Introduction To Pemphigus Vulgaris And The Immune System Biology Essay

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The word immunity comes from the word Immunitas that referred to the social immunities that roman senators had during their period of being in the senate. Historically the immune system was known to be involved in the body's protection against microorganisms and "foreign" substances. The protection against microorganisms is accomplished by innate immunity at first. Afterwards the adaptive immunity comes to action. Innate immunity or natural immunity consists of a series of cellular and biochemical mechanisms that are even active and ready before an infection happens. These mechanisms only react against microbes and their response to repeated infection remains constant in every episode. The innate immunity consists of: 1-physical boundaries such as the skin, 2-neutrophils, macrophages and NK cells, 3-the complement system and 4- cytokines. Another more complex and developed immune mechanism is the adaptive or specific immunity. This type's major characteristic is that it is specific for each macromolecule and it also has "memory". The specific immune system has the ability to remember a previous episode of infection and show a greater response in the event of reoccurrence. It is composed of two major parts. One component, B lymphocytes, producing antibodies that help innate immune system to attack "foreign" substances and cause them to be removed from the body; this is also called the humoral immune system. Antibodies recognize microbe antigens specifically and stop infections. They also cause the elimination of microbes from the blood through a series of mechanisms. This immune system is the main protection against outer cellular microbes and their poison. Antibodies are specialized molecules that are programmed for special duties. For example some aggravate the release of inflammation substances from mast cells while others play a role in the clearance of microorganisms from the blood. The other component consists of special white blood cells called T lymphocytes. T lymphocytes are restricted in the recognition of antigens and are only able to recognize peptide antigens joint to MHC molecules. So, T lymphocytes can only recognize and react against antigens on the surface of the cells. These lymphocytes are divided into 3 groups based on their function:1-T helper cells, 2-cytotoxic T cells and 3-regulatory T cells. T helper cells release cytokines in response to antigens. Cytokines have a major role in aggravation of proliferation and differentiation of T cells and other cells such a B cells, macrophages and other leukocytes. Cytotoxic T cells destroy infected cells with viruses or other intracellular microbes. There is a debate about the role of regulatory T cells. The start of the specific immune reaction is due to antigen presentation by special cells. These cells are called "antigen presenting cells (APC). The most professional APCs are dendritic cells that present the antigens to T lymphocytes in secondary lymphoid organs.

Normally, the immune system can distinguish between "self" and "non-self" and only attacks those tissues that it recognizes as "non self." This is usually the desired response, but not always. For example when a person is given an organ transplant, the immune system will correctly recognize the new organ as "non self" (unless it is from an identical twin) and will attack it in a process called rejection. Another situation is autoimmune diseases that generate from an overactive immune response of the body against substances and tissues normally present in the body. In other words, the body actually attacks its own cells. In fact, the immune system mistakes some parts of the body as a pathogen and attacks it. This may be limited to certain organs (e.g. in thyroiditis) or involve a particular tissue in different places (e.g. Goodpasture's disease which may affect the basement membrane in both the lung and the kidney).

1.2 The skin

The skin is considered the largest organ of the body and has many different functions. The skin functions in thermoregulation, protection, metabolic functions and sensation. The skin is divided into two main regions, the epidermis, and the dermis as seen in fig(1), each providing a distinct role in the overall function of the skin. The dermis is attached to an underlying hypodermis, also called subcutaneous connective tissue, which stores adipose tissue and is recognized as the superficial fascia of gross anatomy.

Fig(1). Anatomy of skin

The epidermis is the most superficial layer of the skin and provides the first barrier of protection from the invasion of foreign substances into the body. The principal cell of the epidermis is called a keratinocyte. The epidermis is subdivided into five layers or strata, the stratum germinativum (SG), the stratum spinosum (SS), the stratum granulosum (SGR), the stratum lucidum (not seen in this photomicrograph) and the stratum corneum (SC) in which a keratinocyte gradually migrates to the surface and is sloughed off in a process called desquamation.

Fig(2). Histology of epidermis

Demonstrated in fig(2) it can be seen that the stratum germinatum (SG) provides the germinal cells necessary for the regeneration of the layers of the epidermis. These germinal cells are separated from the dermis by a thin layer of basement membrane. After a mitotic division a newly formed cell will undergo a progressive maturation called keratinization as its migrates to the surface.

The cells that divide in the statum germinativum soon begin to accumulate many desmosomes on their outer surface which provide the characteristic prickles of the stratum spinosum, which is often called the prickle-cell layer. The progressive maturation of a keratinocyte is characterized by the accumulation of keratin, called keratinization. The cells of the stratum granulosum accumulate dense basophilic keratohyalin granules. These granules contain lipids, which along with the desmosomal connections, help to form a waterproof barrier that functions to prevent fluid loss from the body. Epidermis varies in thickness throughout the body depending mainly on frictional forces and is thickest on the palms of the hands and soles of the feet. The stratum lucidum is normally only well seen in thick epidermis and represents a transition from the stratum granulosum to the stratum corneum. As a cell accumulates keratinohyalin granules, it is thought that rupture of lysosomal membranes release lysosomal enzymes that eventually cause cell death. The dead and dying cells filled with mature keratin form the stratum corneum. The deeper cells of the stratum corneum retain their desmosomal junctions, but as they are pushed to the surface by newly forming cells of the stratum germinativum, the dead cells gradually break apart and are lost, a process called desquamation. The dermis assumes the important functions of thermoregulation and supports the vascular network to supply the avascular epidermis with nutrients. The dermis is typically subdivided into two zones, a papillary dermis and a reticular layer. The dermis contains mostly fibroblasts which are responsible for secreting collagen, elastin and ground substance that give the support and elasticity of the skin. Also present are immune cells that are involved in defense against foreign invaders passing through the epidermis. The papillary dermis contains vascular networks that have two important functions. The first being to support the avascular epidermis with vital nutrients and secondly to provide a network for thermoregulation. The vasculature is organized so that by increasing or decreasing blood flow, heat can either be conserved or dissipated. The vasculature interdigitates in areas called dermal papillae. The papillary dermis also contains the free sensory nerve endings and structures called Meissner's corpuscles in highly sensitive areas. The reticular layer of the dermis consists of dense irregular connective tissue, which differs from the papillary layer, which is made up of mainly loose connective tissue (note the difference in the number of cells). The reticular layer of the dermis is important in giving the skin it overall strength and elasticity, as well as housing other important epithelial derived structures such as glands and hair follicles.

1.3 Pemphigus vulgaris

1.3.1 introduction to Pemphigus vulgaris

Pemphigus vulgaris (PV) is one of the diseases that is categorized as an autoimmune disease. Pemphigus is derived from the Greek word pemphix meaning bubble or blister.

Fig(4): Intact bullas in PV

Pemphigus is the general name for a group of chronic bullous diseases. Pemphigus used to be a general term that included most bullous eruptions of the skin, but by improvement of diagnostic tests, bullous diseases have been reclassified more specifically. Pemphigus vulgaris is the most common form of pemphigus [1]. Oral erosions are usually seen weeks or months before the onset of skin blisters fig(5). In one study, the soft palate was involved in 80% of cases at initial presentation [2].

Fig(5):oral ulcers

The skin blisters are non pruritic and vary in size from 1 to several cm fig(4). They appear gradually and may be persistant for a considerable length of time, but if no treatment is administrated they become generalized.The fragility of the vesicle roof, which consists of only a thin layer of the upper epidermis causes the blisters to rupture easily. Nikolsky's sign is positive in pemphigus vulgaris. Applying pressure to the bullae causes the fluid to be divided laterally into the midepidermal areas altered by bound IgG. Healing takes weeks after the bullae are ruptured. As it is shown in fig(6) brown hyper pigmentation without scarring remains in the site of the healed erosion. Blisters, erosions, and lines of erythema may appear in the esophageal mucosa.

Fig(6): PV lesions on the body

1.3.2 Epidemiology of Pemphigus Vulgaris

PV is an autoimmune, intraepithelial, blistering disease that has been reported to occur worldwide. PV incidence varies from 0.5-3.2 cases per 100,000. PV incidence is increased in patients of Ashkenazi Jewish descent and those of Mediterranean origin. Most pemphigus cases are middle aged and elderly men and women. It has the same prevalence in both genders.

PV is a potentially life-threatening autoimmune mucocutaneous disease with a mortality rate of approximately 5-15%. Complications secondary to the use of high-dose corticosteroids contribute to the mortality rate. Morbidity and mortality are related to the extent of disease, the maximum dose of systemic steroids required to induce remission, and the presence of other diseases. Prognosis is worse in patients with extensive disease and in older patients.

1.3.3 Association of Pemphigus Vulgaris with other diseases

Erythematosus and Vulgaris forms of pemphigus have been reported to be in close relation with myasthenia gravis and thymoma [3,4]. In these patients the clinical course is variable, but most patients present by myasthenia gravis features first. Detection of thymoma usually precedes the onset of pemphigus. Usually the lymphoid or reticuloendothelial system neoplasms , have a higher incidence in patients with pemphigus than in normal persons.

Paraneoplastic pemphigus (PNP) is a disease with clinical and histologic characteristics of both Stevens-Johnson syndrome and pemphigus vulgaris. PNP is in association with non-Hodgkin's lymphomas and other malignant neoplasms [5-7]. This condition is presented by painful mucosal ulcerations [8], conjunctival reactions [9], and polymorphous skin lesions [10-11] on the trunk and extremities that usually progress to blisters. The treatment is dependent upon the remission of the malignancy and generally this condition has a poor prognosis. Hence the eruption of the blisters is not always concomitant with the underlying cancer the term Neoplasia-associated pemphigus may be more accurate for this disorder [12]. In this condition antibodies against epithelial proteins are present in desmosomes and hemidesmosomes in the epidermis

1.3.4 Pathogenesis

PV is mediated by circulating auto antibodies directed against keratinocyte cell surfaces molecules. Clinical and experimental observations indicate that the circulating auto antibodies are pathogenic. Blisters in PV are caused through the process of binding IgG auto antibodies to keratinocyte cell surface molecules. These intercellular or PV antibodies bind to keratinocyte desmosomes and to desmosome-free areas of the keratinocyte cell membrane. The binding of autoantibodies results in a loss of cell-cell adhesion. This process is called acantholysis. Pemphigus antibody binds to keratinocyte cell surface molecules desmoglein 1 and desmoglein 3. This binding process can cause acantholysis both directly or by triggering a series of reactions that causes acantholysis indirectly. Pemphigus antibody also fixes components of the complement system to the surface of epidermal cells. Recently it was seen that antibody binding may activate complements with the release of inflammatory mediators and recruitment of activated T cells. Different transmembrane desmosomal cadherins, including Dsg 3, are associated with submembranous plaque proteins that anchor to the keratin network. One of these plaque proteins is plakoglobin. Some studies have investigated the role of this protein in pemphigus vulgaris. Korman et al. studied the importance of plakoglobin as an autoantigens in pemphigus foliaceus and pemphigus vulgaris [13]. In 2001 a group of researchers in Switzerland also demonstrated that plakoglobin plays a central role in PV [14]. In this study, the researchers by establishing a calcium-dependent differentiation model to study the molecular pathogenesis of PV using PG+/+ and PG−/− keratinocytes, discovered a central role for plakoglobin in the disease process. The absence of keratin retraction after binding of PV IgG to PG−/− keratinocytes demonstrates that steric hindrance alone is not sufficient to induce PV lesions, and that this process is dependent on plakoglobin.[14]

1.3.5 Diagnosis

Diagnosis is maid through 3 different procedures: 1- Skin biopsy for light microscopy. In a blister biopsy an intraepidermal bulla, acantholysis and a mild-to-moderate infiltrate of eosinophils is usually seen fig(7). Acantholysis is defined as separation of epidermal cells near the blister following dissolution of the intercellular cement substance.

Fig(7): Histology in PV (H&E)

2- Direct immunofluorescence of skin. For this purpose usually two biopsies are recommended that is usually helpful in evaluation of oral lesions. One biopsy should be taken from the edge of a fresh lesion and the second from normal skin. Specimens are put in transport media unrefrigerated available from specialized laboratories. IgG and, in most cases C3 are found in the intercellular substance areas of the epidermis fig(8). Direct immunofluorescence study on the esophagus was reported positive in all patients [15].

Fig (8): Direct immunofluorescence in PV

3- Indirect immunofluorescence:

Serum IgG antibodies can be verified by indirect immunofluorescent and are present in approximately 3 out of 4 of patients with active disease in all forms of pemphigus . Patients with pemphigus foliaceus have antibodies to a complex of three polypeptides 260-kd, 160-kd (desmoglein), and 85-kd (plakoglobin). This is called the foliaceus complex; patients with pemphigus vulgaris have antibodies to a complex of 210-kd, 130-kd, and 85-kd (plakoglobin) polypeptides (the vulgaris complex) [16].. By testing on two tissue substrates in most cases the antibodies of pemphigus vulgaris can be differentiated from pemphigus foliaceus. In these cases titers are reported on two substrates. In most cases by activation of disease the level of circulating intercellular IgG antibody elevates and decreases respectively in remission [17]. So periodic serum detecting variations in titers are recommended to evaluate the clinical course [18]. This testing is usually done every 2 to 3 weeks until remission, and every 1 to 6 months afterwards.

1.3.6 Treatment

In the treatment of this disease both high-dose corticosteroid (more than 120 mg/day) [19-20] therapy and low-dose (60 mg/day) [21] therapy have been promoted. A recent study demonstrated that the only advantage that high dose therapy has is a more prompt initial control of the disease and this regimen did not decrease the frequency of relapse or the complications in long run.

In low dose therapy a single daily dose of 1 mg/kg/day of prednisolone or prednisone is given. Patients are treated until remission. This state is defined as no new blisters for at least l week. Then prednisolone is tapered with the dose of 10 mg monthly until the daily dose of 20 mg prednisolone is achieved. Afterwards adjuvant therapy of 15 mg methotrexate weekly or 100 mg cyclophosphamide [22] daily is added. Prednisolone is then gradually decreased 2.5 mg per month. The adjuvant therapy is gradually decreased after one year if the patient is in good condition. In an episode of blisters involving more than 50% of body surface area that is considered as a major relapse treatment with the original starting dosage should be started. Otherwise treatment is advocated by doubling the dosage of prednisolone. The value of adjuvant therapy has been questioned for bullous pemphigus by the authors of this regimen [21] and by others [23].

Adjuvent therapy is important because it can help lower the prednisolone dosage needed for therapy. This can assist decreasing the risk for potential toxicity of systemic corticosteroids. Cyclophosphamide (1.5 to 2.5 mg/kg/day) [22], chlorambucil, azathioprine (1.5 to 2.5 mg/kg/day) [24], dapsone [25], cyclosporine [26], and gold [27-28], plasma exchange [29], and extracorporeal photopheresis [30], have been used for adjuvant therapy [23, 31]. Hydroxychloroquine 200 mg twice a day was reported to be an effective adjuvant in patients with persistent and widespread pemphigus foliaceus. It is shown that this was especially seen when photosensitivity was present [32]. Adjuvent therapy could either be started at the same time as corticosteroids or afterwards.

Conventional long-term, high-dose corticosteroid therapy for pemphigus causes many side effects. Pulse treatment regimens have been reported. On 3 consecutive days, 100 mg dexamethasone is administered intravenously and, on day 1, 500 mg cyclophosphamide is administered intravenously. Between pulses patients receive 50 mg cyclophosphamide orally per day. Initially the pulses are repeated every 2 weeks and later at 10-week intervals. In this report, after 6 months (14 to 48 treatments) 50% to 100% of patients with various forms of pemphigus were clear [33, 34] .A similar program using only monthly pulse doses of cyclophosphamide 500 to 1000 mg per treatment also provided control for cases that had failed to respond to oral steroids and other agents [35].

The combination of nicotinamide (1.5 gm/day) and tetracycline (2 gm/day) or minocycline (100 mg twice a day) was found to be an effective alternative to steroids in superficial pemphigus (pemphigus foliaceus and pemphigus erythematosus) and a steroid-sparing adjuvant, rather than a steroid alternative for pemphigus vulgaris [36].

Direct immunofluorescence should be performed before therapy is discontinued. A negative direct immunofluorescence finding is a good indicator of remission [37].

1.4 literature review

The cause of PV remains unknown; however, several potentially relevant factors have been identified. Potent B-cell survival factors, are usually elevated in patients with Ab-dependent systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis and systemic sclerosis [38]. Although, the cellular processes that initiate and maintain B cell dysregulation in Ab-dependent autoimmune diseases are not well understood, however, it is widely accepted that mechanisms involved in survival and activation of self-reactive B cells are disturbed.

In this regard, recently identified two TNF family molecules, B cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL), have received increasing attention as key regulators of normal B cell functions and the induction and development of B-cell dependent autoimmune diseases [39]. BAFF is an essential factor for B cell survival, and its three receptors, transmembrane activator and calcium modulator ligand interactor (TACI), B cell maturation antigen (BCMA), and BAFF receptor (BAFF-R), are variably expressed on B cells during their differentiation [40]. APRIL is homologous to BAFF, but binds only to TACI and BCMA. APRIL shares many functions in common with BAFF, while they also have still distinct functions. Notably, serum levels of BAFF and APRIL are increased in autoimmune diseases, including SLE and rheumatoid arthritis[41]. Moreover, blockade of BAFF and APRIL prevents autoimmunity in animal models of disease and is being developed for human use [42]. Accordingly, it is considered important to examine the correlation between bullous diseases and BAFF/APRIL system to infer the function of these TNF family molecules in the immunopathogenesis of these skin blistering diseases, and to suggest the efficacy of BAFF and/or APRIL-targeting therapy in these skin disorders.

A number of studies have investigated the relation of BAFF and/or APRIL levels in patients and the occurrence of autoimmune diseases. Tangye et al. worked on BAFF and APRIL in human B cell disorders in 2006 [43]. This was a review study that noted that in previous studies BAFF levels had significant relation with activity of SLE, RA, wegner disease and Bcell related malignancies such as CLL and also CVID and IgA deficiency. Also Bossen et al. in 2006 reviewed BAFF, APRIL and their receptors: structure, function and signaling [44]. Some studies have also been carried out specifically in autoimmune diseases. Among them Zhu et al. conducted a research on the effects of BAFF and BAFF-R-Fc fusion protein in immune thrombocytopenia in 2009 [45]. In this study the effects of recombinant human BAFF (rhBAFF) and BAFF-R-Fc fusion protein (BR3-Fc) on B cells, T cells, platelets, secretion of interferon gamma (IFN-gamma), and interleukin-4 (IL-4) were measured by flow cytometry and ELISA. Patients with active disease had higher levels of plasma BAFF and BAFF mRNA than patients in remission and controls In the same year another study was done in the field of dermatological diseases by Jee et al. on the B cell-activating factor level in children with atopic dermatitis [46]. In this study, levels of serum BAFF, a proliferation-inducing ligand (APRIL) and total serum IgE level, and total eosinophil count were measured in 245 children. Serum BAFF level in children with atopic dermatitis was significantly higher than in healthy controls. Serum APRIL level was not different between the groups. Serum BAFF level significantly correlated with total serum IgE level and total eosinophil count [46]. However, the results of another study showed the elevated APRIL levels in atopic dermatitis patients while BAFF levels remained in normal range [47]. Interestingly, a few studies have also been carried out on bullous diseases specially PV. In one study that was conducted by Asashima et al. increased levels of BAFF in bullous pemphigoid but not in PV have been shown [48]. This study did not include APRIL levels. Another study that took place in Tokyo University indicated increased levels of BAFF and APRIL in bullous pemphigoid but didn't investigate these levels in PV [49]. As stated, only a few studies have been done on this particular subject and neither one has investigated both BAFF and APRIL levels in PV. Therefore, comparison of both proliferation inducing ligands (BAFF & APRIL) levels between PV and normal controls is important and is considered as the goal of the present study.