Increased expression of eotaxin and its specific receptor CCR3 in bullous pemphigoid

ARTICLE

Bullous pemphigoid (BP) is an autoimmune skin disease characterized by blister formation as a result of the interaction of autoantibodies with hemidesmosomal antigens, followed by complement activation and leukocyte infiltration [1, 2]. A variety of activated inflammatory cells, such as mast cells, eosinophils and lymphocytes has been detected in the lesional skin of patients with BP [3-6]. All these cellular types actively participate in blister formation through local production and release of several cytokines and soluble factors involved in the amplification and maintaining of tissue damage. Early cutaneous infiltration of activated CD4⁺ T cells and eosinophils seems to be a crucial event in the development of bullous lesions; cytokines and soluble mediators released by these cells (T lymphocyte-derived interleukin (IL)-2, soluble IL-2 receptor (sIL-2R), IL-4, IL-5, IL-6 as well as eosinophil-derived eosinophil cationic protein (ECP), major basic protein (MBP), 92 kDa gelatinase) have been extensively detected in blood and blister fluids of patients with BP [7-12]. We recently proposed that activated T lymphocytes involved in the pathomechanism of BP may belong to the Th2 cell subset, since we documented high levels of circulating IL-4 and soluble CD30 (sCD30), an activation marker preferentially expressed and released by Th2 lymphocytes, in sera of BP patients. We further suggested a role for sCD30 as a serological marker of disease activity, due to its significant reduction after immunosuppressive therapy [13]. Taken together, these data make it clear that the trafficking of circulating leukocytes through the sites of inflammation, their activation and cytokine release play a crucial role in the pathogenesis of BP.

The superfamily of chemokines, small (8-10 kDa) inducible chemoattractant cytokines, has been shown to be responsible for chemotaxis, adhesion and activation of leukocytes at inflammatory sites [14-16]. The two main subclasses of alpha- and beta-chemokines are respectively associated with acute and chronic inflammation, since alpha-chemokines act primarily on neutrophils chemotaxis and degranulation, whereas beta-chemokines are able to activate the other leukocyte types such as T cells, eosinophils, monocytes/macrophages, basophils and dendritic cells. Human eotaxin, a member of the beta-chemokines, has been reported to preferentially attract and activate eosinophils during inflammation and allergic reactions and a variety of cells, including endothelial cells, macrophages, epithelial cells, eosinophils and T cells are known to be a source of this chemokine [17, 18]. Extensive studies performed on animal models of allergic inflammation have demonstrated the pivotal role of eotaxin, in synergism with IL-5, in inducing rapid tissue eosinophil recruitment [19-21]. It is known that eotaxin specifically binds to only one receptor, named CCR3, which is however shared with other chemokines, including eotaxin-2, eotaxin-3, MCP-4 (with high affinity) plus MCP-3 and RANTES (with lower affinity) [14, 15]. The presence of CCR3 was first documented on eosinophils and basophils [22, 23]. More recently, several experimental studies have also shown that T lymphocytes producing Th2 cytokines (IL-4, IL-5) are able to express CCR3 both in vivo and in vitro, thus suggesting this receptor as an additional marker to reveal Th2-mediated reactions [24-27]. Increased expression of both eotaxin and CCR3 has been documented in inflammed tissue during Th2-related immunological disorders such as asthma, allergic rhinitis, atopic dermatitis, chronic sinusitis, and their presence is often associated with the inflammatory infiltrate and/or the severity of the disease [28-33]. Recently, a correlation between the presence of eotaxin and both IL-5 and tissue eosinophilia has been documented in patients with BP, confirming the role of these chemotactic factors in activating and recruiting eosinophils during tissue damage [34, 35]. In this study we have investigated the expression of both eotaxin and CCR3 in patients suffering from BP, in order to confirm a prevalent activation of Th2 immune response during the disease.

Materials and methods

Skin samples

Skin specimens including lesional and perilesional skin were obtained from 10 patients affected with active BP, aged from 52 to 71 years, attending our department. Diagnosis was confirmed by the common clinical and histopathological findings; indirect immunofluorescence (IIF) on monkey oesophagus and direct immunofluorescence (DIF) revealed that all subjects had circulating and tissue-bound autoantibodies against the basement membrane zone (BMZ). IIF performed on 1 M NaCl human split-skin demonstrated epidermal site autoantibody binding in all patients. All skin samples were taken from lesions of early appearance (< 48 hrs), histologically characterized by the presence of an inflammatory infiltrate mainly consisting of eosinophils and lymphocytes. No patient was undergoing any therapy at the time of the study and concomitant allergic and neoplastic pathologies were excluded. Skin samples from 3 patients with pemphigus vulgaris (PV) were studied as controls.

Immunohistochemistry

Serial cryostat sections (5 mum) were cut frozen, coated on polylysine pretreated slides and air-dried overnight at room temperature. Specimens were fixed in 100% cold acetone 10 min at 4° C and washed 10 min in phosphate buffered saline (PBS). Skin specimens were pretreated 10 min with peroxidase blocking reagent (DAKO Corp., Carpinteria, CA, USA) to suppress endogenous peroxidase and pseudo-peroxidase activity, washed again in PBS and then incubated for 1 hour at room temperature in a humid chamber with the following anti-human primary monoclonal antibodies (mAbs): anti-Eotaxin (1:100; R&D System, Minneapolis, MN, USA), anti-CCR3 (1:500; kindly provided by Dr. Charles Mackay, LeukoSite, Inc. Cambridge, MA, USA), anti-EG₂ (1:100; Pharmacia, Uppsala, Sweden) to identify activated eosinophils and anti-CD3 (1:100; Becton Dickinson, San Josè, CA, USA) to detect the presence of T lymphocytes. As negative control, the primary mAb was substituted with buffer. The slides were rinsed twice and then incubated 30 min with goat anti-mouse immunoglobulins conjugated to a peroxidase labelled polymer (EnVision + TM, Peroxidase, DAKO Corp., Carpinteria, CA, USA). After washing, revealing reaction was performed using 3,3'-diaminobenzidine (Liquid DAB⁺ Substrate-Chromogen System, DAKO Corp., Carpinteria, CA, USA) as chromogen substrate. Slides were then washed again, counterstained with hematoxylin and viewed microscopically using a Leitz Dialux 20 microscope.

Sera and blister fluids

Serum samples for quantitative evaluation of circulating eotaxin were obtained from all subjects with BP (n = 10); sera from PV patients (n = 5) and healthy donors (n = 10) were studied as control groups. Blister fluids for the same investigation were collected from bullae of early onset in 5 out of 10 BP patients enrolled into the study, compared to suction blisters raised on the forearm of three healthy volunteers selected from the serum sample healthy donor group. Immunoenzymatic assay was performed using a commercially available ELISA kit (R&D System, Minneapolis, USA), according to the manufacturer's instructions. The detection limit of the assay was estimated to be 5 pg/ml. Serum concentrations of total IgE were also measured in all subjects enrolled in the study using a fluoroimmunoenzymatic method (FEIA; UniCAP System, Pharmacia, Sweden) considering positive a concentration higher than 100 KU/l. All sera and blister fluids, stored at - 80° C until use, were evaluated in duplicate.

Statistical analysis

Data were evaluated by nonparametric statistical tests; Mann-Whitney U test was used to compare BP patients and control groups and Wilcoxon signed rank test revealed differences between BP blister fluids and corresponding sera; probability values of p < 0.05 were considered to be significant.

Results

Immunohistochemical evaluation of eotaxin and CCR3

Qualitative analysis of immunohistochemical staining showed high expression of both eotaxin and CCR3 in 8 out of 10 skin specimens from BP patients. As shown in Figure 1 a strong immunoreactivity for eotaxin was found to be localized mostly in the upper dermis of lesional and, to a lesser extent, perilesional skin. Eotaxin immunostaining was observed to be expressed by both eosinophils (EG2) and lymphocytes (CD3) at perivascular sites and was also widespread throughout the upper and middle dermis. The analysis of CCR3 staining documented a similar pattern of distribution when compared to eotaxin expression. Double staining with anti-CCR3 and anti-EG2 or anti-CD3 was not feasible in this study, but their distribution in serial sections was reliable for providing evidence of the expression of CCR3 on both EG2⁺ and CD3⁺ cells, mainly at perivascular sites. In the other two BP skin samples, histologically characterized by scanty eosinophils and the prevalence of infiltrating lymphocytes, we could document CCR3 expression only on CD3⁺ cells, in both lesional and perilesional dermal infiltrate; no immunostaining was observed either for eotaxin or EG2. In skin specimens derived from PV patients and studied as controls, we only observed perivascular CD3⁺ cells, without staining for EG2⁺ eosinophils, eotaxin and CCR3 (data not shown).

Detection of eotaxin in sera and blister fluids

Large amounts of eotaxin were detected both in sera and blister fluids of BP patients. As shown in Figure 2, mean serum levels of eotaxin in BP were significantly increased when compared to both healthy donors (169 ± 69 vs 71 ± 27 pg/ml, p = 0.003) and PV patients (75 ± 17 pg/ml, p = 0.01). In a subset of BP subjects (5 out of 10) mean eotaxin levels in blister fluids were significantly higher than those detected in corresponding sera (2,087 ± 681 vs 196 ± 35 pg/ml, p = 0.003) and in blister fluids from three normal donors (166 ± 49; p = 0.003) (Fig. 3).

Total IgE serum levels were high in 7/10 patients with BP (856 ± 240 KU/l) and in 1/5 PV patients (320 KU/l) while normal values of IgE were detected in all healthy donors. Furthermore, no significant correlation was found between eotaxin and IgE serum levels (p = 0.273).

Discussion

It is now well known that, in addition to autoantibody deposition and complement activation, cell-mediated immune reactions are key events in the pathogenesis of BP. Bullous lesions are infiltrated by inflammatory cells, mainly consisting of activated T lymphocytes and eosinophils from the early stages of blister formation. Large scale production and release of cytokines and soluble mediators from these cells has been extensively documented both in bullae and sera of patients affected with BP, often related to disease activity and skin involvement [8]. Several studies have shown tissue deposition and high concentration of eosinophil cationic protein (ECP) and major basic protein (MBP) in BP sera and blister fluid, and the presence of a strong eosinophil-colony stimulating activity seems to be derived from lesional infiltrating T lymphocytes [10, 11]. Furthermore, our previous studies performed on BP patients demonstrated high levels of circulating IL-4 and sCD30, a Th2 activation marker, in relation to the disease activity [13]. On the whole, these data suggest that activation of Th2 lymphocytes able to recruit and activate eosinophils in inflammed skin is a major step in the pathomechanism of tissue damage during BP. In the present study, we documented that eotaxin and its specific receptor CCR3 are significantly expressed in lesional skin of patients affected with active BP, both associated with the presence of CD3⁺ T lymphocytes and eosinophils.

Eotaxin is able to induce, in cooperation with IL-5, eosinophil accumulation into inflammatory sites via CCR3 interaction. High expression of mRNA and protein for both eotaxin and CCR3 has been found in bronchial mucosal biopsies from atopic asthmatics, and eotaxin immunoreactivity seems to precede the influx of activated eosinophils in bronchial tissue and bronchoalveolar lavage fluid [30]. Experimental data have shown that, other than macrophages, mast cells, epithelial cells and endothelium, both eosinophils and T cells influxing inflammed tissue significantly contribute to eotaxin production and release, thus providing an autocrine mechanism involved in local recruitment of inflammatory cells during Th2-mediated allergic reactions [28, 30]. Recent studies have also documented an increased expression of eotaxin in BP, associated with the presence of IL-5 and tissue eosinophilia, thus accounting for the role of this chemokine in eosinophil activation during the disease [34, 35]. Our study confirms the pathogenic relevance of eotaxin in tissue damage and provides the first evidence for CCR3 expression in BP lesional skin. In parallel, the high eotaxin release observed in the bloodstream, and, at highest levels, in blister fluids, could be regarded as a systemic sign of chemokine production at sites of skin inflammation. We have been unable to detect the presence of eotaxin and CCR3 in the skin and/or in sera of PV patients studied as a blistering disease control group, indicating that this chemokine may play a specific role in BP. Recent studies document that T cell differentiation under conditions that favor Th1 or Th2 polarization induces a different pattern of chemokine receptors, with CCR5 and CXCR3 preferentially expressed on Th1s, and CCR3, CCR4 and CCR8 expressed, both in vivo and in vitro, on Th2 cells [25, 27]; in addition, CCR3⁺ T cell clones are able to produce IL-4 and/or IL-5 and to migrate in response to eotaxin [36, 37]. Gerber et al. have demonstrated the expression of CCR3 on CD3⁺ cells co-localizing with eosinophils in tissues from contact dermatitis, nasal polyps and ulcerative colitis, suggesting eotaxin/CCR3 interaction as an additional pathway of T cell recruitment [38]. All these data account for CCR3 as a marker of Th2 cells involved in immune reactions leading to leukocyte migration to sites of inflammation. According to our results, we may suggest a further mechanism in the pathogenesis of BP in which, from the early stages of the disease, eotaxin is responsible for chemotaxis of both eosinophils and Th2 cells that, through local production and release of cytokines such as IL-4, IL-5 and eotaxin itself, are able to maintain and amplify the immunological process underlying blister formation. Furthermore, the expression of CCR3 on infiltrating CD3⁺ lymphocytes seems to confirm Th2 activation in BP, as suggested by our previous data.

Article accepted on 16/10/01

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