Invited Review

Mediators of Inflammation 4, 5-15 (1995)

DURING the last few decades it has been recognized that

cell death is not the consequence of accidental injury, but
Apoptosis and inflammation
is the expression of a cell suicide programme. Kerr et aL
(1972) introduced the term apoptosis. This form of cell
death is under the influence of hormones, growth factors C. Haanen and I. Vermes c*
and cytokines, which depending upon the receptors
present on the target cells, may activate a genetically
controlled cell elimination process. During apoptosis the Department of Clinical Chemistry, Medical
cell membrane remains intact and the cell breaks into
apoptotic bodies, which are phagocytosed. Apoptosis, in
Spectrum Twente, P.O. Box 50.000, NL-7500
contrast to necrosis, is not harmful to the host and does KA Enschede, The Netherlands
not induce any inflammatory reaction. The principal
event that leads to inflammatory disease is cell damage,
induced by chemical/physical injury, anoxia or starva- ca Corresponding Author
tion. Cell damage means leakage of cell contents into the
adjacent tissues, resulting in the capillary transmigration
of granulocytes to the injured tissue. The accumulation of
neutrophils and release of enzymes and oxygen radicals
enhances the inflammatory reaction. Until now there has
been little research into the factors controlling the accu-
mulation and the tissue load of granulocytes and their
histotoxic products in inflammatory processes.
Neutrophil apoptosis may represent an important event
in the control of intlamtnation. R has been assumed that
granulocytes disintegrate to apoptotic bodies before their
fragments are removed by local macrophages. Removal
of neutrophils from the inflammatory site without re-
lease of granule contents is of paramount importance for
cessation of inflammation. In conclusion, apoptotic cell
death plays an important role in inflammatory processes
and in the resolution of inflammatory reactions. The facts
known at present should stimulate further research into
the role of neutrophtl, eosinophil and macrophage
apoptosis in inflammatory diseases.

Key words: Apoptosis, Cytokines, Growth factors, Hormones,

Inflammation, Integrins.

Introduction phenomenon, and a dynamic balance between cell

proliferation and cell elimination. ,4 Programmed cell
During the last few decades it has been recognized death is responsible for the deletion of useless,
that cell death is generally not the consequence of unwanted or crippled cells, remodelling of organs,
ageing or accidental injury, but that cells die as the canalization of ducts, fashioning of the body, forma-
expression of a genetically programmed active cell tion of digits, and fusion of palatal shelves.
suicide. This physiological form of cell death, which Necrosis has been the traditional term for acciden-
is profoundly influenced by the extracellular tal cell death. Necrosis occurs in response to harmful
microenvironment, is of fundamental significance in insults such as physical damage, hypoxia,
embryogenesis, morphogenesis, and in the pro- hyperthermia, starvation, complement attack, and
cesses that govern tissue shape and cell renewal. chemical injury. The earliest morphological changes
Active elimination of cells was observed originally in that occur during necrosis, are swelling of the cyto-
1951 by GlOcksmann and in 1966 by Saunders as an plasm and organelles. These changes are the expres-
essential feature in the development of vertebrates sion of the loss of selective permeability of the
and invertebrates, respectively. They recognized pro- cytoplasmic membrane. This demolition results in
grammed cell death as a mechanism by which un- dissolution of the organelles, leaking of the cellular
wanted or useless cells are eliminated. In the past ten contents into the extracellular space, and finally in
years it has become increasingly apparent that pro- the disappearance of the cytoplasmic membrane.
grammed cell death occurs not only during embry- Necrosis affects tissue areas or groups of contiguous
onic development, but appears to be a widespread cells and elicits an inflammatory reaction in the

1995 Rapid Communications of Oxford Ltd Mediators of Inflammation Vol 4 1995 5

C. Haanen and I. Vermes

adjacent viable tissues in response to the released haematopoietic system. Neutrophils undergo
cell debris. apoptosis during resolution of the inflammatory re-
Apoptosis shows, morphologically, a distinct pat- action. Apoptosis of lymphocytes is an essential
tern of cell resolution. The earliest changes include mechanism in the regulation of the immune system.
the loss of cell junctions and specialized membrane It occurs during evolution of hormone dependent
structures such as microvilli. The cytoplasm shrinks organs (prostate, endometrium, mammary tissue).
and the nucleus coalesces into condensed masses, Cell injury due to a variety of agents (viral infection,
which later break up into fragments. The physical injury, chemical insults) may also lead to
mitochondria initially remain intact. The apoptosis. In general, any event that can produce
endoplasmic reticulum transforms into vesicles that necrosis by cell destruction (toxins, radiation) can
fuse with the cytoplasmic membrane. These pro- induce apoptosis if the cell initially survives. Finally
cesses result in contraction of the cytoplasmic vol- apoptosis has been demonstrated in premalignant
ume, associated with the loss of intracellular fluid and malignant tissues. Comprehensive reviews have
and ions. The cell adopts a convoluted outline and recently been given by Ellis et al., Alison and.
subsequently breaks into several membrane-en- Saraf, Cohen, Schwartzman and Cidlowski, 1
closed apoptotic bodies containing well-preserved Gewitz, 11 and Vermes and Haanen. 12
organelles and nuclear fragments. The apoptotic The most prominent morphological features and
bodies are phagocytosed and digested by nearby biochemical differences between apoptosis and
resident cells. The engulfing cells belong mostly to necrosis are summarized in Table 1. This review
the mononuclear-phagocyte system, but epithelial or covers present knowledge about mediators and
endothelial cells, and even tumour cells, may also be modulators of apoptosis, and the biochemical
involved. It has been recognized that apoptotic bod- mechanisms and its genetic control. Special emphasis
ies themselves provide a stimulus for phagocytosis is given to the influence of hormones, cytokines and
by changes on their cytoplasmic membrane. Once growth factors in the occurrence of apoptosis and to
ingested the apoptotic bodies undergo rapid degra- the role of apoptosis in inflammatory disease.
dation. Apoptosis does not induce any inflammatory
reaction. A characteristic biochemical feature of the
process is double-strand cleavage of nuclear DNA at Mediators/Modulators of Apoptosis
the linker regions between nucleosomes, leading to Apoptosis is induced by stimuli that trigger
the production of oligonucleosomal fragments of intracellular responses that result in a characteristic
+ 200 kDa or multiples of it. Apoptosis is not harmful type of cell death. This concept implies the existence
to the host and is in fact necessary for a normal of specific stimuli and the presence of target cells. An
existence. Distinctive morphological and biochemi- important characteristic of apoptosis is that it is in-
cal characteristics have been reviewed by Wyllie et duced by withdrawal of cell specific stimuli and can
al.3 and Trump et al.5 be inhibited by the action of cell specific hormones,
In 1972 Kerr et al. described the ultrastructural and growth factors and mitogens. We may speculate
morphological differences that can be observed be- about the advantages of celt survival, depending
tween necrosis and programmed cell death6. They upon signals produced by other cells. One possibility
introduced the term apoptosis as opposite to that of is that it could provide a mechanism for eliminating
necrosis. Apoptosis is derived from the Greek term cells that end up in an abnormal location. For exam-
0trtort’mmg that indicates the fall of leaves from trees ple, when a tissue is lacerated, cells may become
or the shedding of petals from flowers. displaced or transported by the circulation into
At present, the term apoptosis is often used syn- places where they could cause trouble if they sur-
onymously with programmed cell death. The vived and proliferated. The same mechanism may
melding of the two concepts may lead to confusion. prevent haematopoietic stem cells from proliferating
Programmed cell death is the definition of a ’con- outside the bone marrow, or cancer cells from estab-
certed programme’ of cell elimination during meta- lishing metastases. Another advantage of cell survival
morphosis, embryogenesis and morphogenesis. being independent of the presence of specific
Apoptosis indicates the phenomenon of ’physiologi- growth factors, is that control of the total number of
cal cell death,’ which does not include any program- distinct cell types, because they have to compete for
ming. limiting amounts of growth factors, establishes a
Apoptosis takes place during embryogenesis, dur- continuous selection for the most competitive and
ing the development of the nervous system and the vital cells.
immune system, in the course of normal tissue turn
over, and after withdrawal of trophic hormones or Hormones: In mammals apoptosis is prominent in
cytokines from target tissues. Apoptosis is implicated certain tissues undergoing atrophy as a result of
in the homeostatic cell balance in normal adult tis- withdrawal of hormones. It is observed during tissue
sues such as intestinal crypts, the skin, the involution, for example in the uterus after delivery
6 Mediators of Inflammation. Vol 4. 1995
 Apoptosis and inflammation
Table 1. Morphological and biochemical differences between apoptosis and necrosis

Features Apoptosis Necrosis

Stimuli Physiological Pathological

Origin Lack of growth factor Anoxia, Starvation
Hormonal influence Physical insult
Mild toxic stimulus Chemical injury
Occult phase Minutes to hours None
Adhesion property Immediately lost Initially intact
First manifestation Cell shrinking Cell ,swelling
Nuclear changes Condensation, karyorrhexis Karyolysis
Nuclear chromatin Margination, segmentation Nuclear folding
Nucleolar changes Intact, later degraded Granulated
Membrane integrity Persists for some time Early failure
Surface morphology Smoothing, blebbing Lysis
Surface changes Expression vitronectin None
thrombospondin None
Cytoskeletal changes Surface protrusions Fragmentation
Cytoplasmic budding Leakage of cellular contents
Formation of apoptotic bodies
Mitochondria Initially unaffected Swelling, Ca2/ uptake
ER/Golgi apparatus Initially unaffected Dilated
Organelles Structurally intact Swollen, leaky
Protein synthesis Process can be blocked by Not affected by antibiotics
actinomycin D,
cycloheximide
Ca2/ T, endonuclease T
Cytoplasmatic changes

Nuclear changes
,
transglutaminase 1"
p53 bcl-2,1,, c-myc T
Internucleosome cleavage
Rupture lysosomes
Release of content
Diffuse degradation
DNA laddering DNA smear
Cells affected Individual cells Groups of contiguous cells,
Scattered cells tissue areas
Cell elimination Engulfment by macrophages Exudative inflammation in
and endothelial cells adjacent tissues
Scar formation Absent Present

and in the breast gland after weaning. prevented when exogenous NGF is provided. 19,2
Hypophysectomy induces apoptosis in the adrenal There is evidence that the targets of neurons may not
cortex and in the thyroid gland. Apoptosis occurs in be the only source of NGF and that other peptides
endometrial cells deprived of steroid hormones 13,14 may act as a survival signal for neurons and glial
and in the prostatic glandular acini after cells. Such peptides include platelet derived growth
orchidectomy. 15 While some hormones prevent factor (PDGF), insulin-like growth factor (IGF),
apoptotic death of susceptible cells, others trigger fibroblast growth factor (FGF) and ciliary
apoptosis; for example, corticosteroids in thymocytes neurotrophic factor (CNTF). ’1-25 Cells that are in cycle
and lymphocytes (see Table 2). ,16 The mechanism of are much more dependent upon the presence of
lymphocyte killing by glucocorticoids is unclear. growth factors, than are non-cycling cells. Fibroblasts
Transcriptional regulation is implicated since an in- engineered to express c-myc and which therefore
tact transcriptional transactivation domain of the constitutively remain in cycle, undergo apoptosis
glucocorticoid receptor gene is required. 17 In all these when deprived of a restricted group of cytokines,
examples the signals that either trigger or prevent principally IGF an PDGF. 26 The death of
apoptosis also control other aspects of growth and haematopoietic cells, such as normoblasts,
development of susceptible cells. In other words neutrophils and megakaryocytes, has the morpho-
both cell replication and cell death are regulated in logical and biochemical characteristics of apoptosis.
concert to produce growth and cell regression, and The suppression of apoptotic cell death of
to balance the size of organs. haematopoietic (precursor) cells requires the con-
tinuous presence of haematopoietic growth factors
Growth factors: It has been suggested that all animal such as erythropoietin (EPO) and colony stimulating
cells are dependent on the presence of at least one factors (CSFs). 27 Next to CSFs, IGF also prevents the
survival factor. 18 These survival factors include occurrence of apoptosis in IL-3 dependent
growth factors. The nerve growth factor (NGF) was haematopoietic cells. 28 Apoptosis of erythroid pro-
the first growth factor to be identified and character- genitor cells is suppressed by erythropoietin
ized. Neurons die if they receive inadequate (EPO). 29, Williams et al. 27 observed that occurrence
neurotrophic support; conversely neuronal death is of apoptotic death of myelo-monocytic progenitor
Mediators of Inflammation. Vol 4. 1995 7
C. Haanen and I. Vermes

Table 2. Enhancement or suppression of apoptosis by tissue specific hormones, growth factors and cytokines
Cells/tissues Apoptosis
Enhance Suppress
Hormones
Adrenal cortex Adrenocorticotropic hormone
Prostrate Testosterone
Endometrium Oestrogen hormone
Progesterone
Thymocytes Corticoids

Growth factors
Neurons NGF, PDGF, IGF, FGF, CNTF
Primordial germ cells SCF
Endothelial cells FGF
Haemopoietic cells IGF
Myeloid progenitor cells CSFs, G-CSF, GM-CFS
Neutrophils NGF
Mast cells IGF-1
7-M12 leukaemia cell line TGF-II
Tumour cells Gfs (autocine production)

Cytokines
Neurons LIF
Haemopoietic(precursor)cells IL-3
Activated precursor cells TNF(
Neutrophils IL-3
TNFo
Eosinophils IL-5
Mast cells IL-3
Monocytes/macrophages IL-11, TNF(z
Stimulated monocytes/ IL-4
macrophages IFN3,
Thymocytes TNF
T-lymphocytes IL-2, IL-4
CD4+/CD8+ T-lymphocytes IL-2
Megakaryoblasts IL-11
B-chron. lymph, leukaemia IL-4
blc2 + chronic lymphatic leukaemia IL-4
7-M12 leukaemia cell line IL-3, IL-6
Tumour cells Cytokines (autocrine production)
Abbreviations: CNTF, ciliary neutrophic factor; CSF, colony stimulating factor; G-CSF, granulocyte CSF; GM-CSF, granulocyte/monocyte
CSF; FGF, fibroblast growth factor; IFN, interferon; IL, interleukin; IGF, insulin like growth factor; LIF, leukaemia inhibiting factor; NGF, nerve
growth factor; PDGF, platelet derived growth factor; TGF, transforming growth factor; TNF, tumour necrosis factor; SCF, stem cell factor.

cells was suppressed when either granulocyte colony late the viability of the cells. Apoptosis of sympa-
stimulating factor (G-CSF) or granulocyte- thetic neurons is inhibited by NGF, but is enhanced
macrophage colony stimulating factor (GM-CSF) was by leukaemia inhibitory factor (LIF). 5 The same
added to the culture medium. Just like the mechanism applies for cycling haematopoietic pro-
granulocyte-macrophage precursors, which are lost genitor cells, which undergo apoptosis under the
during culturing unless exposed to GM-CSF or G- influence of tumour necrosis factor-ot(TNF(,), 6 or
CSF, the survival of neutrophils is dependent upon when certain cytokines (e.g. IL-3) are removed. 7,8
the continuous presence of these factors.1.32Recently Many authors have shown that apoptotic death of
it has been described that nerve growth factor (NGF) haemopoietic progenitor cells is prevented by addi-
has an inhibitory effect on the occurrence of apop- tion of interleukin-3 (IL-3). 9-4 The cytokine IL-3
tosis in murine peritoneal neutrophils. 3 Endothelial regulates the development and differentiation of
cells in culture undergo apoptosis when deprived of neutrophils in concert with CSFs. 31-42 Erythropoietin
FGF. 4 A mechanism by which various tumour cells (EPO) retards DNA breakdown and prevents
evade apoptosis is by the autocrine production of apoptosis in erythroid progenitor cells. 44 The
various survival factors, such as IGF or cytokines. haematopoietic cytokines EPO, IL-3 and stem cell
In conclusion, as indicated in Table 2, various factor (SCF) are necessary during the mid-gestation
types of growth factors, such as CSFs, FGF, PDGF, haematopoietic development in the foetal mouse
NGF and IGF inhibit apoptosis of susceptible cells. liver to protect these cells from apoptosis in liver
during ontogeny. 45 Mature neutrophils undergo
Cytokines: Not only growth factors control cell pro- apoptosis on ageing, 4<47 a process that is enhanced
liferation and maturation, for cytokines also modu- by tumour necrosis factor-o(TNFo0. TNF also en-
8 Mediators of Inflammation. Vol 4. 1995
 Apoptosis and inflammation
hances cAMP induced apoptosis in mouse an antigen-presenting cell. This is a positive selection
thymocytes 48’49 and in the human histiocytic procedure, known as MHC restriction. Consequently,
lymphoma cell line U937. 5 TNF secreted by immature T cells that bind to self-antigens or to
lymphocytes and macrophages, triggers apoptosis in antigens not presented in association with the MHC
many cell types. Apoptosis induced in the murine complex are eliminated by apoptosis. 5-7 T cells
leukaemia cell line 7-M12 by transforming growth which survive the selection procedure go on to reach
factor (TGF-II) or cytotoxic agents, can be counter- maturity and leave the thymus.
acted by IL-3 and IL-6. 51 The MHC molecules consist of two types: (1) Class
Mature T lymphocytes undergo apoptosis when MHC displays peptides from proteins made inside
deprived of IL-2, 52,53 whereas IL-2 primes CD4 + and a cell (e.g. virus infected cells, cancer cells); and (2)
CD8+ mouse T lymphocytes to undergo apoptosis Class II MHC displays peptides from proteins that
when, subsequently, they are exposed to antigen have entered the cell from outside (e.g. foreign,
which triggers the TCR. 54’55 IL-2 and IL-4 selectively abnormal, altered proteins, bacterial toxins).
rescue T cell subsets from glucocorticoid-induced T-lymphocytes occur in two varieties, depending
apoptosis. 56 IL-4 inhibits apoptotic cell death in B-cell upon the structure of a co-receptor next to TCR:
chronic lymphocytic leukaemia cells in vitro. 57 (1) T cells with co-receptor CD8+ recognize antigens
The rate of apoptosis of human eosinophils, which only when displayed by Class MHC and be-
is slower than that reported for neutrophils, is de- come cytotoxic lymphocytes (CTL) or natural
layed in vitro by the eosinophil differentiation factor killer (NK) cells. CTLs and NK cells recognize
IL-5, which appears to have no effect on neutrophil foreign antigens displayed by aberrrant, malig-
apoptosis. 24,58,59 Therefore, despite the close nant or virus-infected cells, which they then
haematopoietic origin of eosinophils and destroy by inducing apoptosis.
neutrophils, apoptosis and longevity, like their (2) T cells with co-receptor CD4+ recognize antigens
growth and differentiation, are controlled by differ- only when presented by Class II MHC and
ent specific growth factors and cytokines. become T helper cells (TH cells). TH cells
During apoptosis of monocytes/macrophages IL-1 produce interleukins (ILs); factors that spur other
is released, which binds with high affinity to specific T or B cells into action, such as gamma
receptors on target cells. 6 TNFot and IL-113 prevent interferon (IFNT) <---TH-1 cells and IL-4 <--TH-2
apoptotic death in human monocytes/ cells. 8
macrophages. 1 Apoptosis of stimulated monocytes/ A CTL with a TCR that fits onto an MHC/antigen
macrophages is inhibited by IFNq, (from TH-1 cells complex presented by a macrophage, brings IL-2
and enhanced by IL-4 (from TH-2 cells). 62 Murine receptors to expression. In case of TCR activation
mast cells undergo apoptosis on removal of IL-3. 63 under the influence of IL-2, the IL-2 receptors en-
Human megakaryoblastic cell lines produce IL-11, hance the TCR/CD-3 signal to the nucleus to start CTL
which acts as an autocrine growth factor in these cell reduplication. Otherwise, when deprived of IL-
cells. 4 2, the CTLs undergo apoptosis. After antigen clear-
In conclusion, as summarized in Table 2, cytokines ance, IL-2 production ceases because TH cells are no
released by lymphocytes, monocytes or longer stimulated and consequently the antigen re-
macrophages, which bind to cell receptors, modulate sponsive T cells undergo apoptosis. This mechanism
the viability and occurrence of apoptotic death in has the physiological role of terminating the immune
susceptible cells. response and reducing the increased cell numbers to
original proportions.
Killer cells/cytotoxic lymphocytes: During the matura- Unlike T cells, B cells secrete their receptors, which
tion of T-lymphocytes in the thymus, a rearrange- then circulate in the blood as antibodies. These
ment takes place in the T-cell receptor genes, which antibodies lock onto antigens and help to destroy
resultg in the production of T cells with a tremendous them. B cells express the same variety in antibody
variety of T-cell receptors (TCRs). This random pro- production as do the T cells in their TCRs. If antibod-
cess results in many T cells that bear TCRs and can ies present on B cells engage with an antigen, these
react against components of their own tissues. Self- cells are stimulated to proliferate and to secrete large
reactive T cells are potentially dangerous and must amounts of the same kind of antibodies. One of the
be eliminated before they enter the circulation. In the remarkable qualities of immunoglobulin production
thymus the T cells that react with self-antigens un- is that the affinity of the antibodies to antigen im-
dergo apoptosis, a negative selection procedure. T proves during the period of immune reaction. This is
cells must not only learn to avoid attacking self- due to the fact that the B cells, which carry the most
antigens, they must also learn to detect their targets avidly binding antibodies, are stimulated to prolifer-
only when encountered as a peptide presented as ate at the expense of those bearing the poorer
localized in the groove of the major antibodies, which are deleted by apoptosis. 9, Ger-
histocompatibility complex (MHC) on the surface of minal centre B cells, which do not receive antigen
Mediators of Inflammation. Vol 4. 1995 9
C. Haanen and I. Vermes

stimulation of their surface immunoglobulin, un- RNA and protein synthesis. Administration of RNA
dergo apoptosis. 71 and protein synthesis-inhibitors results in suppres-
Comprehensive reviews about apoptosis in sion or delay or apoptosis. Knowledge about the
immunogenesis have been given by Golstein et aL, 72 genes that regulate the intracellular biochemical
Von Boehmer, 73 Cohen and Duke, 74 Fesus 75 and processes is still fragmentary. A number of genes and
Kabelitz et aL 6 Summarizing, the immune system is gene products* have been implicated in the control
’taught’ during its development and in further life, to of apoptosis. In mammalian systems these include:
distinguish between self and non-self and to improve Fas/APO=I, bcl-2, (c-myc c-myb, c-fos, c-jun), p53,
the quality of the immune response, where apoptosis ced-3/ICE, TRPM-2/SGP. In the following sections
is their ’teacher’. details are given of those genes and gene products
where a role in the apoptotic process has been
Cell receptors.. There are a number of similarities
demonstrated.
between apoptotic cell death and CTL or NK cell
mediated cytolysis, which suggest that these cells Fas gene/APO-1 molecule. Fas is a gene, whose
activate an endogenous cell death programme in the product (Fas) is a membrane-spanning protein, ho-
target cell. From available data it is supposed that mologous to tumour necrosis factor (TNF) receptor
CTL-mediated cytolysis involves two mechanisms: and nerve growth factor (NGF) receptor. 79,8 TNF
(1) engagement of the CTL-TCR with an antigen/ induces surface receptor apoptosis in ta.rget cells via
receptor on the surface of the target cell results in this cell. The Fas product is identical to the cell
activation of the cell death programme in the target surface molecule APO-1. 81 Fas/APO-1 cross-linking
cell; and (2) the same engagement stimulates the by antibody induces apoptosis, which indicates that
release from the CTL of lytic granules that contain Fas product, APO-1, mediates an apoptotic signal
perforins and fragmentins, which damage the cellular into the cells. Apo-1 has been shown to be expressed
membrane of the target cell. The relative contribu- in human B lymphocytic leukaemia cells. Bcl-2 ex-
tions of membrane and apoptotic damage in CTL pression is down-regulated and APO-1 expression
mediated cytolysis are unknown. NK cells, together upregulated by exposition to IL-2, which prepares
with TNF, secreted by TH cells/macrophages, are the cells for anti-Fas/Apo-1 mediated apoptosis. 2
able to trigger apoptosis in many cell types. When T The Fas/APO-1 system has also been shown to be
cells are deprived of cytokines, then mature T cells the target molecule for cytotoxic T cells. Mice bear-
undergo apoptosis. T cells also undergo apoptosis, ing the lpr mutation are defective in Fas, and the
when the triggering of the auxiliary surface molecule thymoma, that they develop is not due to T cell
CD4 of T cells occurs, uncoupled from TCR trigger- proliferation but to cell accumulation, because the
ing. 77 For instance, by this mechanism, CD4 trigger- thymocytes die less readily than their normal coun-
ing of T lymphocytes by the HIV-1 viral envelope gp- terparts. 84
120 may induce apoptosis of T lymphocytes that are
Bcl-2 gene. The bcl-2 oncogene was first identified
not even infected, thus explaining the T-cell deple-
as a gene over-expressed in human follicular B-cell
tion in AIDS patients. TM
lymphomas, following reciprocal chromosomal
Genes: Several genes have been associated with translocation t(14; 18)(q32; q21). In this aberration a
apoptosis, either because they are expressed or not translocation of the proto-oncogene bcl-2 (B Cell
during apoptosis and because their expression/sup- Lymphoma-2_) on chromosome 18 to the IgH locus
pression affects the process of apoptosis. A very on the JH segment of the immune gene on chromo-
characteristic biochemical event in apoptosis is the some 14 has occurred. When over-expressed bcl-2
double strand cleavage of nuclear DNA at the linker inhibits apoptosis in a variety of cells including
regions between the nucleosomes. This results in a cytokine/growth factor-deprived haematopoietic
number of 180-200 base pair fragments and multi- cells and neurons. 5-7 Bcl-2 does not stimulate cell
ples of it, which become visible as a ladder pattern proliferation, but promotes survival of cells in a non-
on agarose gel electrophoresis of the DNA extracted cycling state. Bcl-2 co-operates with disregulated c-
from apoptotic cells. In contrast, in necrosis the DNA myc expression to promote haematopoietic tumour
breakdown occurs randomly, which results in DNA development in transgenic mice. Since bcl-2 inhibits
fragments that present as a smear in electrophoresis induction of apoptosis by c-myc, the basis of this co-
of DNA extracted from necrotic cells. DNA damage operation is presumably the preservation from
induced by radiation or cytotoxic drugs can lead to apoptosis by bcl-2 of c-myc-expressing target cells,
apoptosis. It is even likely that most which then can proliferate. Bcl-2 renders cells less
chemotherapeutic drugs induce tumour cell sensitive to radiation and to cytotoxic drugs; only
apoptosis rather than inhibit tumour cell prolifera- CTL killing is not inhibited. 6 The mechanism by
tion. 37 It has been shown that there is a lag period of
several hours between the triggering and the occur- In the text, genes are given in italic characters and gene
rence of apoptosis, suggesting the probable need for products in roman characters.
10 Mediators of Inflammation. Vol 4. 1995
 apoptosis and inflammation
which bcl-2 blocks apoptosis is unknown, but it may the involved cell. ml
be related to the fact that the gene product resides at The role of the p53 protein can be summarized as
the inner side of the mitochondria, which remain follows:
intact during a large part of the apoptotic process. An
DNA damage -+ p53 protein
attractive hypothesis is that bcl-2 protects cells by
--+ increases survival factor dependence
inhibiting lipid peroxidation, even in the presence of --+ arrest in G1 until repair is completed
reactive oxygen intermediates. 88 Recently, a number
of bcl-2-related genes have been identified, includ-
ing bax, bcl-x, mcll, E1B (in adenovirus), LMW5-HL
(in African swine fever virus), BHRF1 (in
Epstein-Barr virus).
C-myc gene. Growth factors induce expression of
the oncogene c-myc, which indicates that expression
of c-myc is associated with cell proliferation. 89 Other-
-
--> apoptosis if repair fails
DNA damage --> p53 protein
mitotic failure, cell death
-+ progress to malignancy
ced-3/ICE. Recently a cell suicide gene responsible
for programmed cell death in Caenorhabditis elegans
(ced-3) has been cloned. 12 It was demonstrated that
the gene product is a protein, very similar to
wise, deprivation of growth factors causes down-
regulation of c-myc and is accompanied by growth interleukin-l-converting enzyme (ICE). ICE, a
arrest or induces in cells with a high expression of c- cysteine protease, cleaves the inactive precursor of
myc apoptosis. 9 The c-myc protein has apparently a IL-1 to generate an active cytokine. ICE causes
dual role: (1) promoting proliferation in the presence apoptosis in rat fibroblasts in which the ICE gene was
of a relative abundance of appropriate growth fac- introduced and brought to expression, m3 In addition
tors; and (2) inducing apoptosis when c-myc is ex- it was found that bcl-2 could inhibit the cell death
pressed in cells during growth arrest. 91 Vaux et al. 5 brought on by over-expression of ICE gene. The
showed that expression of the bcl-2 gene in pre-B researchers concluded that members of the ced-3/
cells of transgenic mice with a high expression of c- ICE gene family might function in apoptotic cell
myc promotes survival of these cells, which means death in vertebrates.
that bcl-2 co-operates with c-myc to immortalize pre-
MTS1. MTS1 (multiple tumor suppressor 1) en-
B cells. The role of growth factor in relation to c-myc codes for a previously identified inhibitor (p16) of
expression can be summarized as follows
cyclin-dependent kinase 4 (cdk-4). This gene prod-
Growth factor (GF) uct prevents the activation of c-myc by blocking an
--+ c-myc expression q" --+ cell proliferation enzyme in the pathway that allows messages from
GF deprivation the cell surface receptors to switch on c-myc. In 46%
-+ c-myc expression $ --+ cell in GO of 290 different human cancers functional copies of
GF deprivation MTS1 are missing leaving c-myc unchecked and the
+ c-myc expression "I" "1" --+ apoptosis pathway to cell division open. TM
p53 protein. Many studies concerning the p53
protein have shown that this protein has a tumour
Apoptosis and Inflammation
suppressor activity, which is illustrated by the fact
that p53 is the most commonly disrupted gene in Bacterial infections: The principal event that leads to
human malignancies. 92 It has been demonstrated that inflammatory disease is cell damage, which can be
the p53 protein forms a complex with the cell cycle induced by bacterial or viral toxins, chemical or
regulating protein cdc2-kinase after which the cell physical injury, anoxia, starvation, or allergic auto-
becomes arrested in the G 1-phase or may switch to immune attack. Massive cell damage and necrosis
a differentiation mode. % In cells where DNA damage result in leakage of cell contents into the adjacent
has occurred, e.g. after UV or gamma irradiation or tissues. Dissipation of enzymes and mediators of
after chemotherapy, the p53 protein accumulates and inflammation, released from the necrotic cells, results
cell proliferation is arrested. 94-9a Possibly this mecha- in expression of adhesive molecules on endothelial
nism is important for preventing reproduction of cells and on the neutrophils. Receptors on
DNA damage and to give the cell time for DNA repair. granulocytes and monocytes (and lymphocytes),
This capacity of p53 may have the function of elimi- such as Mac-l, LFA-1 and P150.95 (VLA-4), with high
nating those cells in which DNA repair has not been affinity for the adhesion peptides (integrins), interact
successful. Cells that lack active p53 protein, because with their counterparts on the endothelium (ELAM-
of mutation or binding onto other cellular or viral 1, ICAM-1, ICAM-2 and VCAM-1). This mechanism
proteins, are not arrested in the Gl-phase and do not promotes the capillary transmigration of these cells to
undergo apoptosis. 99 Such cells are consequently the injured tissue. The expression of adhesins is
genetically less stable and may develop malignant upregulated by IL-1, TNF and by exposure to
cell clones. 1 Circumstantial evidence exists to pos- chemotaxins such as C’5a, IL-8. The accumulation of
tulate that p53 induces survival factor dependence in neutrophils and release of enzymes and oxygen
Mediators of Inflammation Vol 4 1995 11
C. Haanen and I. Vermes

radicals from the attracted granulocytes underlie the in chronic inflammation. Neutrophil apoptosis may
inflammatory reaction. represent an important event in the control of inflam-
An inflammatory reaction is not always a salutory mation, eliminating the neutrophil for disposal and
response to injurious events. Neutrophil for its capacity to generate and release histotoxic
granulocytes have been implicated in the products. 47 The efficient removal of neutrophils from
pathogenesis of a wide variety of diseases. 15 They inflamed sites, once their biological purpose (killing
contain a large number of agents with the capacity to of invading pathogens, breakdown of cell debris) is
injure tissue and to degrade matrix proteins into completed, is a highly desirable and effective mecha-
chemotactic fragments. 16 Persistent accumulation of nism on the road to reconstitution of the normal
inflammatory cells is associated with destruction of situation. Neutrophils can undergo apoptosis and the
tissue matrix or deposition of scar tissue and can lead associated changes of the cell surface aid their
to the loss of organ function. Examples of such phagocytosis by macrophages. 46 It has been assumed
catastrophic organ damage are given by lung emphy- that granulocytes disintegrate to apoptotic bodies
sema, respiratory distress syndrome, fibrosing before their fragments are removed by local
alveolitis, pneumoconiosis, liver cirrhosis, forms of macrophages. 112 Removal of neutrophils from the
glomerulonephritis, and rheumatoid arthritis. inflammatory site, without release of granule con-
For inflammatory tissues to return to normal, all the tents, is of paramount importance for cessation of
events involved in the evolution of inflammation inflammation. Neutrophil apoptosis is accelerated by
must be reversed. These must include removal of the TNF. 13 This process operates wherever a need
inciting stimulus, cessation of neutrophil accumula- exists to curtail a suppurative exudation, be it the
tion, cessation of further release of histotoxic and resolution of an exsudative infection or the
pro-inflammatory mediators, return of microvascular obturation of an abscess cavity.
permeability to normal and cessation of monocyte The rate of apoptosis of human eosinophils, al-
emigration from blood vessels. The tissue monocytes ready slower than that reported for neutrophils, is
must transmutate into macrophages, which remove delayed in vitro by the eosinophil differentiation
extravasated fluid, proteins, bacterial and cellular factor IL-5. 24’58’59
debris and accumulated neutrophils. Cessation of The processes involved in the control of monocyte
neutrophil and monocyte emigration may occur after recruitment have not yet been elucidated, although
dissipation of chemotactic factors from the inflamed very likely they are governed by similar principles as
tissue, but the mechanism for the loss of chemotaxins described under neutrophil emigration.
has not yet been identified. Macrophages play an important role in reconstitution
Recent reviews dealing with the general aspects of of normal haemodynamics and recovery of tissue
inflammation have been given by Saukkonen et integrity after inflammation. Monocytes are mobi-
al., 17 Haslett, 18 Dinarello and Wolff, 19 and Bonta lized to the site of inflammation by C’5a, MLP, MCP-
and Ben-Efraim. 11 The following paragraph is re- 1 and TGF[. Their elimination from the inflammatory
stricted to the role of apoptosis in the clearance of site presumably takes place mostly by apoptosis,
granulocytes from the ’battlefield’ of inflammation by which is promoted by IL-4 (<---TH-2 cells) and inhib-
macrophages. ited by GM-CSF/M-CSF, IL-3, IFN(<--TH-1 cells), IL-
Granulocytes, where they have infiltrated, release l and TNFO. TM
enzymes, oxygen radicals, cytokines and mediators
of inflammation, locally. All these products may trig- Viral infections: Viruses have developed strategies to
ger potentially dangerous responses within the or- inhibit apoptosis following virus infection, which
ganism. This aspect of inflammation has led to much serves to prolong the life of their target cells and
research into enzyme inhibitors, oxygen radical scav- promote virus replication. Epstein-Barr virus induces
engers and membrane stabilizers, but little attention the expression of bcl-2 related genes in the infected
has been paid to factors controlling the accumulation cells and encodes for a bcl-2 homologue, 15 including
and the tissue load of granulocytes and their EIB in adenovirus, LMW5-HL in African swine fever
histotoxic products. virus, BHRF1 in Epstein-Barr virus infection. 115-118
Tissue kinetics of neutrophils depend upon the Presumably, adenovirus, human papilloma viruses
half-life of the neutrophils, the amount of and SV40 modulate apoptosis also by interaction of
neutrophils that enter the tissue per time unit, and viral proteins with cellular P53.
the speed by which they are removed. Lee et al. TM It has recently been suggested that both qualitative
have shown that bacterial products such as and quantitative defects in CD4+ T cells in patients
lipopolysaccharide, the chemotactic peptides C5a with HIV infection may be the result of activation-
and GM-CSF, all exert an inhibitory effect on the rate induced cell death by apoptosis. 19-22 Since apoptosis
of neutrophil apoptosis. Concentrations of these can be induced in mature murine CD4+ T cells after
mediators are likely to be high in acutely infected cross-linking of CD4 molecules, uncoupled from TCR
tissues, but vary considerably during resolution and triggering, there has been speculation that cross-
12 Mediators of Inflammation Vol 4 1995
 Apoptosis and inflammation
linking of the CD4 molecule by HIV gp120 or by Concluding remarks
gp120/anti-gp120 immune complexes prepares the
cell for apoptosis that occurs when MHC class II Cell death plays an important role in
molecule presents an antigen to the TCR. 77,121,122 immunogenesis, during inflammation and in the
Thus, the mere activation of a prepared cell by a resolution of inflammatory reactions. The mecha-
specific antigen or superantigen could lead to the nisms and mediators involved have not yet been fully
death of the cell, without direct infection by HIV. elucidated, but there is evidence that surrounding
Apoptosis of CD8+ cells has been obscured in vitro cells adjacent to inflammation kill themselves by
even in the absence of an antigenic stimulus. 123,124 apoptosis, increasing the damage caused by inflam-

TISSUE INJURY
bacterial products, toxins, immune complexes, physical injury, cytokines

Chemotaxis
PG 1-2. PGD/E/F vasodilatation
5-HETE chemotaxis
PAF (<--endothelial cells) chemotaxis, increased permeability, leukocyte aggregation
IL-8(<--monocytes) attractant of granulocytes
Leukotrienes increased permeability/oedema
FXll activation kinin formation, coagulation, fibrinolysis
Complement activation C’5a -) chemotaxis, C’3b -) phagocytosis

Cell adhesion
C’5a, TNF -) surface expression on leukocytes of integrins: LFA-1, Mac-l, P150-95
IL-1, TNF -) surface expression on endothelial cells adhesins: ELAM-1, ICAM-1, VCAM-1

Leukocyte margination, Rolling, Adhesion, Emigration, Accumulation

INFLAMMATORY REACTION
Granulocytes release lysosomal enzymes
oxygen free radicals
nitric oxide
Monocytes mobilization, activation, transformation by cytokines
(C’5a, FM LP, F, L- ,MCP- ,TG F-I,TN F)
transformation--)
Macrophages release proteases, collagenases
nitric oxide
arachidonic acid metabolites
fibrinogenic cytokines
angiogenesis (FGF)
growth factors (CSFs,PDGF,FGF,TGF-I)
cytokines (IL-I,IL-8,TNF)
transformation-)
Epitheloid cells induced by IF-y (<--T-cell mediated immunity)

APOPTOSIS of surrounding tissue cells

APOPTOSIS of granulocytes, enhanced by TNF(x, suppressed by CSF, C’5a
APOPTOSIS of monocytes, enhanced by IL-4, suppressed by IL-11,CSF,IFN-y, TNF(x

RESOLUTION INFLAMMATION
FIG. 1. The role of granulocyte recruitment or accumulation and granulocyte/monocyte apoptosis in the evolution and resolution of inflammation.
Abbreviations: C’3b/C’5a, complement factors; CSFs, colony stimulating factors; FXlI, Hageman factor; FGF, fibroblast growth factor; FLMP, formyl-
methionyl-leucyl-phenylalanine; HETEs, hydroperoxy derivatives of arachidonic acid; IFN, interferon; ILs, interleukins; MCP-1, monocyte chemotactic
protein-I; PAF, platelet-activating factor; PDGF, platelet-derived growth factor; PG1,2, D, E, F, prostaglandins; TGF-I, transforming growth factor-I; TNF,
tumour necrosis factor.

Mediators of Inflammation Vol 4. 1995 13

C. Haanen and I. Vermes

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precursors of

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