Apoptosis: Its significance in dermatology

Introduction

The Greek term ′apoptosis′ is derived from apo (=away) and ptosis(=to fall over or to droop) and literally means falling off of petals from a flower or of leaves from a tree. This term was first coined by Kerr et al in 1972 to describe programmed cell death.[1] During the last 5 years, it has become one of the hottest topics in biomedical research because alterations in the process of apoptosis are known to be involved in many disease processes ranging from autoimmune disorders to malignancy. In normal life, apoptosis is important during embryogenesis in carving of various organs and during postnatal life in maintaining the homoeostasis of tissues by a delicate balance with mitosis. Cells die by two ways - necrosis and apoptosis. Necrosis of cells occurs in various disease processes as a passive event while apoptos is an active process which involves synthesis of RNA and proteins by the dying cell. In normal life, it is means of eliminating unwanted, senescent and damaged cells without harming the surrounding normal cells. Defective or excessive apoptosis is involved in many disease processes.[3],[4] Defective apoptosis of autoreactive immune cells may be involved in autoimmune disorders while excessive apoptosis is likely to be a factor in neurodegenerative diseases like Alzheimer′s disease, Parkinsonism and amyotrophic lateral sclerosis.[5],[6] Several dermatological disorders are known to be due to apoptosis of cells either on a limited or massive scale. This article will try to explain the mechanisms involved in apoptosis in some important dermatological conditions.

Morphological changes during apoptosis

Apoptosis can be detected under electron microscope by some characteristic changes. These changes are chromatin condensation on the nuclear membrane, shrinkage of the cell as a whole, blebbing of the cell membrane formation of membrane-bound apoptotic bodies and their phagocytosis and lysis by surrounding cells or macrophages.[1] The process is quite fast and seems to be over in a few hours. The apoptotic bodies, which are membrane-bound structures containing fragments of the nucleus and cellular organelles are either phagocytosed or may be discharged in the lumina of tubular organs. Apoptotic bodies may be phagocytosed by the cells which are normally not known to be phagocytic in function. e.g keratinocytes, hepatocytes etc.

Pathomechanisms

Two main pathways of apoptosis are recognized viz. death receptor pathway and mitochondrial pathway.

Death receptors and apoptosis

Death receptors are transmembrane proteins with parts of the molecules expressed on the cell membrane and inside the cell membrane. They may be expressed on different cells at specific times and their interaction with ligands sets off the process of apoptosis. Six death receptors are recognized. The receptors an their respective ligands are shown in [Table:1].[10]

All the death receptors have a cytoplasmic sequence called ′death domain′ (DD) which is involved in triggering of the so called caspase cascade. Caspases are the enzymes which when activated carry out the actual death sentence by breaking down various structural proteins of the cell. Also, activation of endonucleases is involved in breakdown of nuclear DNA into fragments. These fragments of DNA are important in detecting apoptotic cells by end-labelling or agarose gel electrophoresis which reveals a ladder pattern due to various fragments moving at different rates. Out of these receptors, the Fas receptor (CD 95), first described in 1989 and its ligands are the most studied ones.[10] Reaction of Fas with its ligand (FasL) causes receptor trimerization and recruitment of caspase-8 which subsequently cleaves caspase-3, -6, -7 (downstream caspases) which are executors of death.

The second pathway or mitochondrial pathway is triggered by various stimuli like radiation, stress, withdrawl of growth factors and cytotoxic drugs. These stimuli cause release of cytochrome C from the mitochondria which activates caspase-9 with subsequent activation of downstream caspases leading to apoptosis. All the traditional modalities of cancer therapy like radiation and cytotoxic drugs are thought to act by this pathway.[9]

Genetic control of apoptosis

Several genes are involved in the control of apoptotic process out of which the p-53 and those of Bcl family are important.[2] The p-53 gene is known as the ′guardian of the genome′ and acts by two ways.[11],[12] It detects even a minor damage to DNA and either prevents the division of the abnormal cell till its DNA is repaired or it promotes apoptosis which eliminates the defective cell from the circulating pool of cells. Absence or mutations of p-53 gene are found in almost 50% of malignancies seen in man.

The Bcl family of genes contains about 20 members some of which are pro-apoptotic and the rest anti-apoptotic.[2],[13] BcI-2 is anti-apoptotic while Box is pro-apoptotic. The products of these genes can form dimers in the cytoplasm and exert their control on apoptosis at the level of release of cytochrome C from the mitochondria.

Apoptosis and dermatological disorders

I) Toxic epidermal necrolysis (TEN)Apoptosis of keratinocytes on a massive scale occurs in TEN. This has been well demonstrated by Paul et al by electron microscopy, agarose gel analysis of DNA degradation and by DNA nick end labelling. All the three modalities confirmed the presence of extensive apoptosis of keratinocytes in TEN. Subsequently Viard et al have shown that apoptosis of keratinocytes occurs as a result of interaction between Fas and FasL on keratinocytes.[15] Keratinocytes are normally known to express Fas receptors on their membranes.[16] Fas ligand (FasL) is also known to be expressed on keratinocytes but it is normally not active. Viard et al have shown that keratinocytes from patients with TEN express FasL which is lytically active and induces apoptosis in Jurkat cells (T lymphocyte line) in vitro.[15] They proposed that Fas-FasL interaction between adjacent keratinocytes (which express both) is responsible for the apoptosis. They could evolve a new therapy for TEN by this knowledge. Pooled human intravenous immunoglobulin (IVIG) contains naturally occurring, anti Fas antibodies. Viard et al showed that if keratinocytes in vitro are incubated with IVIG (which blocks Fas receptors), subsequent exposure to FasL does not lead to apoptosis as the Fas receptors are already blocked. This experimental work which suggested a possible benefit of IVIG in treating TEN led them to an open study in which they treated 10 patients with high doses of IVIG (0.2 to 0.75 gm/ kg per day) on 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. This good response was subsequently confirmed by Stella et al.[17] Stella et al also used high doses of methylprednisolone (250 mg 6 hourly) for first 48 hrs along with high doses of IVIG (0.6-0.7 gm/kg/day) for 4 days. Out of 9 patients treated by Stella et al, 8 healed and survived.

(II) Lichen planus and lichenoid reactionsLichen planus is characterized by epidermal basal cell injury and colloid body formation. Colloid bodies or Civatte bodies are apoptotic cells. Bloor et al have shown that in oral lichen planus approximately one apoptotic cell is detected per mm of basal layer. They have also shown that the number of apoptotic cells is proportional to the density of lymphocytic infiltrate. Bcl-2 expression in these lesions is weak or absent and Box expression is localized to the upper prickle cells. Neppelberg et al have shown that in oral lichen planus lesions, apoptotic cells are confined to the basal layer and that there is prominent expression of Fas receptor and its ligand throughout the inflammatory cell infiltrate and in basal cell area.[19]

In cutaneous lichen planus lesions, Shimuzu et al have demonstrated that the infiltrate contains both CD4 and CD8 cells and the perforingranzyme B pathway may be responsible for apoptotic death of basal cells.[20] The major components of the cytoplasmic granules of cytotoxic T cells and natural killer (NK) cells are perforin and granzymes. Perforin is a pore-forming protein which drills a hole in the membrane of the target cell. Granzymes are serine proteases of which four have been identified (granzyme A, B, 3 and H). Granzymes can activate or be a substitute for U.I. L. 1 -converting enzymes (ICE) which can activate the caspase pathway. By immunoelectron microscopy, Shimizu et al could demonstrate that granzyme B molecules were secreted from a lymphocyte into an apoptotic keratinonocyte. They thus suggested that the granzyme -B- positive CD8 cells may be responsible for inducing apoptosis of keratinocytes in lichen planus.[20]

(III) Apoptosis in HIV infectionThe immunodeficiency in HIV infection and acquired immuno deficiency syndrome (AIDS) is characterized by progressive depletion of CD4 lymphocytes. However the features of immunodeficiency cannot be attributed to the dysfunction of HIV infected cells as they are a minority. Along with CD4 lymphocytes, there is also seen a depletion of CD8, CD16 and B lymphocytes. HIV-1 Tat protein, a powerful transcription factor, is a likely candidate which induces apoptosis in various immunocompetent cells to contribute to the immunodeficiency. It has been shown that the recombinant Tat protein induces death in cells uninfected with HIV 1.[21] Also Jurkat cells transfected with Tat gene undergo apoptosis.[22] HIV-1 Tat protein has also been shown to induce apoptosis in peripheral blood mononuclear cells from healthy donors. Thus apoptosis of immune cells is likely to be a mechanism by which these cells are depleted in patients infected with HIV.

(IV) Other conditionsAcute graft versus host disease (GVHD), which occurs as a complication of allogeneic bone marrow transplantation is due to an attack of donor lymphocytes on the tissues of hosts and affects mainly the skin, liver and gut. Apoptosis within these organs has been shown to be the cause of the lesions seen in these organs.[23] The Fas ligand expressed on the donor lymphocytes is responsible for inducing Fas mediated apoptosis in the cells of the target tissues.

Defective apoptosis of malignant cells due to loss of Fas expression is found in basal cell carcinomas and malignant melanomas.[24],[25] Absence of Fas receptors on the cell surface allows the malignant cells to escape Fas mediated apoptosis. Thus defective apoptosis is a feature of malignant conditions. Reinduction of Fas expression on tumor cells of basal cell carcinoma by intralesional injection of interferon-alpha causes regression of the tumour by apoptosis.[24]

In conclusion, apoptosis which is normally a mechanism of eliminating senescent and abnormal cells, goes awry in various pathological conditions understanding the various mechanisms involved in excessive and defective apoptotic processes to help researchers in developing new treatment modalities for these disease processes.