Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1005
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Pamoplantar Pustulosis.
Pathogenetic Studies with Special Reference to the Role of Nicotine
BY
EVA HAGFORSEN
ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001
Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Dermatology and Venereology presented at Uppsala University in 2001 ABSTRACT
Hagforsen, E. 2001.Palmoplantar pustulosis. Pathogenetic studies with special reference to the role of nicotine. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1005. 56 pp.
Uppsala. ISBN 91-554-4955-7.
Palmoplantar pustulosis (PPP) is a chronic disease of unknown pathogenesis. Most of the patients were smokers. High prevalence of a number of autoimmune diseases was observed among the patients (thyroid disease 14%, gluten intolerance 8%, diabetes type 1 3%).
Eosinophils and neutrophils were found in large numbers in the pustules. Massive infiltrates of lymphocytes and mast cells in the dermis below the pustule and an abnormal acrosyringial pattern indicate that the acrosyringium is the target for the inflammation. Immunofluorescence (IF) revealed decreased innervation of the sweat gland, outward migration of substance P-positive granulocytes in the acrosyringium and an increased number of contacts between mast cells and nerve fibres in the dermis.
Distributions of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied, since they regulate the level of acetylcholine, the main inducer of sweating. The most intense AChE-like immunoreactivity (LI) was observed in the acrosyringium in the lowest part of the stratum corneum, corresponding to the site of the pustule in PPP. ChAT-LI in granulocytes and AChE-LI in mast cells were
demonstrated, which may have implications for inflammatory processes in general.
Nicotinic acetylcholine receptors (nAChR) are activated by acetylcholine but also by nicotine. Immunohistochemstry of α-3 and α-7 subtypes of the nAChRs showed that the nAChR expression in healthy skin was influenced by smoking. A highly abnormal α-7 nAChR distribution in PPP skin was observed.
The levels of nAChR antibodies were elevated in 42% of the PPP sera, and 68% of these sera gave specific endothelial IF in the papillary dermis in skin from non- smokers. Positive IF in the acrosyringium was also noted in skin from smokers.
Conclusions: Smoking seems to induce up-regulation of an antigen in palmar skin.
The results indicate that PPP is an autoimmune disease and that nicotine might have a role in the onset of the inflammation.
Key words: Palmoplantar pustulosis, smoking, sweat gland apparatus, neuropeptides, non-neuronal cholinergic system, nicotinic receptor antibodies, autoimmune disease.
Eva Hagforsen, Section of Dermatology and Venereology, Department of Medical Sciences, Uppsala University, University Hospital, SE-751 85 Uppsala, Sweden
Eva Hagforsen 2001 ISSN 0282-7476 ISBN 91-554-4955-7
Printed in Sweden by Fyris-Tryck AB, Uppsala 2001
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CONTENTS
ABBREVIATIONS……….. 7
PAPERS INCLUDED……….. 8
INTRODUCTION……….. 9
Palmoplantar pustulosis………. 9
Inflammation……… 10
Autoimmunity ………. 12
Normal histology of palmar and plantar skin………... 12
The eccrine sweat gland apparatus……… 13
Normal histology and function……… 13
Inflammation and the acrosyringium………. 14
Innervation………..……… 14
Interaction between nervous and immune systems……….. 15
The neuronal cholinergic system……… 15
The non-neuronal cholinergic system……… 17
General………... 17
The non-neuronal cholinergic system in the skin……… 18
Effects of acetylcholine on cells……….. 19
Nicotinic influence - on the neuronal cholinergic system………. 20
- on the non-neuronal cholinergic system………. 20
- on keratinocytes……… 21
- on endothelial cells………... 21
AIMS OF THE STUDY………. 22
PATIENTS AND METHODS………... 23
Patients………. 23
Paper I, II, III and IV……….. 23
Anamnestic data……….. 23
Clinical examination……… 23
Blood samples……….. 23
Biopsies……… 23
Paper V……… 23
Reference persons……… 24
Immunohistochemistry……… 24
Peroxidase and alkaline phosphatase methods……….. 24
Immunofluorescence………... 26
Serum immunofluorescence……… 27
Western blot (paper III)……… 28
Radioimmunoassay (paper V)………. 28
Statistics……… 29
RESULTS AND DISCUSSION………. 30
Paper I………... 30
Anamnestic data………. 30
Clinical findings……….. 32
Inflammatory cells……….. 32
The sweat gland and duct………... 33
Paper II………. 34
PGP 9.5, substance P and calcitonin gene-related peptide……… 34
Nerve fibres and their contacts with mast cells……….. 35
Neuropeptide immunoreactivity in granulocytes……… 36
Paper III……… 37
ChAT and AChE in the epidermis and sweat gland apparatus……….. 37
ChAT and AChE in inflammatory cells………... 39
Paper IV……… 40
Nicotinic receptors in the epidermis and sweat gland apparatus………….. 40
Nicotinic receptors in inflammatory cells……… 42
Paper V……….. 43
Serum antibodies to nicotinic receptors and the immunofluorescence pattern 43 CONCLUSIONS………. 45
ACKNOWLEDGEMENTS……… 46
REFERENCES……… 48
ABBREVIATIONS
ab antibody
ABC avidin-biotin complex
ACh acetylcholine
AChE acetylcholinesterase
APAAP alkaline phosphatase-anti-alkaline phosphatase
C5a complement factor 5a
CGRP calcitonin gene-related peptide
ChAT choline acetyltransferase
ECP eosinophil cationic protein
EPO eosinophil peroxidase
EPX/EDN eosinophil protein X / eosinophil derived neurotoxin FcεRI high affinity receptor for immunoglobulin E
FITC fluorescein isothiocyanate
HLA human leukocyte antigen
IF immunofluorescence
Ig immunoglobulin
IL interleukin
LI like immunoreactivity
mAChR muscarinic acetylcholine receptor
MC mast cells
MHC major histocompatibility complex
MNL mononuclear leucocyte
mRNA messenger ribonucleic acid
nAChR nicotinic acetylcholine receptor
PAP peroxidase anti-peroxidase
PBS phosphate buffered saline
PGP 9.5 protein gene product 9.5
PPP palmoplantar pustulosis
SP substance P
TRITC tetramethyl-rhodamine-isothiocyanate VIP vasoactive intestinal peptide
PAPERS INCLUDED
I. Eriksson MO, Hagforsen E, Pihl-Lundin I, Michaëlsson G: Palmoplantar pustulosis-a clinical and immunohistochemical study. Br J Dermatol. 1998;
138: 390-398.
II. Hagforsen E, Nordlind K, Michaëlsson G: Skin nerve fibres and their contacts with mast cells in patients with palmoplantar pustulosis. Arch Dermatol Res.
2000; 292: 269-74.
III. Hagforsen E, Aronsson F, Einarsson A, Nordlind K, Michaëlsson G: The distribution of choline acetyltransferase- and acetylcholinesterase-like immunoreactivity in palmar skin from patients with palmoplantar pustulosis.
Br J Dermatol. 2000; 142: 234-42.
IV. Hagforsen E, Edvinsson M, Nordlind K, Michaëlsson G: Expression of α-3 and α-7 subunits of nicotinic acetylcholine receptors in the skin of patients with palmoplantar pustulosis. (submitted)
V. Hagforsen E, Mustafa A, Lefvert AK, Nordlind K, Michaëlsson G:
Antibodies against nicotinic receptors in serum from patients with palmoplantar pustulosis. (submitted)
Reprints were made with permission from the publishers.
Cover photograph: Immunofluorescence pattern with PPP sera on normal palmar skin from a non- smoking control. Note the pattern in the papillary dermis. A photograph of a palm from a PPP patient is inserted.
INTRODUCTION Palmoplantar pustulosis
Palmoplantar pustulosis (PPP) is a chronic skin disease with an unknown
pathogenesis. It may be a localised form of pustular psoriasis, and occasionally the patients have psoriasis-like lesions, particularly on the forearms and legs, but the relationship is controversial. PPP is characterised by sterile intra-epidermal pustules and usually also erythematous, scaly skin on the palms and soles. It is more common in women than in men and is also more common in smokers than in non-smokers (Eriksson et al 1998). The age at onset is usually between 20 and 60 years, 40-60 being most common.
Associations between PPP and autoimmune diseases such as autoimmune thyroid disease have been reported (Rosén 1988). However, in that study no improvement of the palms and soles occurred when the thyroid disease was treated. A diabetic pattern has been found in 22% of Japanese PPP patients at oral glucose tests (Uehara 1983) but the clinical relevance of these tests is difficult to evaluate, since abnormal glucose tolerance tests are not uncommon in middle-aged and elderly persons. Furthermore, symptoms resembling rheumatoid arthritis have been noted in 13% of PPP patients, which may be compared with a figure of 2.7% in the general population (Enfors and Molin 1971).
It is known that the pustules in PPP contain neutrophil granulocytes, but why these cells are so abundant here is still unknown. There is a report, however, of intercellular expression of interleukin-8 (IL-8) in the epidermis (Anttila et al 1992) of PPP skin and IL-8 is a chemoattractant for neutrophils (Baggiolini et al 1989). Anttila et al also observed strong IL-8 immunoreactivity in the whole eccrine sweat gland apparatus in palmar skin from both PPP patients and control subjects. IL-8 has also been found in sweat, and both this protein and its mRNA have been detected in sweat gland
epithelium in abdominal skin (Jones et al 1995), indicating that IL-8 is produced in situ.
There is no curative treatment for PPP today. Usually the patients with mild
symptoms are treated with emollients. Topical steroids are used in patients with more severe forms and the most severe cases are treated with systemic drugs (retinoids, cyclosporin). All these drugs are potent anti-inflammatory agents, which are virtually independent of the cause of inflammation.
Inflammation
There are two fundamentally different types of defence against infection and tissue damage, namely the innate response and the adaptive response.
Neutrophils are called “the first line of defence”, since within minutes of tissue damage or pathological invasion they adhere to the endothelium of vessel walls and migrate into the involved tissue. These cells form part of the innate immune response.
Other cells involved in the innate response are monocytes and macrophages
(phagocytic cells), basophils, mast cells and eosinophils (which release inflammatory mediators), and natural killer cells. Neutrophils and macrophages have receptors for antibodies and complement, a fact which enhances phagocytosis of microorganisms coated with immunoglobulin and/or complement. Phagocytes also remove the body’s own dead or dying cells. The most important chemotactic factors for neutrophils are C5a (derived from complement), bacterial products (such as N-formyl-methionyl- leucyl-phenylalanine), leucotriene B4 (product of arachidonic acid metabolism) and IL-8.
Eosinophils are mainly tissue cells, and are most abundant in the gastrointestinal tract, skin and lungs. Eosinophils are involved in processes in, for example, allergy and parasitic infections. They contain different granular structures, of which specific granules are most numerous. The specific granules, in turn, contain eosinophil
cationic protein (ECP), eosinophil protein X (EPX) (also known as eosinophil derived neurotoxin (EDN)) and eosinophil peroxidase (EPO). Activated eosinophils probably kill parasites mainly by releasing ECP, instead of by phagocytosis. Eosinophils may also induce neurotoxic effects by secreting EPX, and EPO has antibacterial
properties. In addition, eosinophils secrete prostaglandins, leukotrienes and various cytokines. Lipids, complement components, cytokines and chemokines are known eosinophil chemoattractants. Eotaxin is an example of a more recently discovered eosinophil chemoattractant (Garcia-Zepeda et al 1996). Increased amounts of ECP in the serum or in tissues have been shown to reflect increased turnover and/or increased tissue activity of the eosinophils.
Basophils and mast cells (MC) are important in atopic allergies. Allergen binding to IgE bound to high-affinity IgE receptors (FcεRI) cross-links the FcεRI, leading to secretion of inflammatory mediators such as histamine, prostaglandins and
leukotrienes. Mast cells migrate into tissues, where they mature. They seem to be
localised in organs that are potential ports of entry of foreign agents, such as the skin, lungs and gut. Mast cells have been found in tissues of patients with allergic diseases, but these cells are also linked to chronic inflammatory disorders with a hitherto unknown pathogenesis, for example psoriasis. They are divided into subsets on the basis of their content of neutral serine proteases (Irani et al 1986). One subset, MCTC, contains tryptase, chymase, cathepsin G and carboxypeptidase, whereas the other subset, MCT, contains only tryptase. MCTC is found predominantly in the skin and in the bronchial, nasal and intestinal mucosa, whereas MCT is localised mainly in mucosal surfaces (Irani et al 1986; Irani et al 1989).
B and T lymphocytes (helper and cytotoxic) are involved in the adaptive (cell- mediated) response. When T cells develop (in the thymus), there is a positive selection in which cells that are able to interact usefully with peptides presented by major histocompatibility complex (MHC) molecules survive, and a negative selection in which cells reactive to self-proteins undergo apoptosis. Only 1% of the immature precursor cells develop to immunocompetent T cells and are passed into the
circulation. The antigen-presenting cell is another important cell, which displays the antigen to the T-cell receptor on the surface of helper T lymphocytes. The antigen is presented by MHC class II molecules on the surface of dendritic cells. There are three main kinds of class II molecules, HLA-DR, -DP and -DQ. When the receptors of the T cells bind to antigens, the antigen-specific T cells proliferate (clonal selection).
Cytotoxic T cells have an antigen-specific T cell receptor, which recognises antigens bound to MHC class I molecules (HLA-A, -B or -C), which are expressed on virtually all cells. Self-cells that have been altered or infected are recognised and destroyed by cytotoxic T cells. Mature B cells (plasma cells) produce antibodies against specific antigens presented to them by helper T cells. Five different classes of antibodies (immunoglobulins) are produced. One of them, immunoglobulin A (IgA), has an important role in the first defence of the mucosal surfaces.
Cytokines, small soluble proteins, play a central part in the communication between cells in the immune system and are also important for growth and differentiation of haematopoietic, epithelial and mesenchymal cells. They regulate cell function in pico- to nano-molar concentrations through specific receptors. There are pro-inflammatory cytokines, e.g. IL-1, tumour necrosis factor α, IL-6 and granulocyte-macrophage colony stimulating factor, inflammatory cytokines, e.g. IL-2, IL-4 and interferon-
gamma (IFNγ) and cytokines that are anti-inflammatory, e.g. IL-10, transforming growth factor β and IL-1 receptor antagonist. All these cytokines are involved to varying degrees in different types of skin inflammation, including autoimmune reactions, and are thus believed to have a role in the pathogenesis of psoriasis and related skin diseases.
Autoimmunity
A disease is defined as autoimmune if the tissue damage is shown to be caused by an immune response to self-antigens. Autoimmune disease can be triggered by
autoreactive T cells or by autoantibodies. The tissue may be damaged as a result of a direct attack on the cells bearing the antigen, of immune complex formation or of local inflammation. Another type of autoimmunity occurs when autoantibodies bind directly to cellular receptors, causing either excess activity (e.g. Graves’ disease) or inhibition of receptor function (e.g. myasthenia gravis).
Autoimmunity can be triggered by a variety of mechanisms, in most of which infectious agents are involved. As a result of a tissue injury, antigens that are not normally present in the circulation may be exposed to the immune response and not recognised as a self-antigen. Infectious agents may induce either T- or B-cell
responses that can cross-react with self-antigens (molecular mimicry). Superantigens produced by bacteria or viruses bind directly to the MHC class II molecule and induce polyclonal T-cell activation leading to an autoimmune process.
Many human autoimmune diseases show HLA-linked associations, as may be
expected, since the ability of T cells to respond to a particular antigen depends on the MHC type. It also seems as if sex hormones are involved in the pathogenesis of autoimmune diseases. For example, systemic lupus erythematosus is more common in women.
Normal histology of palmar and plantar skin
It is not known why PPP is localised to the palms and soles. However, the skin in these locations differs from that in other parts of the body. Glabrous (non-hairy) skin is characterised by a thick epidermis divided into several well-marked layers. There are no hair follicles in glabrous skin, nor are there any sebaceous glands. In the dermis of glabrous skin there are encapsulated sense organs, whereas the sensory
nerve endings in hairy skin are sometimes free, or terminate in hair follicles, and others have expanded tips. The density of the sweat glands on the palms and soles is 600 to 700/cm2, compared to only 64 glands/mm2 on the back. Furthermore, the stratum corneum is much thicker on the palms and soles, as a result of which the outermost part of the sweat duct (the acrosyringium) has a well-developed coil structure there, which is not so apparent in other sites.
The eccrine sweat gland apparatus Normal histology and function
The eccrine sweat gland apparatus consists of a secretory coil and a duct (Fig.1). The coiled part is made up of the secretory coil and the proximal duct. The distal duct is straight and connects the coil with the epidermis. In the epidermis and the stratum corneum the duct forms a spiral (the acrosyringium) leading up onto the skin surface.
There are two main types of sweating: thermoregulatory and emotional (mental) sweating. Thermoregulatory sweating occurs especially on the upper part of the trunk and the face, but also on the palms and soles. Emotional sweating is provoked by anxiety or pain and is characteristically associated with the palms, but its underlying mechanisms are not known. The nerves surrounding the sweat glands are sympathetic post-ganglionic fibres, which consist of non-myelinated class C nerve fibres, but
acetylcholine (ACh) is the principal neurotransmitter, acting via muscarinic receptors.
However, adrenaline may also induce palmar eccrine sweating (Wolf and Maibach 1974). Nicotine has been found capable of inducing axon reflex sweating by iontophoresis (on the foot and leg) and produced a stained area (iodine – starch method) similar to that after ACh iontophoresis (Riedl et al 1998).
Inflammation and the acrosyringium
The acrosyringial epithelium possesses specialised keratinocytes, which are immune- competent. Accordingly, expression of MHC class II HLA-DR, which play a critical role in cell-mediated immune responses, has been observed on acrosyringial
epithelium in normal human skin (Murphy et al 1983). In a study of the antigenic profile of the acrosyringium in normal skin (from the abdomen), McGregor el al (1991) found expression of HLA-DR, -DP and -DQ on the acrosyringial epithelium, and on the keratinocytes surrounding the acrosyringium they observed CD 36 (monocyte/platelet-specific molecule). Furthermore, expression of CD 68 (monocyte/macrophage-specific molecule) was detected on the acrosyringial epithelium, but not on dermal ducts or sweat glands. Immunohistochemically,
Reitamo et al (1990) demonstrated IL-1 throughout the eccrine sweat gland apparatus.
The distal part of the acrosyringial epithelium showed intense staining. Didierjean et al (1990) reported the presence of IL-1β in sweat from both truncal and palmar- plantar regions, whereas IL-1α was detectable only in sweat from palms and soles, indicating a site-dependent difference in the secretion of the two IL-1 molecules.
Furthermore, the IL-1 concentrations were much higher in the sweat during jogging and sauna bathing than during spontaneous sweating, which they suggested could be due to a stress-induced increase in the production of IL-1 by sweat gland cells. IgA, which forms a defence barrier against microbial antigens on mucosal surfaces, has been detected in sweat secreted onto the skin surface (Imayama et al 1995), indicating involvement of this immunoglobulin in the local immune defence of the skin.
Innervation
Most nerve fibres in the skin are sensory, and most of them are unmyelinated C and myelinated Aδ fibres that end as free nerve endings. Sensory nerve fibres express several neuropeptides, which are biologically active polypeptides, and most
neuropeptide-containing fibres are located around blood vessels, sweat glands and hair follicles or are present as free nerve endings. Neuropeptides can induce
neurogenic inflammation. Substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP), among other peptides, have been
demonstrated in nerve fibres in human skin (Wallengren et al 1987). Substance P- containing fibres are most densly located in the palms, soles and axillary skin (Eedy 1993).
Human sweat glands are entangled with nerve fibres. Along the sweat duct, from the gland to the surface of the skin, one or two nerve fibres are oriented. The nerve fibres
around the sweat gland apparatus are reported to express CGRP, VIP and sparsely SP (Kennedy et al 1994).
Interaction between nervous and immune systems
There have been many reports indicating that the nervous system interacts with the cutaneous immune system to mediate local inflammation. For instance neuropeptides might be involved in skin diseases such as psoriasis [CGRP (Artemi et al 1997), SP (Al'Abadie et al 1995); (Naukkarinen et al 1996), VIP (Anand et al 1991)], atopic dermatitis [CGRP (Pincelli et al 1990), VIP (Ostlere et al 1995; Pincelli et al 1991)]
and eczema [VIP (Anand et al 1991)].
Some neuropeptides are able to degranulate mast cells (for example SP and VIP;
(Ebertz et al 1987; Lowman et al 1988) and also to induce vasodilatation and may stimulate chemotactic and phagocytic activity of neutrophils and stimulate IgA production by B cells [reviewed in Ansel et al (1996)].
It has been shown that neutrophil granulocytes contain VIP (O´Dorisio et al 1980), that mast cells in patients with atopic dermatitis contain SP (Toyoda et al 2000) and that activated T cells also contain SP (De Giorgio et al 1998).
The neuronal cholinergic system
Acetylcholine is the neurotransmitter of the cholinergic system. The synthesis of ACh from coenzyme A and choline is catalysed by choline acetyltransferase (ChAT).
Acetylcholinesterase (AChE) is the cholinergic enzyme that hydrolyses ACh to acetate and choline.
Acetylcholine acts on cells via two different classes of receptors, nicotinic (nAChR) and muscarinic acetylcholine receptors (mAChR). Receptors of both classes are found in the central nervous system.
Nicotinic receptors are also present in autonomic ganglia and at neuromuscular junctions, while muscarinic receptors are found on autonomic effector cells
innervated by post-ganglionic parasympathetic nerves, and in blood vessels, where they modulate vasoconstriction and dilatation (Furchgott and Zawadzki 1980).
The nicotinic AChRs are ligand-gated ion channels that mediate influx of Na+ and Ca2+ and efflux of K+ and are formed by various combinations of transmembrane α, β, γ, δ and ε glycoprotein subunits. Each nAChR consists of five such subunits, different combinations of which determine the functional and pharmacological characteristics of the receptor (Conti-Tronconi et al 1994). The α1, β1, γ, δ and ε subunits have been found at the neuromuscular junction. Neuronal nAChR consists of combinations of the α-2 to -9 and β-2 to -5 subunits. Nicotinic AChRs formed by the α-4 and β-2 subunits are the major subtype in the brain (Whiting et al 1991). The α-3 subunit generally forms nAChRs together with the α-5, β-2, and β-4 subunits (Conti- Tronconi et al 1994). The α-7, -8 and -9 subunits can form functional nicotinic receptor channels of their own. The ACh-binding sites are believed to reside
primarily on the α subunits (Papke 1993). However, α-5 subunits, which are closely related to β-3 subunits, are believed not to be capable of forming an ACh-binding site (Wang et al 1996). Alpha-7 nAChRs, which have five α subunits, thus have five putative binding sites for ACh (Fig. 2). It has also been demonstrated that α-7 nAChRs are highly permeable to Ca2+ (Bertrand et al 1993).
Fig. 2. Subunit arrangements of two nAChR types around the central cation channel. To the left, the subtype with α3 as binding site with two putative ACh-binding sites; to the right, α subtypes (e.g. α7, α8 or α9) forming functional homomers with five putative ACh-binding sites.
The muscarinic AChRs are glycoproteins with seven α-helical transmembrane segments, and they are coupled to G proteins (Hulme 1990; Hulme et al 1990). There are five known subtypes, m1 to m5. The muscarinic receptor subtypes m1, m3 and m5 mediate, upon stimulation, an activation of phospholipase C activity, resulting in an increase in the Ca2+ concentration; m2 and m4 mediate, upon stimulation,
inhibition of adenyl cyclase activity, resulting in decreased cyclic AMP formation (Hulme 1990; Hulme et al 1990).
Neuronal nAChRs may be involved in neuronal diseases. It has been suggested, for example, that α-7 nAChRs may be associated with some aspects of schizophrenia.
Freedman et al (1995) found a decrease in α-7 nAChR in the hippocampus of brains from schizophrenia patients, and a decreased level of the α-7 nAChR subunit protein has also been observed in the frontal cortex of schizophrenic brain (Guan et al 1999).
A high proportion of schizophrenic patients are intensive tobacco users (Lohr and Flynn 1992), and it has been proposed that they may be attempting to self-medicate (Dalack et al 1998). In patients with Alzheimer’s disease a decrease in high-affinity nicotine binding sites is one among other changes in the brain (Nordberg and
Winblad 1986; Whitehouse et al 1986). Parkinson’s disease is also associated with a large loss of high-affinity nicotine binding sites in the brain (Perry et al 1995).
The non-neuronal cholinergic system General
The term “non-neuronal cholinergic system” is based on the fact that ACh is found in cells other than neurones. Dale (1914) and Ewins (1914) obtained the first evidence of the presence of ACh in plants.
The occurrence of acetylcholine has been analysed in human placenta (Rowell and Sastry 1981), bronchial epithelial cells (Klapproth et al 1997; Wessler et al 1995) mononuclear cells (Fujii et al 1996), sperm (Sastry and Sadavongvivad 1978), retina (Hutchins and Hollyfield 1986) and keratinocytes (Grando et al 1993b; Klapproth et al 1997).
A variety of non-neuronal tissues synthesise and degrade ACh. Expression of the ChAT protein has been found in non-neuronal cells such as bronchial epithelial cells (Klapproth et al 1997; Reinheimer et al 1996), keratinocytes (Grando et al 1993b),
cells of the human small and large intestine (Klapproth et al 1997) and placental cells (Rowell and Sastry 1981).
AChE has also been found in the placenta (Rowell and Sastry 1981) and in heamatopoietic cells, i.e. red blood cells (Herz and Kaplan 1973), platelets and T lymphocytes, but not B lymphocytes (Szelenyi et al 1982). In a colonic biopsy specimen, expression of AChE mRNA has been detected in mast cells with high- affinity receptors for IgE (Nechushtan et al 1996).
Furthermore, some of these cells have been shown to express ACh receptors.
Macklin et al (1998) have reported that human vascular endothelial cells express the subunits that form functional nAChR similar to the nAChR expressed by ganglionic neurones (α-3, α-5, β-2 and β-4). These subunits have also been detected in human bronchial epithelial cells (Maus et al 1998), where Zia et al (1997) found the α-3, α- 4, α-5 and α-7 subunits. The alpha-7 subunit of nAChR is also expressed by
endothelial cells (Conti-Fine et al 2000), and the α-3 and α-4 subunits have been found on lymphocytes (Hiemke et al 1996).
The muscarinic receptor subtypes m2 and m3 have been found in human mononuclear leucocytes (MNLs) (Bronzetti et al 1996). Hellström-Lindahl and Nordberg (1996) found the mRNAs for the m3, m4 and m5 muscarinic subtypes in MNLs and also in purified T cells. Fujino et al (1997) observed expression of the m1 and m2 subtypes in human lymphocytes. Human skin fibroblasts have been reported to express m2, m4 and m5 mAChR subtypes (Buchli et al 1999).
The non-neuronal cholinergic system in the skin
Grando et al have made detailed investigations of the non-neuronal cholinergic system in the skin. They demonstrated that human keratinocytes express the ChAT protein and also synthesise and secrete ACh (Grando et al 1993b). Furthermore, they showed that the human epidermis expresses AChE and also nicotinic and muscarinic AChR (Grando 1997; Grando et al 1995a). The ion channels on keratinocytes are similar to those expressed by ganglionic neurons, since the α-3, α-5, β-2 and β-4 subunits, which are found on keratinocytes, are known to form functional receptors in several combinations among themselves (e.g. α3β2, α3β2α5, α3β4, α3β4α5 or α3β2β4α5) on ganglionic neurons, and the α-7 subunit, which is also present on keratinocytes, can form functional nAChRs of its own (Grando et al 1995a).
Keratinocyte ACh, like neuronal ACh, uses Ca2+ as a second messenger (Grando et al 1996). Ion fluxes mediated by nAChR channels are essential for maintaining
keratinocyte viability, as demonstrated in experiments using muscarinic and nicotinic blocking agents, where interruption of nicotinic, but not muscarinic, pathways of ACh signalling was found to inhibit keratinocyte division and result in premature cell death (Grando et al 1993a). Both nicotinic and muscarinic AChRs regulate cell adhesion and motility, since blocking by κ-bungarotoxin and mecamylamine, specific AChR antagonists, caused cell detachment and abolished cell migration (Grando et al 1995a).
High affinity mAChRs have been found on keratinocyte cell surfaces in a high density (Grando et al 1995b). These receptors mediate effects of muscarinic drugs on keratinocyte viability, proliferation, adhesion, lateral migration and differentiation.
The mAChR subtypes m1, m3, m4 and m5 have been found in the epidermis (Ndoye et al 1998). Keratinocytes expressed a unique combination of mAChR subtypes at each step of their development in the epidermis and it was proposed that each receptor may regulate a specific cell function (Ndoye et al 1998).
Effects of acetylcholine on cells
Non-neuronal ACh can exert its effects through different pathways. Acetylcholine released from non-neuronal cells activates the membrane-bound AChR (nicotinic and muscarinic) localised on the same (autocrine effects) or on neighbouring cells
(paracrine effects). Klapproth et al (1997) reported that the mitogenic effect of ACh on cultured human bronchial epithelial cells was counteracted by antagonists of nicotinic and muscarinic receptors. Thus, the effect of ACh is mediated by the classical extracellular membrane-bound receptors. Interestingly, in the same study it was found that the ChAT blocker, bromoacetylcholine, had a stronger
antiproliferative effect than a combination of the two antagonists blocking the
nicotinic and muscarinic receptors respectively, which may suggest that ACh also has cytosolic action.
Experimental evidence has been presented that ACh is involved in the regulation of the mitotic cycle of epithelial cells in humans (Cavanagh and Colley 1989; Grando et al 1995a; Grando et al 1993b; Klapproth et al 1997). Furthermore, detection of ACh and its receptors in immune cells indicates that it may take part in the immune system
by activation and proliferation of these cells [reviewed in Kawashima and Fujii (2000)].
Nicotinic influence
−−−− on the neuronal cholinergic system
Nicotine acts agonistically on nAChR, and is thus able to reproduce the same effects as ACh on cells expressing these receptors, but in contrast to ACh, nicotine is not degraded by AChE. The nAChR channel opens in response to the binding of agonist (activation) but also becomes refractory to activation during prolonged exposure to nicotinic agonists (desensitisation) (Peng et al 1994). The nicotine concentrations required to desensitise the receptor are nearly 1000 times lower than those required for activation [reviewed by Changeux (1990)]. It has been reported that smoking increases the number of nAChRs in the human brain (Breese et al 1997). It is proposed that nicotine-induced up-regulation of neuronal nicotinic receptors results from a decrease in the rate of receptor turnover (Peng et al 1994). The increase may also be due to an adaptive response of neurones to accumulation of chronically desensitised receptors. Alpha-3 nAChRs are more resistant to desensitisation than other receptor subtypes, such as those containing the α-4 and α-7 subunits (Olale et al 1997).
−−−− on the non-neuronal cholinergic system
Nicotine is present in high concentrations in the blood of smokers (Russell et al 1980) and might contribute to desensitisation of the nAChRs and in this way influence their normal function. There are reports that smoking increases the number of nAChRs on human bronchial epithelial cells in vivo and that nicotine increases the number of nAChRs in vitro on these cells (Zia et al 1997), and that long-term exposure of respiratory epithelial cells to nicotine increases their Ca2+ concentration, which could lead to cell damage. Smoking also increased the number of nAChRs on
polymorphonuclear cells in vivo (Benhammou et al 2000; Lebargy et al 1996) Lebargy et al (1996) reported that the increase in binding sites persisted for several months and did not return to the non-smoking level until one year after cessation of smoking.
Alpha-7 nAChRs have been found in small cell lung carcinomas, and the growth of cell lines derived from these tumours was inhibited by an α-7-specific antagonist (Chini et al 1992; Codignola et al 1996; Quik et al 1994). Thus, in small cell lung carcinomas in smokers the cancer growth may be facilitated by nicotine stimulation of α-7 receptors.
−−−− on keratinocytes
Short-term exposure to nicotine stimulates cytoplasm motility and lateral migration of cultured keratinocytes (Grando et al 1995a). Other (keratinocyte) functions such as proliferation, adhesion and differentiation may also be affected as a result of accelerated ion exchange through nicotinic channels. Chronic exposure to nicotine abolishes migration of cultured human keratinocytes in a dose-dependent manner (Lee et al 1996). Furthermore, chronic nicotine exposure leads to an increase in the number of keratinocytes forming cornifying envelopes, as well as in the expression of filaggrin, involucrin and transglutaminase type 1 (Grando et al 1996). Zia el al (2000) found that keratinocytes incubated with nicotine in vitro expressed a higher
percentage of the α-7 nAChR.
De Hertog et al (2001) concluded that tobacco smoking is probably a risk factor for cutaneous squamous cell carcinoma. Current smokers were found to be at higher risk than former smokers, and a clear relation to the number of cigarettes currently smoked was also observed.
−−−− on endothelial cells
Nicotine is known to induce vasoconstriction. The cutaneous blood flow, as measured with a laser Doppler flowmeter, was decreased both in habitual smokers and in non- smokers after smoking a single cigarette (Monfrecola et al 1998). The micro- circulation showed a slower recovery phase in the smokers, however, indicating adaptation to smoke.
There are functional nicotinic receptors on the endothelial cells (Macklin et al 1998). Since in tobacco users the concentration of nicotine in the blood is high (Russell et al 1980), these receptors may become desensitised after prolonged
exposure to nicotine, which can make them unable to respond in a normal way to the endogenous ACh.
AIMS OF THE INVESTIGATION
The general aim of this investigation was to study the pathogenesis of palmoplantar pustulosis.
The specific aims were:
- to define the cellular components of the inflammation in PPP.
- to localise the site of inflammation with particular reference to the eccrine gland and duct.
- to study the distribution of the general nerve marker PGP 9.5 and of the neuropeptides substance P and calcitonin gene-related peptide in PPP skin.
- to study contacts between sensory nerve fibres and mast cells in PPP skin.
- to study the distributions of choline acetyltransferase and acetylcholinesterase in palmar skin from healthy non-smokers and smokers and from PPP patients.
- to study the distributions of the α-3 and α-7 subunits of the nicotinic
acetylcholine receptor in palmar skin from healthy non-smokers and smokers, and from PPP patients.
- in view of the association between PPP and autoimmune diseases, to address the question of whether PPP itself might be an autoimmune disease by measuring serum antibodies to nAChR and by screening PPP sera for immunofluorescence in healthy palmar skin.
PATIENTS AND METHODS Patients
Paper I, II, III and IV Anamnestic data
Fifty-nine patients (52 women, 21-79 years old, and 7 men, 43-71 years old) with typical PPP of the palms and/or soles answered a questionnaire. Their smoking habits over the years was also investigated.
Clinical examination
Thirty-nine of the patients (35 women, 4 men) who answered the questionnaire were examined clinically (GM). The degree of erythema and scaling was graded from 0 to 4 and the number of fresh pustules was counted. The patients were only using
emollients at the time of the examination. None of the patients were taking beta- blockers or lithium.
Blood samples
Sera from the patients were analysed by routine methods for: triiodothyronine, thyroxine, thyroid stimulating hormone, immunoglobulins (IgG, IgA, IgM and IgE), eosinophilic cationic protein and antibodies (ab) to thyroglobulin, thyroid peroxidase, parietal cells and gliadin (IgA and IgG).
Biopsies
After intradermal injection of xylocaine-adrenaline, one to three 3-mm punch biopsy specimens were taken from involved skin and in some patients also one from
seemingly non-involved skin.
The specimens were either fixed in buffered 4% formalin and embedded in paraffin, or snap-frozen in 70oC, or fixed in 4% paraformaldehyde with 0.2% picric acid (Lanas fixative) for one hour and then rinsed in 0.1 M Sörensen’s buffer containing 10% sucrose for at least 24 h before they were frozen.
Paper V
In this study sera from the 39 patients described above and six new sera were used (39 women, 19-71 years old; 6 men, 36-70 years old). At the onset of PPP 43 patients were smokers. At the time of the present study 9 had stopped smoking or had reduced the number of cigarettes in recent years.
Reference persons
The reference group consisted of two subgroups: smokers and non-smokers. All of the smokers had been smoking for many years.
The number of smokers and non-smokers varied in the different studies:
Papers I and II: 2 smokers (1 woman, 1 man) and 7 non-smokers (6 women, 1 man), all of them healthy.
Papers III and IV: 7 smokers (5 women, 2 men) and 8 non-smokers (7 women, 1 man), all of them healthy.
Three punch biopsy specimens, which were handled in the same way as the specimens from the patients, were taken from palmar skin in all of these persons.
Paper V: In this study serum samples were taken from 23 patients with palmar eczema. Of these patients, 15 had smoked for many years, but six of them had stopped smoking in recent years.
One 3-mm skin punch biopsy specimen was taken from healthy non-smoking and smoking persons, from the hypothenar region after intradermal injection of xylocaine- adrenaline. For comparison, biopsy specimens were also taken from the dorsal aspect of the forearm and from the gluteal region. These specimens were snap-frozen at -70oC.
Immunohistochemistry
Peroxidase and alkaline phosphatase methods
Detailed descriptions of the different methods used in these studies are given in the respective papers.
Table 1a presents an overview of the different antibodies used, the fixatives, and the staining techniques employed.
In all specimens endogenous peroxidase activity was blocked by incubation in 0.3%
H2O2 in phosphate buffered saline (PBS) for 15 min. Between the incubations the sections were rinsed in PBS twice for 5 min.
Controls with IgG of the same isotype and in the same dilution as the primary monoclonal antibodies were negative. Polyclonal antibodies gave no staining when they were preabsorbed with their corresponding peptides.
Antibodies used in peroxidase and alkaline phosphatase methods. er ber AntigenAntibodyVisualizingDilutionSourceFixativeTechnique Eosinophil cationic proteinAnti-EG2Eosinophils§1/200Kabi PharmaciaAcetonePAP APAAP* Human neutrophil lipocalin (HNL) (Seveus et al 1997)Anti-HNLNeutrophils§10 ug/mLPharmacia DiagnosticsMethanolAPAAP* CD3Anti-CD3Lymphocytes§1/100Becton-DickinssonAcetonePAP TryptaseMAB1222Mast cells§1/5,000Chemicon Int. Inc.FormalinPAP Keratins (Watanabe et al 1993)AE1/AE3§§Sweat gland apparatus1/1,000Boehringer Mannheim Corp.FormalinPAP ChATAnti-ChATChAT1/5Boehringer Mannheim Corp.LanaABC** ChATMAB 305ChAT1/250Chemicon Int. Inc.AcetoneABC AChEMAB 303AChE1/600Chemicon Int. Inc.AcetoneABC α-3 nAChR subunitmAB 313nAChR1/3,000RBILanaABC** α-7 nAChR subunitAChRα-7§§§nAChR1/50Santa Cruz Inc.AcetoneABC lkaline phosphatase –antialkaline phosphatase (APAAP) technique was used, as neutrophils contain peroxidase, which could give false positive staining. tions used were 14 um, in other stainings 6 um sections were used. number of stained cells was counted in the epidermis, papillary dermis (below the pustule when applicable) and in the reticular dermis. §§ The sections stained E1/AE3 were treated with 0.05% protease for 10 min before staining. AE1/AE3 contains antibodies against cytokeratins 1-8, 10, 14/15, 16, and 19. Sections tylotic eczema were also stained with AE1/AE3 for comparison. he AChRα-7 antibody was the only one that was polyclonal.
To reduce the possible variations in staining intensities, all specimens used for one antibody were stained on the same day. All immunohistochemical and
immunofluorescence evaluations were made on coded slides.
In the vital epidermis the staining intensity was estimated in the different strata as follows: unstained = 0, weak = 1, medium = 2 and strong = 3. The numbers of unstained and of ChAT-, AChE-, α-3- andα-7-positive ducts in the reticular dermis, in the papillary dermis and in the vital epidermis and coils were counted. All visible dermal ducts were counted as one duct each. The proportions of weakly and strongly stained coils and ducts in the reticular dermis were calculated by dividing their number by the total number (unstained and stained) of coils and ducts in the reticular dermis. The numbers of immunoreactive cells in the papillary dermis and reticular dermis and below the pustules were counted and classified as very few (0-3), few (4- 10) or many (>10).
One immunohistochemical double staining was performed (Table 1b).
Table 1b. Double staining with ABC and APAAP techniques.
Paper number Antigens Visualising Techniques
III AChE and chymase1 AChE in mast cells ABC – APAAP
1The chymase antibody was used to verify that only mast cells were AChE+, since this antibody
worked better than the tryptase antibody in this double staining. Both antibodies were monoclonal.
Sections 6 µm thick were used.
Immunofluorescence
Table 2a shows the different antibodies used, the fixatives, and the staining techniques employed. Five non-adjacent sections were placed on each slide.
Table 2a. Antibodies used in immunofluorescence stainings.
Paper number
Antigen Antibody Visualising Dilution Source Fixative Secondary antibody conjugated with
II PGP 9.5 Anti-PGP 9.5 Nerve fibres 1/800 Biogenesis Lana TRITC
II Substance P Anti-SP Substance P 1/400 Peninsula Lana TRITC
II Calcitonin gene- related peptide
Anti-CGRP CGRP 1/400 Peninsula Lana TRITC
PGP 9.5 = protein gene product 9.5; TRITC = Tetramethyl-rhodamine-isothiocyanate; All antibodies were polyclonal and 14 µm thick sections were used.
Six compartments in all specimens were analysed: the epidermis, dermo-epidermal junction, papillary dermis, reticular dermis, eccrine sweat glands and their ducts and, wherever applicable, beneath pustules. Each separate fragment of nerve fibre was considered as one fibre. All five sections were analysed and the mean values per square millimetre or millimetre of epidermal length were calculated. Image analysis of the nerve fibres around the sweat glands was performed. The area of the positive nerves was expressed in per cent of the total sweat gland area.
Three immunofluorescence double stainings were performed (Table 2b).
Table 2b. Double stainings with immunofluorescence techniques.
PGP 9.5 = Protein gene product 9.5; HNL = human neutrophil lipocalin
FITC = Fluorescein isothiocyanate; TRITC = Tetramethyl-rhodamine-isothiocyanate Sections were 14 µm thick.
*All close contacts between mast cells and nerve fibres in the papillary dermis were counted and the number of such contacts per mm of epidermal length was calculated.
Serum immunofluorescence (paper V)
Sections from palmar skin of healthy controls (non-smokers and smokers), 6 µm thick and fixed in acetone, were incubated overnight at +4°C with serum (dilution 1/150) from 45 patients with PPP and 23 patients with palmar eczema. Immunofluorescence stainings were also performed with sera from 7 patients with myasthenia gravis, all of whom had elevated serum concentrations of nAChR antibodies. Fluorescein-
isothiocyanate (FITC)-anti-human IgG (dilution 1/40; Dakopatts, Glostrup, Denmark) was used as secondary antibody. Control with FITC anti-human IgG omitting the patient serum was negative. All parts of the sweat gland apparatus (duct and gland), epidermis and dermis were studied for the presence of staining. The staining intensity was classified as weak (+), medium (++) or strong (+++).
Double staining: endothelium – palmoplantar pustulosis serum (paper V)
This double staining was performed to confirm that the immunofluorescence obtained with the PPP sera was localised on endothelial cells. Sections 6 µm thick and fixed in
Paper number
Antibodies against Visualizing Secondary antibody
conjugated with II Tryptase and PGP 9.5 Contacts mast cells - nerve fibres* Texas Red - FITC II Substance P and HNL Neutrophils containing substance P TRITC - FITC II Substance P and EG2 Eosinophils containing substance P TRITC - FITC
acetone were incubated with two mouse monoclonal anti-human endothelial
antibodies, Q bend 10 (dilution 1/40; Skybio, Bedfordshire, UK) and CD 31 (dilution 1/40; Dakopatts), overnight at 4°C. Biotinylated horse antimouse IgG (dilution 1/200;
Vector, CA, USA) was used as secondary antibody. Subsequently the sections were incubated with Texas Red Streptavidin (dilution 1/100; Vector) for 30 min and then with 10% normal mouse serum (Dakopatts) for 60 min. The sections were then allowed to react with 10% normal rabbit serum for 10 min and thereafter with serum from the patients and FITC-anti-human IgG as above. To rule out non-specific staining, including overlapping between the fluorescence filters, three control
stainings were performed: one using mouse IgG of the same isotypes and dilutions as the primary endothelial antibodies plus patient serum, another with mouse IgG and omitting the patient serum, and as a third control the endothelial antibodies were used without the patient sera.
Western blot (paper III)
Western blot analysis was run on cell extracts from pure preparations of neutrophils and eosinophils from peripheral blood from healthy donors. Granulocytes from a Ficoll preparation were incubated with supermagnetic particles coupled to a monoclonal antibody against CD 16, a molecule present on neutrophils but not on eosinophils (Hansel et al 1991). The cell preparations were kindly provided by Associate Professor Lena Håkansson, Section of Clinical Chemistry, Department of Medical Sciences, University Hospital, Uppsala. Protein extract from human placenta was used as a positive control (Rowell and Sastry 1981).
The proteins were separated on 10% SDS-PAGE ready-gels (Bio Rad, CA, USA), and blotted on a nitrocellulose membrane. ChAT was visualised with a polyclonal rabbit-anti-ChAT antibody (dilution 1/1,000; Biogenesis, Poole, UK) and an Immun- Blot kit with an alkaline phosphatase conjugate (dilution 1/3,000; Bio Rad). As a negative control, placenta extract was used as above, but without the primary antibody.
Radioimmunoassay (paper V)
Serum nAChR antibodies were measured in sera from 45 PPP patients and, for comparison, in sera from 10 patients with palmar eczema, with a radioimmunoassay
(Lefvert et al 1978). In brief, a preparation of cholinergic receptors from human skeletal muscle was incubated with radiolabelled alpha-bungarotoxin, serum was added and the toxin-receptor-IgG complex was precipitated using anti-human IgG.
The precipitate was separated and washed by centrifugation. Radioactivity (CPM) was determined and the concentration of receptor antibodies in arbitrary units was calculated.
Statistics
The statistical significance of differences was calculated by the Mann-Whitney U-test (papers I, II, III, IV and V) or Fisher’s exact test (paper V).
RESULTS AND DISCUSSION Paper I
Anamnestic data
The worsening effect of warm weather and stress in a high proportion of patients indicated that the sweat gland apparatus might be a possible target for the
inflammation. The fact that 95% of the patients were smokers at the onset of the disease (at a mean age of 42 years, range 15-66 years) pointed to nicotine as a possible precipitating factor for the disease (Table 3).
Table 3. Anamnestic data in 59 patients (52 women, 7 men) with palmoplantar pustulosis.
Per cent Heredity for
-palmoplantar pustulosis 14
-psoriasis 22
-thyroid disease 22
-gluten intolerance 3
Patients with history of
-psoriasis 10
-thyroid disease 14
-gluten intolerance 8
-diabetes 7
-vitiligo 5
-alopecia areata 3
Stress preceding onset of PPP 25 Smoker at onset of PPP 95 Worsening associated with
- hot weather 36
- stress 46
Pruritus 95
Arthralgia 42
There was a high prevalence of a number of autoimmune diseases among the PPP patients, as shown in Table 3.
The association between autoimmune thyroid disease and PPP is well known and has been investigated in detail by Rosén (1988). The majority of the PPP patients with
thyroid disease had hypothyroidism, the prevalence of which in Swedish women is 1.9 percent (Hallengren 1998).
The increased prevalence of coeliac disease in PPP patients has not been reported previously. The prevalence figures for coeliac disease in the Swedish population have increased in the last few years since the introduction of screening for antibodies to gliadin and endomysium and recently also to tissue transglutaminase. Silent coeliac disease has been diagnosed in 0.3% of Swedish blood donors (Grodzinsky 1996).
Recent data from a screening study of children in Northern Sweden indicate a prevalence of at least 1% (Carlsson et al 2001). Since January 2001 anamnestic data have been available for 82 patients with PPP. The prevalence of previously diagnosed coeliac disease in this extended group is 6%. One of our patients who was found to have coeliac disease had disabling PPP, but since the introduction of a gluten-free diet, her PPP has totally cleared, indicating that gluten intolerance might be of pathogenetic relevance in PPP.
There was also a high prevalence of diabetes among the PPP patients. This has become even more evident since the number of patients has increased (to 82)
compared with the number at the start of the study. Twelve of the 82 patients (14.6 %) had diabetes; 9/12 were < 50 years old. At screening for diabetes in the community of Laxå, Sweden, 0.8% of the screened women aged 25-44 years and 2% of those aged 45-54 years were found to have diabetes (type 1 or type 2) (Andersson et al 1991).
Four of the patients (4.8%) had type 1 diabetes. The prevalence of type 1 diabetes among women in the community of Laxå was 0.3-0.4%. Thus there is a marked increase in the prevalence of diabetes in PPP, which has not been reported previously.
However, a predisposition to diabetes in PPP was discussed in a Japanese study, as a diabetic pattern at an oral glucose tolerance test was found in 22% of the patients (Uehara 1983), but there are no other reports on such an association. There are, however, previous reports of an increased prevalence of type 2 diabetes in psoriasis (Binazzi et al 1975). A significantly raised prevalence of psoriasis and vitiligo has been reported in children with type 1 diabetes (Montagnani et al 1985).
The high prevalence of associated autoimmune disease in PPP gave us reason to consider the possibility that PPP itself might be an autoimmune disease affecting the skin and also the joints.
Clinical findings
Erythema and scaling were present in all but one patient. Fresh pustules were observed in 26 patients (range 1-100). Patients with the highest cigarette consumption had the largest mean number of pustules (but there were large variations and the groups of patients were small).
The association between PPP and autoimmune disease was further strengthened by the presence of antibodies to thyroglobulin/thyroperoxidase in 25% of the patients.
IgA antibodies to gliadin were present in 25%, compared to 9% in female healthy blood donors (Lindquist et al, unpublished data). Among patients with psoriasis vulgaris 16% had IgA antibodies to gliadin (Michaëlsson et al 1993), thus the prevalence of IgA antibodies to gliadin is even higher in PPP.
The significantly elevated mean serum IgA and decreased IgM are similar to the pattern present in coeliac disease and dermatitis herpetiformis (O'Mahony et al 1990) and also in psoriasis (Michaëlsson et al 1995) and psoriatic arthritis (Lindqvist el al, unpublished data), indicating that the intestinal mucosa might also be involved in PPP.
Elevated serum ECP in PPP was previously reported by Lundin et al (1990) and suggested that the eosinophil granulocyte was activated. The results of the present study further confirm that eosinophil granulocytes are involved in the inflammation (see below).
Inflammatory cells
The pustules were found to contain large numbers of eosinophils, an observation not made previously, as well as neutrophils, indicating that the eosinophils participate in the pustule formation together with neutrophils. Furthermore, numerous eosinophils were present in the papillary dermis below the pustules. Another previously
unreported feature was the massive infiltrates of mast cells in the upper dermis, especially in specimens with pustules. One important chemoattractant for both neutrophils (Baggiolini et al 1989) and activated eosinophils (Burrows et al 1991) is IL-8, which has been shown to be present in the eccrine duct in general and in the epidermis in PPP skin (Anttila et al 1992; Jones et al 1995). Furthermore, mast cells have been found to secrete IL-8 (Ansel et al 1997).
There was a massive infiltrate of lymphocytes in the papillary dermis, with a tendency to accumulation below the pustule. The accumulation of the participating
inflammatory cells below the pustules may indicate that there is an epidermal target for the inflammation that is not evenly distributed in the epidermis.
Fig. 3. Schematic drawing of the participating inflammatory cells in PPP.
The sweat gland and duct
In the specimens from involved PPP skin no acrosyringia were visible with the keratin antibody AE1/AE3, reported to give staining in the sweat gland apparatus (Watanabe et al 1993), in contrast to the findings of acrosyringia in the control specimens, indicating that the intraepithelial duct may be destroyed in PPP, which might reflect an inflammatory process at this site. This feature may have pathogenetic relevance in PPP, since there were no changes in the appearance of the acrosyringia in specimens from our tylotic eczema patients or in those from patients with
dyshidrotic eczema (Kutzner et al 1986). We also stained the sweat pores in the palmar skin of PPP patients with the iodine-starch method and compared the pattern with that in the palms of healthy persons (no data shown). In the PPP patients a diffuse pattern was observed, whereas in the control persons there was a distinct pattern of black spots in even rows over the entire palm.
