Recent Advances in Dermatology Sanjay Ghosh, Dinesh Hawelia, Susmit Haldar
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Apoptosis: Its Role in Different Dermatoses1

Anil H. Patki
 
INTRODUCTION
There are two ways in which an eukaryotic cell can die: necrosis and programmed cell death. Apoptosis represents an important type of programmed cell death.1 Since necrosis occurs as a result of some external insult and apoptosis occurs due to an innate cellular programme, they may as well be described as ‘cell murder’ and ‘cell suicide’ respectively.
The term ‘apoptosis’, which is pronounced with a short ‘a’ and a silent second ‘p’, is a Greek word literally meaning falling of leaves from a tree or of petals from a flower (Apo = away, ptosis = to droop). It was first used by Kerr et al2 in 1972 to describe the mechanism of controlled cell deletion little described till that time. The process of apoptosis is important in both physiological and pathological contexts. During the embryonic life, various organs and limbs are carved by controlled death of certain cells. In adult life, apoptosis is necessary to maintain the homeostasis by a balance with mitosis. The size of organs like liver remains the same as the number of cells produced by mitosis are balanced by the number of cells eliminated by apoptosis.3 Also, autoimmune responses are normally prevented by apoptotic death of auto-reactive lymphocyte clones. Thus, apoptosis is important in eliminating the unnecessary or senescent cells without causing any damage to the surrounding normal cells. Necrosis, by contrast, attracts inflammatory cells which lead to tissue damage.
Apoptosis, if gone awry, can lead to certain disorders. If auto-reactive immune cells or cancer cells are not eliminated, an autoimmune or malignant disorder may be the result. Also, excessive and uncontrolled apoptosis may lead to disorders like Parkinsonism, Alzheimer's disease and amyotrophic lateral sclerosis.4,5 It has also been found that certain viral infections like that due to the human immunodeficiency virus [HIV] induce apoptosis in CD4 lymphocytes.6
Thus, apoptosis is an important phenomenon in both health and disease. This essay will discuss the mechanisms of apoptosis and the importance of apoptosis in different dermatoses.2
 
MORPHOLOGY OF APOPTOSIS
As opposed to necrosis, where the cell swells up and lyses, apoptosis involves reduction in volume, blebbing of the cell membrane and the margination of chromatin along the nuclear membrane.2 This is followed by the collapse of the nucleus into apoptotic bodies surrounded by a membrane. The cytoplasmic organelles are much less affected during this process. An important difference between necrosis and apoptosis is that apoptotic cells do not invoke any inflammatory reaction unlike the necrotic cells which release chemotactic cell contents. The apoptotic bodies consist of membrane bound fragments of nucleus and cell organelles. They are phagocytosed by surrounding cells or macrophages. In case of tubular organs, the apoptotic bodies are released in the lumina. Thus, apoptosis is a special type of programmed cell death which can occur in health or disease. The death of keratinocytes in the stratum corneum is also an example of programmed cell death which is slightly different from apoptosis and is termed ‘differentiation to death’.1
 
PATHOMECHANISMS OF APOPTOSIS
To die actively, a cell must receive some ‘message’ to initiate the process. This message or death signal is received by some transmembrane proteins of the TNF (Tumor necrosis factor) family of the receptors called the death receptors. Each death receptor has two or four cysteine-rich extracellular domains and a cytoplasmic sequence termed ‘death domain’. Six human death receptors have been identified along with their respective ligands which bring the death signal (Table 1.1).7 When a death ligand combines with a death receptor, a signal is transmitted in the cytoplasm to a system of enzymes called caspases. Initially, caspase 8 and 10, which are initiator caspases, are activated and later, akin to the complement cascade, the effector caspases viz., caspases 3,6, and 7 are activated. The caspases in turn activate a variety of endonucleases and proteases which cause breakdown of cellular contents and ultimately apoptosis.
Besides the death receptor pathway, there is another pathway inducing apoptosis can be: mitochondrial pathway.8 Triggered by factors like radiation, withdrawal of growth factors and cytotoxic drugs, release of cytochrome C from the mitochondria activates caspase 9 with further activation of downstream caspases and finally leads to apoptosis.
Thus, apoptosis is a process by which a cell dies through activating its own system of serine proteases and endonucleases which breakdown cellular contents. The nuclear DNA is broken down into various fragments. This phenomenon is useful in the detection of apoptosis, as agarose gel electrophoresis reveals a ‘ladder’ pattern as different DNA fragments move at different rates3.
Table 1.1   Death receptors and ligands7
Death receptors
Death ligands
1. Fas (CD 95, Apo 1)
Fas-L (CD 95L) (Fas ligand)
2. TNF-R1 (Tumor necrosis factor receptor 1)
TNF
3. TRAMP (Apo 3) (TNF related apoptosis mediating protein)
Apo 3 L, TWEAK (TNF-like weak inducer of apoptosis)
4. TRAIL-R1 (TNF–related apoptosis inducing ligand receptor 1)
TRAIL
5. TRAIL-R2
TRAIL
6. DR 6
?
 
GENES AND APOPTOSIS
Several genes are important in either promoting or inhibiting apoptosis. Amongst them, the p53 gene and those of Bcl-2 (B cell lymphoma) family are important. The p53 gene is a tumor suppressor gene whose product, the p53 phosphoprotein, has a molecular weight of 53000 and is important in protection against malignancy.9 It acts in two ways. Whenever there is a mild damage to the cellular DNA, the p53 protein arrests the cell in G1 phase, allowing the cell to repair the damage before it proceeds to the S phase. If the damage to the DNA is irrepairable, the p53 protein eliminates that cell by inducing apoptosis. Thus it is a guardian of the genome. Absence or abnormalities of the p53 gene are found in almost 50 percent malignancies found in man. p53 mutations are associated with aggressive malignancies and poor prognosis.
The Bcl family of genes consists of about 20 members, some of which are pro-apoptotic and the rest anti-apoptotic. The products of these genes can form either homodimers or heterodimers in the cytoplasm and can exert their control on apoptosis at the level of release of cytochrome C from the mitochondria.10
 
APOPTOSIS AND SKIN DISORDERS
Excessive or defective apoptosis of epidermal cells or those of the immune system is an important feature of many dermatological conditions. The dyskeratotic and eosinophilic keratinocytes, seen in the epidermis after exposure to ultraviolet radiation (UVB and UVC), are apoptotic cells.11 These, so called ‘sunburn cells’, appear in the epidermis within 30 minutes of the exposure and reach their maximum number in 24 hours. These cells then move upwards within the epidermis to be shed off. Another 4common physiological example of apoptosis in the skin is that of the regression of the portion of hair follicle during catagen.12 Besides these common examples, apoptosis is an important feature of many hypersensitivity reactions, autoimmune disorders, malignancies and graft-versus-host disease.
 
Toxic Epidermal Necrolysis
Toxic epidermal necrolysis (TEN) is a severe drug reaction resulting in extensive epidermal damage and separation of sheets of epidermis from the underlying dermis. The common drugs causing TEN are antibiotics and antibacterials, non-steroidal anti-inflammatory drugs and anticonvulsants. Some cases of TEN also show lesions suggestive of erythema multiforme and this group of patients is considered to have an overlap of TEN and Stevens-Johnson syndrome (SJS). The keratinocyte death in TEN and TEN-SJS overlap conditions was shown to be due to apoptosis by Paul et al in 1996.13 They demonstrated the apoptosis of keratinocytes by electron microscopy which revealed the typical changes of apoptosis; by DNA-nick end labeling which stained apoptotic cells and by agarose gel electrophoresis of DNA extracted from the epidermis which revealed the ladder pattern due to DNA fragmentation. TEN is a life-threatening condition with a high mortality rate and requires intensive care. Paul et al conjectured that agents inhibiting apoptosis may play an important part in management of TEN.13
Subsequently, an important advance in the therapy of TEN was reported by Viard et al in 1998.14 It was known that keratinocytes normally express Fas (CD 95) receptor on their surfaces. Viard et al showed that in TEN, keratinocytes also express Fas ligand which is lytically active and induces apoptosis in Jurkat cells (a T-lymphocyte line) in vitro. Intravenous immunoglobulin (IVIG), prepared from multiple donors, contains naturally occurring anti-Fas antibodies. If keratinocytes are treated with IVIG (leading to blockade of Fas receptors), futher exposure to Fas ligand does not lead to apoptosis. Armed with this knowledge, Viard et al treated 10 patients of TEN with high doses of IVIG (0.2 to 0.75 gm/kg per day) for 4 successive days. The disease process halted in 1–2 days and epidermal regeneration was complete in 7–12 days with a favourable outcome in all patients.14 In a subsequent study, Stella et al treated 9 patients of TEN with high doses of IVIG (0.6 to 0.7gm/kg per day) for 4 days out of which 8 healed and survived.15 However, whether systemic problems in TEN (hepatitis, pancreatitis, hematological abnormalities, lung involvement) also involve apoptosis is not known. Treatment of TEN with IVIG denotes a very good example of 5development of a treatment modality on the basis of understanding the pathomechanisms of apoptosis.
 
Lichen Planus and Lichenoid Tissue Reaction (LTR)
The lichenoid tissue reaction (LTR) is characterized by damage to basal cells in the epidermis associated with a massive infiltrate of mononuclear cells in the upper dermis.16 Many clinically diverse skin disorders ranging from lichen planus to lupus erythematosus have in common lichenoid histopathological features. The epidermal cell death, usually found in the LTR, results from apoptosis. The mode of cell death mediated by cytotoxic T cells and natural killer cells represents apoptosis. Cytotoxic T cells mediate apoptosis by atleast 2 ways: i) by perforin and serine proteases (Granzyme A and B), ii) by interaction of Fas receptors on the target cells and Fas ligand on the cytotoxic cells.17
The formation of colloid bodies, also referred to as hyaline, cytoid or Civatte bodies in the lower epidermis and upper dermis is a characteristic histological feature of LTR and is commonly seen in lichen planus. The colloid bodies are nothing but apoptotic cells. Bloor et al have shown that in oral lichen planus, approximately one apoptotic cell is detected per mm of basal layer and the number of apoptotic cells is proportional to the density of the lymphocytic infiltrate.18 Bcl-2 expression (which is anti-apoptotic) is weak or absent in the lesions and Bax expression is localized to the upper prickle cell layer. Neppelberg et al19 using a different method have found a higher number of apoptotic cells than Bloor et al.18 The apoptotic cells in oral lichen planus are mostly confined to the basal layer. Fas and Fas-L are expressed by the basal keratinocytes as well as by majority of the mononuclear cells in the subepithelial infiltrate.19
In cutaneous lichen planus lesions, granzyme pathway seems to be at work. Shimuzu et al have shown that granzyme-B-positive CD8 cells induce keratinocyte apoptosis in cutaneous lichen planus.20 Perforin and granzymes are the main constituents of the granules of cytotoxic T cells and natural killer cells. Perforin is a pore forming protein which drills a hole in the cell membrane of the target cell through which granzyme B is injected in the cytoplasm of the target cell. Granzyme, a serine protease, activates or subsitutes for interleukin1-alpha-converting enzyme (ICE) and promotes apoptosis. Shimuzu et al have shown by immunoelectron microscopy that granzyme B molecules are secreted from a lymphocyte to an apoptotic keratinocyte. They also found the infiltrate to consist of CD4, CD8 cells as well as a few natural killer cells.20 Apoptosis is thus an important feature of lichen planus and lichenoid tissue reaction.6
 
Graft-Versus-Host Disease (GVHD)
GVHD occurs as a result of bone marrow transplantation and an attack by the donor's immunocompetent lymphocytes on the host tissue. The skin, the intestines and the liver are the tissues mostly involved. In the acute phase of GVHD and lichenoid type of chronic GVHD, apoptotic cells are observed in the epidermis.21 These dyskeratotic cells with eosinophilic cytoplasm and small pyknotic nuclei were termed ‘mummified’ cells by earlier workers.22 Various studies have shown that lymphocyte Fas-L, induced during acute GVHD, is involved in inducing Fas mediated apoptosis in the target organs viz., skin, liver and gut.
 
Malignancies
Malignant cells expressing Fas are normally eliminated through apoptosis by cytotoxic T cells expressing Fas-L. However, absence of Fas receptors on the malignant cells allows them to escape Fas-Fas-L mediated apoptosis. This phenomenon is found in basal cell carcinoma and melanoma.23,24 Thus, defective apoptosis is a feature of these malignant conditions. Reinduction of Fas expression on tumor cells of basal cell carcinoma by intralesional injection of interferon-alpha causes regression of tumour by apoptosis.23
Primary cutaneous T cell lymphoma (CTCL) represents a group of T cell neoplasm of skin-homing, CD 4-positive T cells. All the successful therapies in the treatment of CTCL like radiotherapy, electron beam therapy, topical chemotherapy, psoralen and UVA therapy (PUVA) and topical steroids, work by inducing apoptosis of neoplastic T cells.25 Loss of Fas expression on neoplastic cells is found in aggressive types of CTCL but not in indolent types.26 Thus, loss of Fas expression allows tumor cells to escape an effective immune response and may contribute to unfavourable prognosis in some types of CTCL.
 
Autoimmune Disorders
During the development of T cells, the autoreactive T cells are eliminated in the thymus by a process involving apoptosis. Any defect in this process results in persistence of autoreactive immune competent cells which may be an etiological factor in autoimmune disorders of the skin.27
 
Phototherapy: Underlying Mechanism
The narrowband UVB phototherapy of psoriasis has been shown to be causing apoptosis of T cells in the lesions of psoriasis.28 Even UVA phototherapy is known to induce apoptosis of T helper lymphocytes.29
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CONCLUSIONS
In conclusion, apoptosis is an important phenomenon both in health and disease. Excessive and abnormal apoptosis is a feature of disorders like TEN and LTR while defective apoptosis is a feature of conditions like malignancies and autoimmune disorders. Since its description more than 30 years ago, apoptosis has, during the last few years, become one of the hottest topics in biomedical research.
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