Bullous delayed pressure urticaria: pathogenic role for eosinophilic granulocytes?

CASE REPORT

DOI 10.1111/j.1365-2133.2005.06677.x

Bullous delayed pressure urticaria: pathogenic role for eosinophilic granulocytes? ¨cker,* A. Trautmann* and M. Leverkus*§ A. Kerstan,* C. Rose,* D. Simon,
H-U. Simon, E-B. Bro *Department of Dermatology Venerology and Allerology, University of Wu¨rzburg, Germany Departments of
Dermatology and Pharmacology, University of Bern, Switzerland §Department of Dermatology and Venerology, Otto-von Guericke-University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany

Summary Correspondence Martin Leverkus. E-mail: martin.leverkus@medizin. uni-magdeburg.de

Accepted for publication 2 February 2005

Key words: delayed pressure urticaria, eosinophils, interleukin 5, T lymphocytes

Conflicts of interest: None declared.

Bullous delayed pressure urticaria (DPU) is a rare variant of DPU. Treatment of DPU is difficult and the underlying pathogenic mechanism of DPU remains elusive. We report a 72-year-old man with DPU and associated chronic urticaria as well as delayed urticarial dermographism. Pressure challenge gave rise to a deep weal covered by multiple vesicles and bullae after 24 h. Histological examination of a skin biopsy specimen obtained 24 h after pressure challenge demonstrated intraepidermal bullae filled with eosinophils accompanied by a dense, predominantly eosinophilic infiltrate in the dermis. Whereas the numbers and morphology of mast cells were unaltered, the extracellular deposition of eosinophil cationic protein revealed evidence for eosinophil activation. Concomitantly, both CD4+ and CD8+ T lymphocytes were present in the infiltrate and expressed interleukin 5. As bullous DPU may represent the maximal variant of DPU, the investigation of the cellular infiltrate and the chemokines ⁄cytokines released may reveal potential pathogenic mechanisms. A possible effector role of eosinophilic granulocytes, T-cell subsets and mast cells is discussed.

Delayed pressure urticaria (DPU) is a subset of physical urticaria characterized by painful deep, pressure-induced swellings which usually develop 4–8 h after elicitation and frequently last more than 24 h (reviewed by Lawlor and Black1). Concomitantly, mild systemic symptoms such as fever, arthralgia or myalgia may occur. The mean age at onset of DPU is in the third decade2,3 and the mean duration of DPU is 9 years.2 However, the severity of DPU varies in individuals over time.2,4 Of note, DPU is reported to be present in 37% of patients with chronic urticaria (CU),5 while 94% of patients with DPU have associated CU.6 Efficient treatment of DPU is difficult. Various treatment regimens have been proposed in case reports or small series of patients.2,7–13 The pathogenesis of DPU is not clear. The mast cell seems to be the primary effector cell in urticaria14 and was previously postulated also to play a role in DPU.15 Although the pathological features of DPU resemble those of a late-phase reaction,16–18 an antigen has never been identified. The histopathology of DPU is characterized by the sequential appearance of a dense dermal infiltrate with eosinophils and neutrophils in earlier lesions (< 6 h) and eosinophils and lymphocytes in later lesions (> 12 h).18–21 In particular, a deep eosinophil-rich infiltrate in the lower dermis has been reported to be a distinct histopathological feature of DPU, in contrast to other forms of urticaria.19 An increased expression

of vascular endothelial adhesion molecules such as E-selectin, vascular adhesion molecule-122 and intercellular adhesion molecule-1 has been shown in early lesions of DPU.23 These molecules enable the recruitment and migration of eosinophils, neutrophils and lymphocytes into the dermis. Additionally, an increased expression of interleukin (IL)-6,24 tumour necrosis factor-a and IL-325 in the dermal inflammatory cell infiltrate has been demonstrated in lesions of DPU. To our knowledge, only three cases of bullous DPU have been described thus far.26–28 By histological and immunofluorescence studies, we further characterized the eosinophil-rich leucocytic infiltrate in a patient with bullous DPU. Whereas mast cells were normal in number and morphology, we demonstrated high numbers of eosinophil cationic protein (ECP)releasing eosinophils together with IL-5-expressing CD4+ and CD8+ T cells. These findings suggest a pathogenic role for eosinophils in the development of DPU.

Case report A 72-year-old man presented with a large erythematous weal on his abdomen following application of pressure while leaning against an iron bar 24 h previously. He reported a 2-year history of spontaneous urticaria as well as recurrent attacks of large painful hives following pressure to his shoulders, hips,


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435

436 Bullous delayed pressure urticaria, A. Kerstan et al.

hands and knees. These had occurred mainly after cycling and gardening. Weals occurred 4–8 h after pressure elicitation and persisted for 24–48 h. A diagnosis of DPU and CU was made. To verify the clinical diagnosis of DPU we performed a pressure challenge by hanging a 10-kg weight belt of 2 cm in width over his right shoulder for 20 min.26 A painful deep erythematous plaque arose 4 h later, followed by the appearance of vesicles and multiple blisters within the test area 24 h after pressure challenge (Fig. 1A,B). In addition, the patient showed delayed dermographism without blisters after 4 h, which persisted for 8 h. The patient’s personal history revealed no atopy and his family history was unremarkable. Laboratory findings including differential blood count, C-reactive protein, immunoglobulin levels, C3, C4, C1 inhibitor and C1 inhibitor function were normal. Direct immunofluorescence studies revealed cytoid bodies positive for IgG and IgA but no immunoglobulin or complement deposits at the dermal blood vessels or the basal membrane zone. Treatment with cetirizine 10 mg twice daily7 was initiated. At follow-up visits he reported an improvement of the severity of DPU, although he continued to develop nonbullous DPU lesions for the subsequent year; he died 18 months later due to metastatic prostate carcinoma.

Materials and methods After written informed consent, a skin biopsy was obtained from the site of provoked DPU 24 h after pressure challenge. Before skin biopsy, the site was anaesthetized by circumferential injection of 1% scandicaine without vasoconstrictive additives. Haematoxylin and eosin staining was performed on paraffin-embedded sections. Immunofluorescence studies were conducted as recently described,29 using the following monoclonal mouse antibodies: anti-CD4, anti-CD8, anti-CD21 (all Serotec, Oxford, U.K.), anti-ECP (Pharmacia Diagnostics AB, Uppsala, Sweden) and anti-CD1a (Dako, Zug, Switzerland). IL-5 and IL-13 stainings were performed using polyclonal rabbit antibodies (Santa Cruz Biotechnology, Santa Cruz, CA,

A

U.S.A.). Appropriate control antibodies were used as negative controls. Antibody binding was detected with appropriate antimouse and antirabbit tetramethyl rhodamine isothiocyanate-conjugated and ⁄or fluorescein isothiocyanate-conjugated secondary antibodies (Milan Analytica AG, La Roche, Switzerland). Slides were independently analysed by two investigators using confocal laser scanning microscopy (LSM 510; Carl Zeiss, Jena, Germany).

Results Histological examination showed marked spongiosis and intraepidermal blisters filled with predominantly eosinophils accompanied by a dense dermal inflammatory infiltrate (Fig. 2A,B). This infiltrate consisted mainly of eosinophils and a few neutrophils as well as lymphocytes that focally reached the subcutaneous tissue (Fig. 2C,D). There was no vasculitis. Mast cells as investigated by chloroacetate esterase reaction as well as immunohistochemical studies for tryptase expression were normal in number and displayed no detectable degranulation as judged by morphology (data not shown). Further to characterize the cellular infiltrate we performed immunofluorescence studies using markers for eosinophils, Langerhans cells and T and B lymphocytes. ECP staining revealed the predominance of activated eosinophils within the lymphocytic infiltrate located in the upper dermis (Fig. 3A). In contrast, CD1a staining excluded increased numbers or altered localization of Langerhans cells in the epidermis and dermis (Fig. 3B). Both CD4+ (Fig. 3C) and CD8+ (data not shown) T lymphocytes were sparse, and the infiltrate contained only low numbers of B cells (data not shown). Within the infiltrate the relative frequency of T lymphocytes and B cells compared with eosinophils was approximately 1 : 3Æ2 for CD4+ T cells, 1 : 3Æ4 for CD8+ T cells and 1 : 10Æ4 for CD21+ B cells. A key cytokine in eosinophilic differentiation, activation, proliferation and survival is IL-5, which is mainly produced by activated T cells.30 Therefore, we next performed double immunofluorescence staining using a combination of anti-

B

Fig 1. Bullous delayed pressure urticaria. (A) Urticarial plaque on the shoulder 24 h after pressure challenge test. (B) Close-up showing normal skin on the left and pressure urticaria containing multiple blisters and vesicles on the right. Inset shows detail of blister.
2005 British Association of Dermatologists • British Journal of Dermatology 2005 153, pp435–439

Bullous delayed pressure urticaria, A. Kerstan et al. 437

A

B

C

D

Fig 2. Histology of bullous delayed pressure urticaria (DPU). (A) Histological examination of a skin biopsy specimen 24 h after elicitation of DPU on the shoulder. Spongiosis and intraepidermal bullae accompanied by a dense, eosinophil-rich inflammatory infiltrate in the upper and lower dermis. (B) Detail showing extensive infiltration of eosinophilic granulocytes within the bullae. (C,D) Eosinophilic and, to a lesser extent, neutrophilic granulocytes in the reticular dermis (C) that focally reach the subcutis (D). Haematoxylin and eosin; original magnification: (A) · 100; (B) · 200; (C,D) · 400.

A

B

C Fig 3. Immunofluorescence studies. (A) Immunofluorescence analysis of eosinophil cationic protein (ECP) demonstrating the predominance of eosinophilic granulocytes within the infiltrate (lower dermis). (B) Normal distribution of Langerhans cells identified by anti-CD1a antibody. (C) Double immunofluorescence using antibodies to CD4 (red) and interleukin (IL)-5 (green). In the upper dermis CD4+ T cells are present in low numbers and express IL-5. Original magnification: (A–C) · 400; scale bars ¼ 10 lm. Table 1 Quantification of skin-infiltrating T lymphocytes in bullous delayed pressure urticaria Cells mm)2 Upper dermis

Lower dermis ⁄ subcutis

CD4+ % IL-5 CD8+ % IL-5 CD4+ % IL-5 CD8+ % IL-5

positive positive positive positive

60 43Æ3 40 50 21 14Æ3 35 14Æ3

IL, interleukin.

CD4 ⁄anti-IL-5 antibodies or anti-CD8 ⁄anti-IL-5 antibodies, respectively. Interestingly, both CD4+ (Fig. 3C) and CD8+ (data not shown) T cells significantly expressed IL-5. Further quantification revealed that 43Æ3% of CD4+ and 50% of CD8+

T cells located in the upper dermis expressed IL-5, whereas 14Æ3% of both cell types produced this cytokine in the lower dermis (Table 1). It was recently shown that eosinophils are able to produce IL-13 during the course of inflammatory responses.31 However, in bullous DPU IL-13 expression by eosinophils within the dermal infiltrate was largely absent (data not shown).

Discussion We describe a patient with the rare bullous form of DPU and associated CU as well as delayed dermographism. So far only three patients with bullous DPU have been reported.26–28 Of these three patients one also had associated CU.26 The finding of delayed dermographism in our patient is not unexpected, because 55–73% of patients with DPU have this additional symptom.2,3 In contrast to other reported patients with bullous DPU,26,27 concomitant systemic symptoms such as dysphagia or fever were absent in our patient.


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438 Bullous delayed pressure urticaria, A. Kerstan et al.

DPU is unique among the physical urticarias in that the weals appear not until 4–8 h after pressure challenge and may persist for several days. The pathogenesis of DPU remains unclear. Given the rarity of bullous DPU, we considered our case to represent a maximal variant of DPU and set out to characterize the inflammatory infiltrate in more detail. Ryan et al. postulated that mast cells may play a role in DPU because their depletion rendered the tissue refractory to pressure weals.15 Following this line of evidence, increased numbers of mast cells were detected in DPU lesions more recently.18,19,32,33 However, because of the delayed onset, long duration and almost complete failure of H1 antihistamine therapy observed in patients with DPU, the pathogenic role of mast cells in DPU remains controversial.1 In lesional skin of our patient mast cell morphology was intact and mast cell numbers were not obviously increased 24 h after pressure. However, as prior degranulation with consecutive reduction of mast cell numbers cannot be excluded,18,19,32 the precise role of mast cells in bullous DPU remains to be determined, e.g. through sequential biopsy specimens. Histologically, an epidermal reaction pattern is not routinely observed in DPU. In the case reported by Shelley and Shelley,27 an intraepidermal bulla filled with eosinophils accompanied with an eosinophil-rich dermal infiltrate was noted predominantly in the upper dermis. In a case of bullous delayed dermographism, an intraepidermal blister combined with an inflammatory dermal infiltrate rich in eosinophils was observed.34 In line with these findings, we demonstrate a virtually identical histological pattern and hypothesize that blistering probably reflects the intensity of the inflammatory cascade in DPU rather than a distinct reaction pattern. Therefore, we performed immunofluorescent staining for ECP and detected extracellular deposition of ECP, suggesting activation and degranulation of eosinophils (cf. Fig. 3A). We therefore speculate that eosinophils may play a pivotal role in either promoting or maintaining the pathology of DPU. In addition, we hypothesize that activated eosinophils are a major effector cell in inducing spongiosis and subsequent epidermal blistering in bullous DPU. All effector functions of eosinophils require an increase in susceptibility to stimuli able to trigger degranulation and ⁄or inflammatory mediator synthesis. Such priming of eosinophils can be achieved by IL-5.35,36 Here, we show for the first time that CD4+ and CD8+ T cells express IL-5 in DPU, pointing to an additional role for T lymphocytes in bullous DPU. Future studies including serial biopsies are needed to determine the role of activated, cytokine-producing T cells in early stages of DPU. Treatment of DPU remains a major challenge.1 Our patient showed partial relief of his symptoms with cetirizine. In this respect, it remains to be determined if cetirizine interferes with eosinophilic effector function as proposed.7 Successful therapy with dapsone has been reported for DPU.8 This effect has been attributed to inhibition of neutrophil migration or eosinophil function.37 However, a specific treatment interfering with eosinophilic function has not been used in DPU so

far. Because of the pivotal role of IL-5 in eosinophilia such a ‘mechanism-based’ therapy has been reported to be effective in hypereosinophilic syndrome by the use of monoclonal antibodies to IL-5.29 Thus, influencing the activation and survival of eosinophils may be beneficial in severe therapy-resistant cases of DPU. In conclusion, we report a rare case of bullous DPU and present detailed histological and phenotypic characterization of the inflammatory infiltrate. We demonstrate a dense eosinophilic dermal and epidermal infiltrate and extend previous reports characterizing mast cells, eosinophils, T lymphocytes and B lymphocytes. Because of normal numbers and morphology of mast cells in lesional skin, our data appear to question a major role for mast cells in bullous DPU. However, as earlier (< 24 h) mast cell activity cannot be excluded, the precise role of mast cells has to be determined in future studies including control and serial biopsies. None the less, by demonstrating predominantly activated eosinophils in the infiltrate we suggest a major role for eosinophils in the pathogenesis of DPU as well as blister formation. IL-5-expressing T lymphocytes might point to a possible cross-talk between T cells and eosinophils through enhancing eosinophil activation and survival. Our data provide a more detailed view of the nature of the inflammatory infiltrate in DPU and suggest a potential effector role of eosinophils, that might lead to the development of future therapies interfering with eosinophilic effector function.

Acknowledgments We thank Dr Jo¨rn Elsner, Department of Dermatology, University of Hannover Medical School, Germany, for helpful suggestions. Part of this study was supported by a grant from the Deutsche Forschungsgemeinschaft (Le-953 ⁄4–1) to Martin Leverkus.

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