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Keywords:

  • vitamin D;
  • 1α-hydroxylase;
  • extrarenal metabolism;
  • granuloma;
  • slack skin;
  • macrophage;
  • epithelial cell;
  • hypercalcemia

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. REFERENCES

A case of granulomatous slack skin disease is presented in which we studied the possible involvement of extrarenal 1,25(OH)2D in the pathogenesis of the patient's hypercalcemia. Immunolocalization of 1α-OH in skin showed simultaneous dysregulation in epithelial and granulomatous cells.

Introduction: Granuloma-forming diseases such as sarcoidosis are associated with extrarenal synthesis of active 1,25-dihydroxyvitamin D [1,25(OH)2D]. Here we describe a case of granulomatous slack skin disease in which we have studied the possible involvement of extrarenal synthesis of 1,25(OH)2D in the pathogenesis of the patient's hypercalcemia. The aim of the study was to clarify the etiology of hypercalcemia in this patient.

Materials and Methods: This was a case study of a 19-year-old man with a T-cell lymphoproliferative disorder diagnosed as granulomatous slack skin disease who presented with hypercalcemia and raised serum 1,25(OH)2D. Analysis of expression of the enzyme 25-hydroxyvitamin D 1α-hydroxylase (1α-hydroxylase), which catalyzes synthesis of 1,25(OH)2D, was carried out by immunohistochemical analysis of involved and uninvolved skin. Approval was granted by the Mayo Foundation Institutional Review Board and Biospecimens Subcommittee.

Results: In uninvolved skin, expression of 1α-hydroxylase was confined to the basal layer of the epidermis, whereas slack skin showed overexpression of the enzyme in dermal granulomata and basal cells of the epidermis.

Conclusions: Hypercalcemia associated with granulomatous slack skin syndrome seems to be caused by dysregulation of 1α-hydroxylase expression in both epidermal and dermal granulomatous cells. This contrasts with psoriasis and sarcoidosis of the skin, in which overexpression of the enzyme is restricted to keratinocytes and granulomata, respectively.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. REFERENCES

The association between granulomatous disease and abnormal vitamin D metabolism was first observed in patients with sarcoidosis who developed hypercalcemia and/or hypercalciuria after ingesting small amounts of vitamin D or being exposed to UV light.(1,2) Further studies of hypercalcemia in an anephric patient with sarcoidosis indicated that this complication was not caused by aberrant endocrine vitamin D metabolism(3); synthesis of active 1,25-dihydroxyvitamin D [1,25(OH)2D] from its inactive precursor 25-hydroxyvitamin D [25(OH)D] through the enzyme 25-hydroxyvitamin D 1α-hydroxylase (1α-hydroxylase) occurs classically in the proximal tubules of the kidney.(4) This case from Barbour et al.(3) showed that it was possible to maintain normal circulating levels of 1,25(OH)2D in the absence of renal 1α-hydroxylase activity. The explanation for this was provided by data showing that activated pulmonary alveolar macrophages are capable of producing enough 1,25(OH)2D to influence circulating levels of the hormone in some patients with sarcoidosis.(5,6)

Subsequent studies have shown that hypercalcemia associated with dysregulation of vitamin D metabolism is not restricted to sarcoidosis but seems to be a feature of almost every type of granulomatous disease,(7) including tuberculosis(8) and Crohn's disease.(9) Likewise, although macrophages are a potent source of 1,25(OH)2D, expression of 1α-hydroxylase has now been described in many other extrarenal tissues,(10) notably the skin, from which the cDNA for human 1α-hydroxylase was first cloned.(11) Here we describe a case of 1,25(OH)2D-mediated hypercalcemia associated with granulomatous slack skin disease (GSSD). In addition to providing a further example of extrarenal 1,25(OH)2D production caused by granulomatous disease, this study also shows that dysregulation of 1α-hydroxylase expression can occur in both epithelial and granulomatous cells with the involved tissue.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. REFERENCES

A 19-year-old man presented with a 6-year history of an enlarging erythematous plaque on his abdomen and an enlarged inguinal lymph node (Fig. 1A). A biopsy of the skin lesion revealed a clonal T-cell lymphoproliferative granulomatous-like disorder, diagnosed as GSSD. One year prior, the patient had developed 1,25(OH)2D-mediated hypercalcemia for which he was treated with intravenous fluids, pamidronate, and corticosteroids. He had no history of nephrolithiasis or fractures. On this presentation, the patient was once again noted to have hypercalcemia (serum calcium, 14.2 mg/dl; reference range, 8.3–10.7 mg/dl), with an inappropriately elevated serum 1,25(OH)2D concentration relative to the level of hypercalcemia (50 pg/ml; reference range, 22–67 pg/ml). The subject's serum PTH-related peptide was undetectable, and his serum 25(OH)D level was well within the normal range (29 ng/ml; reference range, 8–38 ng/ml). He was again treated with intravenous fluids, pamidronate, and corticosteroids. The granulomatous skin lesion was treated with local proton and electron beam radiation. His hypercalcemia resolved once again after therapy. He remained well for several months until he developed a recurrent skin lesion and subsequent hypercalcemia of 10.3 mg/dl, with a frankly elevated serum 1,25(OH)2D level of 110 pg/ml. Repeat skin biopsies showed advancing granulomatous infiltration of abdominal skin, and the patient experienced periodic episodes of hypercalcemia (10.4–12.0 mg/dl) with concomitant levels of serum 1,25(OH)2D ranging from 69 to 70 pg/ml, with undetectable levels of serum intact PTH (iPTH). Two years after his initial presentation, he underwent an aggressive course of combined therapy with external beam radiation and high-dose oral glucocorticoids. This treatment was efficacious in normalizing his 1,25(OH)2D from a baseline level of 79 to 32 pg/ml. Concurrently, serum calcium declined from a pretreatment level of 11.6 to 10.0 mg/dl, whereas serum iPTH rose from <3 to 4.1 pg/ml.

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Figure Figure 1. Expression of 1α-hydroxylase in skin from a patient with GSSD. (A) Gross anatomy of GSSD lesion at presentation. (B) Immunolocalization of 1α-hydroxylase (brown staining) throughout epidermis in GSSD biopsy (×40). (C) Immunolocalization of 1α-hydroxylase (brown staining) in GSSD granulomata relative to normal skin biopsy and IgG immunohistochemistry controls (×200).

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After approval from the Mayo Foundation Institutional Review Board and Biospecimens Subcommittee, deparaffinized and hydrated paraffin slides were antigen retrieved with 0.01 M sodium citrate in a microwave for 5 minutes. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 10 minutes. Slides were incubated overnight with sheep anti-1α-hydroxylase antibody,(10) followed by a 30-minute incubation with the appropriate biotin-conjugated secondary antibody and a 30-minute exposure to Vectastain avidin-biotin-perioxidase complex (ABC) reagent (Vector Laboratories, Burlingame, CA, USA). The slides were incubated with diaminobenzidine tetrahydrochloride (DAB) developing solution. Sections of skin from patients with sarcoidosis and psoriasis were from archival tissue blocks and were analyzed with Institutional Review Board approval from the University of Birmingham Hospitals Trust, UK (RRK2608). These samples were studied using the same methods as the patient with GSSD but with a different ABC reagent (Binding Site, Birmingham, UK), resulting in a slightly darker brown staining for these samples. Normal sheep IgG was used as negative control antiserum.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. REFERENCES

GSSD is a rare, lymphoproliferative skin disorder, with ∼42 cases reported in the literature to date. As suggested by its name, the disease is characterized by indurated plaques that progress to drooping masses of skin (Fig. 1A). Histological findings typically include lymphocytic infiltration of the papillary dermis along with multinucleated giant cells engulfing phagocytosed elastic fibers. Hypercalcemia and nephrolithiasis have been previously reported with this disease, yet no clear etiology has been described.(12) Here we report a case of GSSD associated with recurrent hypercalcemia and dysregulated overactivation of vitamin D resulting in inappropriately elevated (i.e., in the face of hypercalcemia) circulating concentrations of 1,25(OH)2D. To elucidate the source of the extrarenal 1,25(OH)2D, immunolocalization of the vitamin D–activating enzyme 1α-hydroxylase was performed using skin biopsies of involved and uninvolved (control) skin. Whereas enzyme expression in the uninvolved skin was confined to the basal layer of the epidermis, dermal granulomata and the basal cells of the epidermis both showed increased expression of 1α-hydroxylase in the affected tissue (Figs. 1B and 1C). Overexpression of 1α-hydroxylase activity seems to be common to all granuloma-forming diseases, but the extent to which this results in associated hypercalcemia is less clear.(7) There are several factors that may contribute to this, including the degree of disease involvement, the availability of substrate for extrarenal 1α-hydroxylase [namely circulating 25(OH)D], and the capacity for catabolism of 1,25(OH)2D to biologically inactive metabolites. The functional significance of extrarenal 1α-hydroxylase is also likely to be highly dependent on the pathway involved in its activation. For example, we have shown recently that induction of 1α-hydroxylase in macrophages by a toll-like receptor 2 (TLR2) agonist results in concomitant induction of the vitamin D receptor (VDR), enabling sensitive autocrine response to 1,25(OH)2D.(13) Inflammatory cytokines such as interferon γ also stimulate upregulation of macrophage 1α-hydroxylase(5) but, in contrast to TLR2, this occurs in the presence of cytokine-induced VDR insensitivity.(14,15) Because cytokines such as interferon γ are an essential component of granulomatous diseases, this may help to explain the lack of 1,25(OH)2D responsiveness in the face of enhanced synthesis of the hormone that is characteristic of these diseases.

Another factor that seems to be important in defining the hypercalcemic potential of extrarenal 1,25(OH)2D production is the cellular localization of dysregulated 1α-hydroxylase. For example, we have shown previously that increased expression of 1α-hydroxylase in macrophages associated with B-cell lymphomas results in high circulating levels of 1,25(OH)2D similar to those seen in some patients with sarcoidosis.(16) In contrast, diseases in which there is upregulation of 1α-hydroxylase in both leukocytes and epithelial cells, such as Crohn's disease,(9) dysgerminomas,(17) and breast cancer,(18) seem to be characterized by more modest increments in the serum 1,25(OH)2D concentration. To study this further with respect to the current case, we compared the pattern of 1α-hydroxylase expression in the slack skin disease patient with skin from patients with psoriasis or sarcoidosis (Fig. 2).

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Figure Figure 2. Expression of 1α-hydroxylase in sarcoidosis and psoriasis. Arrows show areas of increased 1α-hydroxylase expression (brown staining) in stratum basale of normal skin; dermal granulomata in sarcoidosis; only epidermis in psoriatic skin (all images: magnification, ×40).

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Immunohistochemical analyses showed that 1α-hydroxy- lase was upregulated in each disease compared with normal epidermal tissue but with clearly distinct patterns of expression. Unlike normal skin in which 1α-hydroxylase is expressed predominantly in the basal layer of the epidermis, psoriatic skin shows enhanced expression of the enzyme throughout the epidermis without any significant immune cell involvement. In contrast, in patients with sarcoidosis of the skin, dysregulation of 1α-hydroxylase occurs only in the immune cell population of the dermis, which is a fundamental feature of these disorders. The fact that hypercalcemia is a key complication of the latter but does not seem to be associated with psoriasis suggests that immune cell expression of 1α-hydroxylase in skin is required for significant elevation of serum 1,25(OH)2D. One possible explanation for this is that macrophages, unlike most epithelial cells, do not exhibit significant levels of activity of the enzyme 24-hydroxylase, which catalyzes conversion of 1,25(OH)2D to less active metabolites. This may be caused in part by interferon γ–induced impairment of VDR-mediated 24-hydroxylase transcription as described above.(14,15) However, we recently described expression of a 24-hydroxylase splice variant peptide in macrophages that lacks metabolic function and may therefore cause perturbation of vitamin D metabolism in these cells.(19) Thus, diseases such as sarcoidosis may be characterized by accumulation of 1,25(OH)2D through decreased catabolism of this metabolite, whereas upregulation of 1α-hydroxylase in psoriasis results in only limited overproduction of 1,25(OH)2D caused by expression of 24-hydroxylase. In the context of the current case of slack skin disease, it seems that dysregulation of 1α-hydroxylase occurs simultaneously in both the immune cell and keratinocyte populations in the skin (Fig. 1B).

Analysis of dysregulated extrarenal 1α-hydroxylase expression in this case of GSSD underlines the capacity for extrarenal expression of 1α-hydroxylase in granuloma-forming diseases and provides an explanation for this particular patient's hypercalcemia. The case study also shows that overexpression of the enzyme need not be restricted to macrophages. Indeed recent data from our group(17,18) suggest that interaction between epithelial and immune cells is likely to play an important role in defining the efficacy of 1,25(OH)2D production and action in peripheral tissues. We postulated that, in tissues such as the skin, lungs, and gastrointestinal tract, expression of 1α-hydroxylase supports a broader role for vitamin D metabolism in maintaining “barrier integrity” through mechanisms that involve both epithelial and immune cells.(20) This case of GSSD may represent the first example of combined dysregulation of these two facets of vitamin D and barrier function.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. REFERENCES
  • 1
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