Killer lymphocytes and how they kill

Killer lymphocytes

and how they kill

E.R. Podack Department

of Microbiology and Immunology, University School of Medicine, Miami, Florida, USA Current

Opinion

in Cell Biology

Introduction

Lymphocyte-mediated cytotoxicity is a fundamental effector mechanism of the immune system. It is thought to be responsible for the elimination of virus infected cells, of transplanted tissues and possibly of tumour cells. Killer cells are thus charged with the task of lysing host cells, if and when they have been altered by intrinsic or extrinsic events. Two types of killer cells have been described. Cytotoxic T cells use the T cell receptor to distinguish self and altered self by the recognition of altered major histocompatibility complex (MI-X) molecules. MHC molecules have the ability to bind short peptides (a process termed antigen presentation). The association of a foreign peptide with self MHC is recognized by T cells as altered self, just as a foreign (allogeneic) MHC molecule of transplanted tissue presents as altered self, and thus is a recognition signal for the host’s T cells. Recognition is a clonal event followed by clonal expansion of the responding T cells and development of certain functional properties including cytotoxicity to the specific target. The second type of killer is known as the natural killer (NK) cell. The recognition structure of NK cells is not known, however, it is not clonally distributed. Generally, NK cells kill rapidly proliferating cells that bear no or low numbers of MHC molecules. Upon stimulation with interleukin 2, a T cell growth factor, activated NK cells increase their cytotoxicity and kill virtually all tumour cells in vitro.

Killing

mechanisms

Lymphocyte-mediated target cell lysis involves plasma membrane and nuclear damage. The plasma membrane becomes leaky to ions and macromolecules. The nuclear structure undergoes chromatin condensation and DNA cleavage to nucleosome size DNA fragments (Russel, Immunol Rev 1983, 72:97-16). These events follow obliga tory killer-target cell contact (conjugation) during which the lethal hit is delivered. Subsequent lysis of the target proceeds in the absence of the killer cell. The molecular mechanisms that represent the lethal hit are controversial. The following three proposals for the mechanism of lymphocyte mediated cytolysis have been

of Miami,

1989, 1:929-933

made (Fig. 1): (1) cytolysis by vectorial secretion (constitutive or induced); (2) cytolysis by membrane-associated toxins, and (3) cytolysis by metabolic events. The secretory

model

for target

cell lysis

Killer lymphocytes contain secretory granules which (Poclack and Konisberg, J Eq Med 1984,160:69>710; MUlard et al, JE3qo Med 1984, X&:75-93), in isolated form, have strong calcium-dependent cytotoxicity for nucleated cells and erythrocytes. Upon killer cell-target conjugation the killer cell re-orients its Golgi complex (Kupfer et al, J Mol cell Immunof 1985,2:37-41) and granules (Yanelli et al, J Immunoll986, 136:337-382) towards the binding site and begins to vectorially secrete individual granules into the intercellular space between the two cells. The re-orientation is preceded by waves of calcium fluxes of both intracellularly mobilized and transmembrane-transported calcium (Engelhjard et al, Ann NYAcadSci 1988, 532303313). On delivery of one or more granules to the target membrane a steep rise in calcium occurs in the target followed by violent blebbing of the membrane and subsequent nuclear disintegration. Composition of cytolytic granules Granules isolated from killer lymphocytes contain cytolytic proteins (perforin; Podack and Dennert, Nature 1982,302:442-445; Dennert and Podack, JE.@ Med 1983, 15714831495; Masson and Tschopp, J Biol Gem 1985, 260:906!%9072; Podack et al, Proc Nat1 Acud Sci USA 1985, 82:8629+%33) and several proteases @-anzymes; Masson and Tschopp, Cell 1987, 49:679-685; Hameed et al, J Immunoll988, 141:3142-3147; Pasternack and Eisen, Nature 1985, 314:743745) as well as proteogfycans (Table 1; Schmidt et al, Nature 1985,318:289-291). Per-for-in is cytoiytic by virtue of its calcium-dependent binding to phospholipids (Tschopp et al, Nature 1989, 337~272-274; Yue et aA, Mol Immunoll987,24:647-654), membrane insertion and intramembmnous polymerization (Podack, Immunol T&y 1986,6:12-16; Podack, J Cell Biocbem 1986,30:133136) to transmembrane channels of functional diameters from 2-16 nm (Figs 2 and 3). These transmembrane pores allow (1) the equilibration of ionic gradients with the loss of the transmembrane potential; (2) the inllux of calcium ions and subsequent nu-

Abbreviations MHC-major

histocompatibility

(Q Current

complex;

Science

NK-natural

killer;

TN&tumour

Ltd ISSN 0955-0674

necrosis

factor.

929

930

Cell-to-cell

contact

n fb)

Fig. 1. Three models for lymphocyte mediated cell lysis. (a) Vectorial secretion of cytolytic granules onto the target membrane. Cytolysis occurs due to pore formation by perforin and due to DNA degradation by calcium influx. This pathway is operative in natural killer and cytotoxic T cells. Cytotoxic T cells may have additional killing mechanisms not dependent on the presence of calcium. fb) Secretion independent lysis through contact with a membrane-bound cytotoxic factor of tumour necrosis factpr-like nature. (c) Cytolysis by metabolic events such as the generation and transport of ATP which can permeabilize cells. Whether this process could also be responsible for DNA degradation is not known. C, granule.

clear disintegration [l], and (3) the entry of other toxic factors through repair endocytosis (Podack et al, Ann NYAcad Sci 1988, 532:292). Perforin is homologous to complement proteins C6, CT, C8 and C9 (Fig. 41, which are the cytolytic channel-fomring proteins of complement [ 21 (Lowrey ef al, Prrx Nut1 Acud Sci USA 1989, 86:247-251; Shinkai et aA, Nature 1988, 334:525-528). Granqmes are homologous to mast cell granule proteases and to cathepsins, proteases contained in granules of neutrophils (Bleackley, Curr T@ Microbiol Immunoll988,140:67; Hershberger et al, Curr Tw Micro biol Immunoll988, 140:81). Their specificity is tryptic or chymotryptic, however, their natural substrates and function is not known. Possible functions include: (1) contribution to cytotoxicity; (2) killer cell recycling by deadadhesion [3] ; (3) killer cell migration by digestion of in-

tercellular matrix proteins, and (4) specific activation of immune functions in bystander cells. The proteoglycan specifically binds the granqmes and per-for-in through its sulfated carbohydrate side chains. Functionally, this complex formation may result in a focusing device for cytolytic factors upon secretion. Proteoglycans may also be involved in the protection of killer cells from their own cytolytic factors. Other granule factors have been less well characterized and it is not clear at present whether and how they contribute to lysis. Other

secreted,

cytotoxic

products

of killer

lymphocytes

Lymphotoxin, tumour necrosis factor (TNF) and natural killer cell cytotoxic factors (S&mid et al, Proc Nat1 Acud Sci USA 1986, 83:1881-1885; Bonavida et al, In Effector mechanisms of the Immune System, volume II edited by

LvmDhocvte

Fig. 2. Electronmicroscopic complexes in side view

appearance appear (inset)

of poly perforin on membranes. as hollow cylindrical complexes

The internal diameter of 16nm length.

of the

ring structure

killinn

Podack

of 16 nm. Poly perforin

931

932

Cell-to-cell

contact

. Table

1. Composition

of human

killer

Protein

Molecular weight fkD)

Perforin

7oooo

Cranzyme

1’

32000

Granzyme

2

3oooo

Cranzyme

3

28000

Chondroitin Others?

sulfate

A

cell granule

Pore

formation

DNA

Protease Tryptase Protease Chymase Protease Tryptase Packaging breakdown, chemotaxis

2ooooo

‘In the murine system seven granzymes chemotactic factors for monocytes and Greenberg, / lmmunol 1989, 142:54%548;

Property

Function

(up

to 16nm)

Calcium-dependent polymerization Disulfide-linked homodimer Monomer

Cytosolin HF HLP

Monomer Proteoglycan

of similar properties have been neutrophils and tumor necrosis Liu et al., Cell 1987, 51:393-403).

described. factor-related

tOther factors include low molecular factors of higher molecular weight

weight (Roussel

and

toxic T-cells generated in vitro and in vivo contain perforin and granules, and secrete these components during the cytolytic attack Reports by Berke and Rosen ( Truns pkant Proc 1987,19:412+6) of the absence of granules and perforin in peritoneal exudate cells were based on insensitive techniques and have been shown to be incorrect (Muller, Eur J Immunol1989, in press; Nagler and Anderson, J Immunoll989, in press). However, evidence has been presented to indicate that the cytolytic granules of T cells, even though present and secreted under physiological conditions of attack, may not be responsible for target ceU lysis. Three lines of evidence against granule mediated target cell lysis in T-cells have emerged:

., 4 ._

(1)

:

Fig. 3. Schematic a phospholipid

Synonym

drawing of a poly perforin complex inserted bilayer. The scheme is drawn to scale.

into

Podack. CRC Press, 1988, pp 91-104) are cytolytic for some cells. Their mechanism of action involves nuclear d&integration ln susceptible target cells. These proteins are apparently not stored in secretory granules and are secreted ln the constitutive pathway similar to other lymphokines. It is Ukely that they are also secreted vectorlally towards the killer-target conjunction site.

Controversies

General agreement exists to suggest that the main pathway by which NK cells kill targets is coupled to their secretion of cytolytlc granules. It is now also clear that cyto-

(2)

(3)

In the absence of extracellular calcium and on (partial) depletion of the killer cells intracellular calcium, the secretion of granzymes during killer ceU attack is greatly diminished whereas target lysis is much less inhibited (Ostergaard and Clark, J Immunol 1987, 139:35733579; Trenn et al, Nature 1987, 330:72-73). It is not clear whether diminished granzyme secretion correlates with diminished perforln secretion. In other words, it is possible that perforln and granzymes reside both in identical and in independent compartments. Cyclosporin A inhibits granule secretion from killer cells and the lysis of erythrocytes (presumably via perforin), however, it has a lesser effect on the lysis of nucleated target cells [4]. Inhibitors of calcium-dependent granule secretion and protein synthesis blockers inhibit both the constitutive and the induced pathway of cellular secretion yet do not or only slightly affect T cellmediated lysis (Clark et al, Immunol Rev 1988, 103:37-52).

The evidence under (3) has been interpreted to rule out any secretory protein of T cells as being responsible for

lymphocyte

killing

Podack

Fig. 4. Homology of perforin with the terminal, channel-forming complement proteins.

c7 C8Ci Perforin

C8P c9

I

Thrombospondin-like

I

Low

m

G-binding

density

sequence

lipoprotein

receptor-like

sequence

?

Membrane-binding growth

sequence

domain factor

receptor-like

sequence

lysis.This would be correct unless a calcium-independent induced-secretion pathway exists.

Annotated reading

Nonsecretory

l l e

models

for

T cell-mediated

lysis

Cytolysis by membrane-associated toxins TNF can exist as a membrane-associated form. The membranes of cytotoxic T cells display m-like factors (5@60 kD) that may be internalized by the target cell upon membrane contact and cause target cell destruction (Liu et al, Proc Natl Acud Sci USA1989, 863286-3290). Cytolysis by metabolic events Extracellular ATP is cytotoxic for most cells by membrane permeabihzation. Cytotoxic T cells are, however, resistant to ATP because they are endowed with membrane-associated extracellular ATFQses. It has been suggested by Sitkovsky therefore, that ATP may represent the lyric effector molecule of cytotoxic T cells. It might be added that other metabolic events could conceivably result in the synthesis of toxins of non-proteinaceous nature. These possibilities are largely unexplored. It will be interesting to observe the development of this area of research and to see what surprises are still to come.

Acknowledgements This

work

was supported

by USPHS

grant

A121999-05

CA39201-05.

references

Of interest Of outstanding

and recommended

interest

1.

HA~IEED A, O&EN KJ, LEE M-K, LICK~ENHEID MG, PODACK ER: Cytolysis by Ca-permeable transmembrane channels. Pore formation caoses extensive DNA degradation and cell lysis. J E@ Med 1989, 169765778. The molecular mechanism of DNA degradation by pore farmers is investigated The role of calcium is established and the dogma that pore fanners don’t cause DNA degradation is challenged. l e

2.

LICHTENHEID MG, OL?EN KJ, LIJ P, LOWWY DM, HAMEED A, HENGARTNER H, PODACK ER (Letter to the Editor) Structure and function of human perforin. Nature 1988, 335:44%452. The sequence of human perforin is established It is shown to be homologous to complement proteins, but not, however, inhibited by homologous restriction factor. l e

3.

HAYES MP, BERREBI GA, HEW PA: Induction of target cell DNA release by the cytotoxic T lymphocyte granule protease graruyme A J Ejgp Med 1989, 170:933946. This paper presents evidence to indicate that granzyme A may be re. sponsible for DNA degredation. The DNA degrading activity copurilies with BLT esterax. .

4.

L~NCKI DW, KAPER BP, FITCH Fw: The requirements for triggering of lysis by cytolytic T lymphocyte clones II cyclosporin A inhibits TCR-mediated exocytosis but only selectively inhibits TCR-mediated lytic activity by cloned CTL. J fmmunol 1989, 142:416-424. Evidence is presented to separate the mechanism of the lysis of nucleated cells and that of erythrocytes mechanism by killer cells. Cyclosporin has inhibitory effects only on the granule mediated pathway of killing l e

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