Mefloquine causes selective mast cell apoptosis in cutaneous mastocytosis lesions by a secretory granule-mediated pathway.

Mastocytosis is a KIT-related myeloproliferative disease characterised by abnormal expansion of neoplastic mast cells (MC) in the skin or virtually any other organ system. The cutaneous form of adult-onset mastocytosis is almost invariably combined with indolent systemic involvement for which curative therapy is yet not available. Here we evaluated a concept of depleting cutaneous MCs in mastocytosis lesions ex vivo by targeting their secretory granules. Skin biopsies from mastocytosis patients were incubated with or without mefloquine, an antimalarial drug known to penetrate into acidic organelles such as MC secretory granules. Mefloquine reduced the number of dermal MCs without affecting keratinocyte proliferation or epidermal gross morphology at drug concentrations up to 40 µM. Flow cytometric analysis of purified dermal MCs showed that mefloquine-induced cell death was mainly due to apoptosis and accompanied by caspase-3 activation. However, caspase inhibition provided only partial protection against mefloquine-induced cell death, indicating predominantly caspase-independent apoptosis. Further assessments revealed that mefloquine caused an elevation of granule pH and a corresponding decrease in cytosolic pH, suggesting drug-induced granule permeabilisation. Extensive damage to the MC secretory granules was confirmed by transmission electron microscopy analysis. Further, blockade of granule acidification or serine protease activity prior to mefloquine treatment protected MCs from apoptosis, indicating that granule acidity and granule-localised serine proteases play major roles in the execution of mefloquine-induced cell death. Altogether, these findings reveal that mefloquine induces selective apoptosis of MCs by targeting their secretory granules and suggest that the drug may potentially extend its range of medical applications.

A gene-centric approach to biomarker discovery identifies transglutaminase 1 as an epidermal autoantigen.

Autoantigen discovery is a critical challenge for the understanding and diagnosis of autoimmune diseases. While autoantibody markers in current clinical use have been identified through studies focused on individual disorders, we postulated that a reverse approach starting with a putative autoantigen to explore multiple disorders might hold promise. We here targeted the epidermal protein transglutaminase 1 (TGM1) as a member of a protein family prone to autoimmune attack. By screening sera from patients with various acquired skin disorders, we identified seropositive subjects with the blistering mucocutaneous disease paraneoplastic pemphigus. Validation in further subjects confirmed TGM1 autoantibodies as a 55% sensitive and 100% specific marker for paraneoplastic pemphigus. This gene-centric approach leverages the wealth of data available for human genes and may prove generally applicable for biomarker discovery in autoimmune diseases.

Autoantibodies Targeting a Collecting Duct-Specific Water Channel in Tubulointerstitial Nephritis.

Tubulointerstitial nephritis is a common cause of kidney failure and may have diverse etiologies. This form of nephritis is sometimes associated with autoimmune disease, but the role of autoimmune mechanisms in disease development is not well understood. Here, we present the cases of three patients with autoimmune polyendocrine syndrome type 1 who developed tubulointerstitial nephritis and ESRD in association with autoantibodies against kidney collecting duct cells. One of the patients developed autoantibodies targeting the collecting duct-specific water channel aquaporin 2, whereas autoantibodies of the two other patients reacted against the HOXB7 or NFAT5 transcription factors, which regulate the aquaporin 2 promoter. Our findings suggest that tubulointerstitial nephritis developed in these patients as a result of an autoimmune insult on the kidney collecting duct cells.

Ablation of human skin mast cells in situ by lysosomotropic agents.

Mast cells are known to have a detrimental impact on numerous types of inflammatory skin diseases such as contact dermatitis, atopic eczema and cutaneous mastocytosis. Regimens that dampen skin mast cell-mediated activities can thus offer an attractive therapeutic option under such circumstances. As mast cells are known to secrete a large array of potentially pathogenic compounds, both from preformed stores in secretory lysosomes (granules) and after de novo synthesis, mere inhibition of degranulation or interference with individual mast cell mediators may not be sufficient to provide an effective blockade of harmful mast cell activities. An alternative strategy may therefore be to locally reduce skin mast cell numbers. Here, we explored the possibility of using lysosomotropic agents for this purpose, appreciating the fact that mast cell granules contain bioactive compounds prone to trigger apoptosis if released into the cytosolic compartment. Based on this principle, we show that incubation of human skin punch biopsies with the lysosomotropic agents siramesine or Leu-Leu methyl ester preferably ablated the mast cell population, without causing any gross adverse effects on the skin morphology. Subsequent analysis revealed that mast cells treated with lysosomotropic agents predominantly underwent apoptotic rather than necrotic cell death. In summary, this study raises the possibility of using lysosomotropic agents as a novel approach to targeting deleterious mast cell populations in cutaneous mastocytosis and other skin disorders negatively influenced by mast cells.

Granzyme H is a novel protease expressed by human mast cells.

BACKGROUND: Many of the functions attributed to mast cells depend on the various pro-inflammatory mediators that are secreted upon mast cell activation. These include a panel of mast cell-specific proteases. In addition, recent studies have indicated that murine mast cells also express granzyme D, a protease previously thought to be confined to cytotoxic lymphocytes. Here, we address the human relevance of the latter findings by investigating whether human mast cells express granzyme H, the granzyme that may represent the functional counterpart to murine granzyme D. METHODS: Cord blood-derived mast cells, LAD2 cells and skin mast cells in situ were evaluated for their expression of granzymes using quantitative PCR, Western blot analysis and immunostaining. Mast cells were activated by either calcium ionophore stimulation or IgE receptor cross-linking. RESULTS: Cord blood-derived mast cells and LAD2 cells were shown to express granzyme H and B mRNA, while granzyme A, K and M expression was undetectable. Mast cell activation by either calcium ionophore or IgE receptor cross-linking caused down-regulated expression of granzyme H. In contrast, granzyme B expression was up-regulated by the same stimuli. Granzyme H expression was also confirmed at the protein level, as shown by both Western blot analysis and confocal microscopy. Further, we show that granzyme H is expressed by human skin mast cells in situ. CONCLUSIONS: The present findings implicate granzyme H as a novel protease expressed by human mast cells and support earlier findings obtained in natural killer cells suggesting that granzymes B and H are reciprocally regulated.

Patients with palmoplantar pustulosis have increased IL-17 and IL-22 levels both in the lesion and serum.

Recent findings about the pathogenesis of pustulosis palmaris et plantaris (PPP), also known as palmoplantar pustulosis, suggest that IL-17 expression in the acrosyringium as well as infiltration of IL-17 positive cells, e.g. Langerhans cells may play important roles. However, to date, it has not been established whether circulating IL-17 related cytokines are involved in PPP. We studied the circulating IL-17 related cytokines as well as the mRNA levels in lesional skin. IL-17 related cytokine mRNAs were increased in the PPP lesions compared with the control tissues (five patients vs five controls). The serum levels of TNF-alpha, IL-17, IL-22 and IFN-gamma also were significantly increased in PPP, but not IL-23 and IL-8 (48 patients vs 20 controls). Our findings document that not only the serum IL-17 but also tissue IL-17 are elevated in PPP and may be in the pathogenesis of this disorder.

Normal and PPP-affected palmoplantar sweat gland express neuroendocrine markers chromogranins and synaptophysin differently.

Earlier findings indicate the acrosyringium as the target for the inflammation in the chronic and intensely inflammatory skin disease palmoplantar pustulosis (PPP). The sweat gland apparatus seems to be an immune-competent structure that probably contributes to the defence of the skin. Furthermore, the sweat gland and duct may be a hitherto unrecognized neuroendocrine organ because it expresses cholineacetyl-transferase and acetylcholinesterase, nicotinic receptors, beta-adrenergic and angiotensin receptors. The aim of this study was to obtain further information about neuroendocrine properties of the sweat gland apparatus by examining the expression of common neuroendocrine markers synaptophysin and chromogranins A and B in healthy palmar skin and in PPP skin. Synaptophysin and chromogranins were expressed in the sweat glands and ducts with some variation in the pattern and intensity of the expression. In PPP skin the expression differed, being higher and lower, depending on the part of the sweat duct. Chromogranins were further expressed in the epidermis, endothelium and inflammatory cells, but its intensity was weaker in epidermis than in the sweat gland apparatus. In most cases, chromogranins in epidermis in involved PPP were weakly expressed compared to healthy controls. The presence of synaptophysin and chromogranins in palmoplantar skin may have marked neuroendocrine effects, and the palmoplantar skin is likely to have important neuroimmuno-endocrine properties. Moreover, the altered chromogranin expression in PPP skin might influence both the neuroendocrine and neuroimmunologic properties of palmoplantar skin in these patients. These results indicate important neuroendocrine properties of the palmoplantar skin.

Patients with psoriatic arthritis have an increased number of lymphocytes in the duodenal mucosa in comparison with patients with psoriasis vulgaris.

OBJECTIVE: To determine if there is evidence of inflammation in the duodenal mucosa in patients with psoriatic arthritis (PsA) and to compare the results with those in patients with psoriasis vulgaris (PsV). METHODS: Nineteen consecutive patients with PsA underwent gastroduodenoscopy, and biopsy specimens were taken from the duodenal and gastric mucosa. In addition to routine processing, the duodenal mucosal specimens were stained for CD3+, CD8+ and CD4+ T lymphocytes, tryptase-positive mast cells, and EG2-positive eosinophil granulocytes. The results were compared with those in duodenal mucosal specimens from patients with PsV and patients with irritable bowel syndrome. RESULTS: Compared with PsV patients (without antibodies against gliadin), patients with PsA had a highly significant increase in intraepithelial CD3+ and CD8+ lymphocytes and also in CD4+ lymphocytes in the lamina propria in the villi. The lymphocyte increase was not related to presence of IgA antibodies against gliadin, endomysium, or transglutaminase, or to concomitant gastritis. Patients with PsA and PsV showed a pronounced increase in mast cells and eosinophil granulocytes. CONCLUSION: The increased lymphocyte infiltration in the duodenal mucosa in PsA, but not in PsV, might indicate different pathogenetic mechanisms in these psoriasis variants.

Women with palmoplantar pustulosis have disturbed calcium homeostasis and a high prevalence of diabetes mellitus and psychiatric disorders: a case-control study.

Palmoplantar pustulosis is characterized by pustule formation in the acrosyringium. Nearly 50% of palmoplantar pustulosis sera produce immunofluorescence of the palmar papillary endothelium from healthy subjects, but also of the endothelium of normal parathyroid gland. With a case-control design the levels of calcium and parathyroid hormone in serum were measured in 60 women with palmoplantar pustulosis and 154 randomly selected population-based control women. One-third of the controls had been smokers, whereas 95% of the cases were or had been smokers. Mean age-adjusted serum calcium was increased in the patients compared with the controls (2.43 vs 2.36 mmol/l; p<0.0001), whereas the parathyroid hormone concentration was suppressed (23.2 vs 31.1 ng/l; p<0.0001). The plasma levels of parathyroid hormone-related protein were normal in patients but there was a strong expression of this protein in the acrosyringium both in palmoplantar pustulosis and control skin. As even a marginal elevation of serum calcium is associated with an increased risk for diabetes, cardiovascular disease and psychiatric disease, we analysed the risk for these disorders in palmoplantar pustulosis patients compared with that in the control group. Both diabetes mellitus and psychiatric disorders were associated with palmoplantar pustulosis with an odds ratio of 8.7 (95% CI 3.3-22.8) and 5.6 (95% CI 2.2-14.4), respectively. Palmoplantar pustulosis is a complex disease with an increased risk for several non-dermatological disorders. The role of the mildly increased serum calcium for the high risk for diabetes and depression deserves to be studied.

Sweat gland morphology and periglandular innervation in essential palmar hyperhidrosis before and after treatment with intradermal botulinum toxin.

BACKGROUND: Intradermal botulinum toxin (Btx) produces long-lasting relief of focal hyperhidrosis, but its mechanism of action is poorly understood. OBJECTIVE: To study the effect of Btx A on the size and innervation of sweat glands in patients with palmar hyperhidrosis. METHODS: Palmar skin biopsy was performed in 26 hyperhidrotic patients before scheduled Btx treatment and in 11 controls. Twelve of the patients also underwent biopsy 1 to 6 months after the Btx injections. Sweat gland morphology was investigated by light microscopy; the cross-sectional area of the secretory tubule and its lumen was measured by image analysis. Immunofluorescence (IF) with antibodies to the neural markers protein gene product 9.5 (PGP 9.5) and growth-associated protein 43 (GAP 43), and to vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP), was used to analyze the periglandular innervation. RESULTS: The gross morphology of the sweat glands was similar in patients and controls, with no significant differences in tubular and luminal areas between the groups. After Btx treatment, the tubular dimensions remained unchanged, but the lumen tended to be smaller ( P = .07). Around the glands, increased GAP 43 staining indicating sprouting was seen within 3 months after Btx treatment ( P = .016); whereas the PGP 9.5 staining was decreased in most specimens ( P = .09) indicating lack of functional nerve growth. No change in VIP or CGRP immunoreactivity was observed. CONCLUSIONS: The sweat glands appear structurally normal in hyperhidrotic patients before Btx therapy, whereas after therapy the luminal area of the gland is frequently diminished. The IF data GAP 43/PGP 9.5 suggest that Btx therapy induces long-standing functional denervation of the sweat glands, which might explain its anti-transpiratory efficacy.

Gluten-free diet in psoriasis patients with antibodies to gliadin results in decreased expression of tissue transglutaminase and fewer Ki67+ cells in the dermis.

Previous studies have shown that 16% of patients with psoriasis vulgaris have IgA and/or IgG antibodies to gliadin, but few have antibodies to endomysium. The increase in duodenal intraepithelial lymphocytes was mild. Still, highly significant clinical improvement was observed after 3 months on a gluten-free diet. This study surveys certain immunohistological aspects of involved and non-involved skin in 28 AGA-positive psoriasis patients before and after 3 months of a gluten-free diet. Staining was performed for CD4+ T lymphocytes, Langerhans' cells, endothelium, proliferating (Ki67) cells and tissue transglutaminase. In the entire group of patients, as well as in those on a gluten-free diet as the only treatment, Ki67 + cells in involved dermis were highly significantly decreased after the diet. There was a significant decrease in Ki67 + cells even in patients without increased intraepithelial lymphocytes. Tissue transglutaminase was highly overexpressed in involved skin in the papillary endothelium, and decreased by 50% after gluten-free diet. The possible role of tissue transglutaminase in the pathogenesis of psoriasis needs further investigation.

Genetic analysis of PSORS1 distinguishes guttate psoriasis and palmoplantar pustulosis.

The PSORS1 locus in the major histocompatibility complex region is the major genetic determinant for psoriasis vulgaris. Within the PSORS1 region reside at least three potential candidate genes for psoriasis susceptibility. Specific allelic variants of the genes HLA-Cw*6, HCR*WWCC, and CDSN*5 are strongly associated with psoriasis vulgaris and are in strong linkage disequilibrium with each other. We have genotyped the three psoriasis vulgaris susceptibility alleles of the PSORS1 locus in two clinical variants of psoriasis (guttate psoriasis and palmoplantar pustulosis) to study whether PSORS1 is also involved in the pathogenesis of these variants. We also asked whether these two clinical subgroups could help us to distinguish the causative gene within the high-risk PSORS1 haplotype. The association of guttate psoriasis with the three PSORS1 susceptibility alleles was similar and even stronger than seen with psoriasis vulgaris. Palmoplantar pustulosis, however, did not show association with any of the three candidate genes at this locus. Finally, no correlation with the age of onset for disease was observed. Our results show conclusively that psoriasis vulgaris and guttate psoriasis have a similar genetic basis for their association to PSORS1, whereas palmoplantar pustulosis appears to be a distinct disorder.

Palmoplantar pustulosis: an autoimmune disease precipitated by smoking?

Ninety-five percent of patients with palmoplantar pustulosis are smokers at onset of the disease. The aim of this study was to determine whether these patients have serum antibodies to nicotinic acetylcholine receptors (nAChR ab) and if their sera induce a specific immunofluorescence in normal palmar skin. Sera from 45 patients with palmoplantar pustulosis and 23 patients with chronic hand eczema were analysed for muscle nAChR ab, and immunofluorescence was performed on healthy palmar skin. Forty-two percent of the patients with palmoplantar pustulosis but none of the eczema patients had raised levels of nAChR ab. Immunofluorescence showed staining on endothelial cells in the papillary dermis in 47% of all sera from patients with palmoplantar pustulosis and in those with nAChR ab in 68%. On palmar skin from smokers there was also a staining of the sweat duct. Sera from patients with chronic hand eczema were negative. Our findings indicate that palmoplantar pustulosis is an autoimmune disease, possibly induced by smoking.