were immunized intracutaneously into the ears with sheep red blood cells (SRBC). Application of a sensitizing dose of the contact allergen dinitrochlorobenzene (DNCB) onto the same ears was shown to suppress or enhance the humoral response to SRBC depending on the time of application. When guinea pigs were sensitized to a contact allergen, application of a sensitizing dose of a non-related allergen on the same ears either had no effect or caused a clear enhancement of the development of delayed type hypersensitivity (DTH). Strongest enhancement was found when both sensitizations were performed on the same day. Further experiments on the effects of a concomitant DTH reaction elicited at the site of application of a contact allergen showed a strong potentiation of DTH when B-cell suppression was minimized by pretreatment with cyclophosphamide (CY). It was considered that CY-DTH-immunopotentiation might be a useful tool for achieving a higher level of sensitivity after epicutaneous sensitization.     

Effect of pollen-mediated oxidative stress on immediate hypersensitivity reactions and late-phase inflammation in allergic conjunctivitis

BACKGROUND: Allergic eye diseases are complex inflammatory conditions of the conjunctiva that are becoming increasingly prevalent and present an increasing economic burden because of direct and indirect health expenditures. OBJECTIVE: We sought to identify factors that may synergize with antigen-induced allergic inflammation and lead to allergic conjunctivitis. We used a murine model of allergic conjunctivitis to test the effect of oxidative stress generated by pollen oxidases using nicotinamide adenine dinucleotide (reduced) or nicotinamide adenine dinucleotide phosphate (reduced) (NAD[P]H) as an electron donor present in pollen grains. METHODS: Reactive oxygen species (ROS) generation by hydrated Ambrosia artemisiifolia pollen (short ragweed pollen; RWP) grains was determined by using 2'-7'-dihydro-dichlorofluorescein diacetate, nitroblue tetrazolium reduction, and Amplex Red assay. The RWP-induced changes in intracellular ROS levels were examined in A549 cells, human primary bronchial epithelial cells, and murine conjunctiva. RESULTS: Ragweed pollen grains contain NAD(P)H oxidase activity, which is diphenyleneiodonium-sensitive and quinacrine-sensitive and sodium azide-resistant. These NAD(P)H oxidases generate a superoxide anion that can be converted to H2O2 by pollen grain-associated superoxide dismutase. These diffusible oxygen radicals from pollen grains increase intracellular ROS levels in cultured epithelial cells and murine conjunctiva. Similar phenomena were observed in sensitized and naive mice, indicating that the RWP-induced oxidative stress in conjunctival epithelium is independent of adaptive immunity. Inactivation of NAD(P)H oxidase activity in RWP decreases the immediate-type hypersensitivity and inflammatory cell infiltration into the conjunctiva. CONCLUSION: Our data suggest that ROS generated by NAD(P)H oxidases in pollen grains intensify immediate allergic reactions and recruitment of inflammatory cells in murine conjunctiva.     

IL-1-induced tumor necrosis factor-alpha elicits inflammatory cell infiltration in the skin by inducing IFN-gamma-inducible protein 10 in the elicitation phase of the contact hypersensitivity response

Contact hypersensitivity (CHS) is a typical inflammatory response against contact allergens. Inflammatory cytokines, including IL-1 and tumor necrosis factor (TNF)-alpha, are implicated in the reaction, although the precise roles of each cytokine have not been completely elucidated. In this report, we dissected the functional roles of IL-1 and TNF-alpha during CHS. CHS induced by 2,4,6-trinitorochlorobenzene as well as oxazolone was suppressed in both IL-1alpha/beta(-/-) and TNF-alpha(-/-) mice. Hapten-specific T cell activation, as examined by T cell proliferation, OX40 expression and IL-17 production, was reduced in IL-1alpha/beta(-/-) mice, but not in TNF-alpha(-/-) mice, suggesting that IL-1 but not TNF-alpha is required for hapten-specific T cell priming in the sensitization phase. On the other hand, TNF-alpha, induced by IL-1, was necessary for the induction of local inflammation during the elicitation phase. We also found that the expression of IFN-gamma-inducible protein 10 (IP-10) was augmented at the inflammatory site. Although IP-10 mRNA expression was abrogated in TNF-alpha(-/-) mice, both CHS development and TNF-alpha mRNA expression occurred normally in IFN-gamma(-/-) mice, indicating that the induction of IP-10 during CHS was primarily controlled by TNF-alpha. Interestingly, CHS was suppressed by treatment with anti-IP-10 mAb, suggesting a critical role for IP-10 in CHS. Reduced CHS in TNF-alpha(-/-) mice was reversed by IP-10 injection during the elicitation phase. Thus, it was shown that the roles for IL-1 and TNF-alpha are different, although both cytokines are crucial for the development of CHS.