45

zmmunology today, August 1980

reported. An example of the first is seen in the work of Schrater and her colleagues 22. Athymic mice made higher a n t i - T N P responses to the thymus-independent antigen, TNP-'FicolI' than did euthymic mice of the same strain. This was because euthymic but not athymic mice made anti-idiotype responses. In another system 23, athymic mice again made no anti-idiotype response. However, in this case the antibody response (to phosphorylcholine) was no higher in athymic mice and lacked a later second peak of antiphosphorylcholine antibody-secreting cells which occurred in euthymic mice. Finally, simultaneous immunization with phosphorylcholine and idiotype in adjuvant had no effect on the n u m b e r of cells synthesizing either anti-phosphorylcholine or anti-idiotype antibodies 24. To summarize, at the time of writing, evidence for physiological idiotype-specific regulation has been derived only from oligoclonal responses, particularly responses to thymus-independent antigens. It is not clear if polyclonal responses to antigens are subject to idiotypic regulation or if the lack of evidence is due to the difficulty in measuring such regulation. As far as antibody responses to autoantigens are concerned at least some are polyclonal (see Ref. 4) and again there is no evidence so far that they are controlled by idiotype-specific mechanisms. Nevertheless, autoantibody responses do appear to be limited by suppressor cells. Whether the harmful autoantibody responses characteristic of some autoimmune diseases are refractory to suppression or if there is a defect among the corresponding suppressor cells remains to be determined. I thank Dr R. B. Taylor for his help. References 1 Low, J.A., Cross, L.M. and Catty, D. (1975) hnmunology 28, 469-478

2 Burnet, F.M. (1962) The Integrity of the Body, Oxford University Press 3 Elson, C.J., Naysmith, J.D. and Taylor, R.B. (1979) Int. Rev. Exp. Pathol. 19, 137-203 4 Nye, L., De Carvalho, L.C.P. and Roitt, I.M. (1980) Chn. Exp. hnmunol. 41 (in press) 5 Parks, D.E. and Weigle, W.O. (1980) J. Immunol. 124, 1231-1236 6 Elson, C.J. and Taylor, R.B. (1977) Immunology 33, 635-641 7 Nossal, GJ.V. and Pike, B.L. (1975)J. Exp. Med. 141,904-917 8 Metcalf, E.S. and Klinman, N.R. (1976) J. Exp. Med. 143, 1327-1340 9 Nossal, G.J.V., Pike, B.L., Teale, J.M., Layton, J.E., Kay, T.W. and Battye, F.L. (1979) Immunol. Rev. 43, 18 -216 10 Bruynes,. C., Urbain-Vansanten, G., Planard, C., DevosCleotens, C. and Urbain, J. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2462-2466 11 Elson, C.J. (1977) Eur. J. Immunol. 7, 6-10 12 Layton, J.E., Teale, J.M. and Nossal, G.J.V. (1979) J. Immunol. 123,709-713 13 Raft, M.C., Owen, j.j.T., Cooper, M.D., Lawton, R., Megson, M. and Gathings, W.E. (1975) J. Exp. Med. 142, 1052-1064 14 Gershon, R.K. and Kondo, K. (1971) Immunology21, 903-914 15 Eardley, D.D., Shen, F.W., Cantor, H. and Gershon, R.K (1979) J. Ea~. Med. 150, 44-50 16 Taniguchi, M. and Tokuhisa, T. (1980) J. Exp. Med. 151, 517-527 17 Basten, A., Miller,J.F.A.P., Loblay, R., Johnson, P., Gamble, J., Chia, E., Pritchard-Briscoe, H., Callard, R. and McKenzie, I.F.C. (1978) Eur.J. Immunol. 8, 360-370 18 Tanigucha, M., Takei, I. and Tada, T. (1980) Nature (London) 283, 227-228 19 Naysmith, J.D., Elson, C.J., Dallman, M., Fletcher, E. and Ortega-Pierres, M.G. (1980) Immunology 39, 469-479 20 Adorini, L., Miller, A. and Sercarz, E.E. (1979) J. Immunol. 122, 871-877 2I Jerne, N.K. (1974) Ann. hnmunol. (Inst. Pasteur) 125C, 373-389 22 Schrater, A.F., Goidl, E.A., Thorbecke, G.J. and Siskind, G.W. (1979)J. Exp. Med. 150, 808-817 23 Kelsoe, G., Isaak, D. and Cerry, J. (1980) ,7" Fxp. Med. 151, 289-300 24 Rowley, D.A., Miller, G.W. and Lorbach, I. (1978) J. Exp. Med. 148,148-157

Surface carbohydrates and lectins in cellular recognition Donald M. Weir Immunology Laboratory, Department of Bacteriology, Edinburgh University Medical School, Teviot Place, Edinburgh, U.K. The problem of cellular recognition is of fundamental importance in many areas of biology, not least to the immunologist who is faced with the need to understand the complex relationships between cells of the i m m u n e system, both phagocytic and nonphagocytic, and their ability to recognize and react to foreign materials. Two main types of recognition mechanism are involved: one depends upon the ability of cells to read gradients of diffusabte substances such

as chemotactic factors and the other on cell-surface characteristics such as the glycoproteins of the major histocompatibility system. It is not clear which mechanism is involved in many of the activities of the cells of the i m m u n e system - for example the passage of progenitor (or pre-committed) lymphocytes to the primary lymphoid organs and their final distribution in T-cell or B-cell domains of the secondary lymphoid tissues. It is noteworthy, how© Elsevler/North-Holland Biomedmal Press 1980

46 ever, that lymphocyte differentiation is accompanied by the expression of various glycoproteins at the cell surface 1 and T-lymphocyte differentiation, in particular, by an increase in the sialic-acid content of the Thy-1 antigen 2. This article focuses on certain cell-surface characteristics which a p p e a r to determine the ability of particular phagocytes to recognize and bind foreign material such as bacteria and t u m o u r cells expressing tumour-specific surface molecules.

Surface carbohydrates Surface molecules characteristic of both prokaryotic and eukaryotic cells are carbohydrates and are often associated with proteins or lipids. As informational molecules carbohydrates have m a n y advantages over proteins or nucleic acids, particularly their ability to exist in a variety of spatial configurations, to be linked in a number of different ways (e.g. a and [3 linkages), and readily to form branched structures (e.g. by O and N glycosidic linkages). There are, for example, 1024 possible structures for a twelve-residue oligosaccharide of three mannose, three N-acetylglucosamine, three galactose and three sialic-acid residues. Cell-membrane carbohydrates are made up of nine basic subunits - four 6-carbon sugars (glucose, galactose, mannose, fucose), two 5-carbon sugars (arabinose, xylose), two amino sugars (N-acetylglucosamine, N-acetylgalactosamine) and sialic acid - and these are joined by glycosyltransferases of different specificities to form chain and branched oligosaccharides. Glycoprotein and glycolipids are inserted asymmetrically into the cell m e m b r a n e with the carbohydrate determinants on the outer surface.

immunology today, August 1980

or isolectins with very similar chemical and biological properties 3. In this article some of the binding activity described is referred to as 'lectin-like' and m a y fall outside the strict definition of lectins proposed by Goldstein et al. 4. Lectin-like binding proteins can include sugar-specific enzymes, transport proteins, hormones and toxins. Some of these have multiple binding sites and can agglutinate cells and thus act as a lectin while others have only one site and cannot do so, thus falling outside the strict definition of a lectin. A biological role for the lectin produced by the roothairs of white clover has been suggested by its ability to bring about the binding of the nitrogenfixing rhizobial symbiont Rhizobium tnfollii. Lectins may help to protect plants from plant pathogens and insect predators and they are important in mating systems in lower plants 4. Recognition based on carbohydrates has been described in a variety of primitive species such as the slime mold Dictyotelium discoideum 6. The organism begins its life cycle as a non-social amoeboid vegetative form which, when its food supply is exhausted, beTABLE I. Some plant and animal sugar-specific lectins and lectin-like materials. Source

Carbohydrate specificity

Plant Phytohaemagglutinin (red kidney bean) Wheat germ agglutinin Ridnus commums

i>Gal Nac (~Glc NAc)z, sialic acid 13- D-Gal

(castor bean) Soy bean agglutinin Concanavalin A (jack bean)

~Gal NAc, i>Gal a-D-Man, a- D-GIc

Lens culinarzs

Man, Glc

(lentil) Arachzs hypogaea

Carbohydrates and recognition Cell-surface-expressed carbohydrates or carbohydrate moieties present on other molecules in the tissue fluids are involved in recognition phenomena in a wide variety of plant and animal species. Carbohydrate molecules are recognized and complementary binding interactions instituted by protein or glycoprotein molecules termed lectins. The term was originally used to describe carbohydrate-recognizing molecules derived from plants such as phytohaemagglutinin (PHA) from red kidney beans and concanavalin A from j a c k beans. The carbohydrate specificity of these and some other lectins is shown in Table I. The main characteristics of lectins are their ability to bind sugars, to agglutinate cells, and to stimulate lymphocytes. Most lectins interact specifically with a single sugar although for some the specificity is broader and includes closely related sugars. Certain lectins interact only with complex carbohydrate structures such as those that occur in glycoproteins or cell surfaces. Lectins are usually made up of subunits and occur as a group of closely related glycoproteins

Gal

(peanut)

Bacterial Escherichm coil Salmonella typhzmurzum Pseudomonas ceruginosa

Cholera toxin Diptheria toxin

Man

Man Gal Ganglioside GM1 Oligosaccharide of cell wall Salmonella cholera suis and ovalbumin glycopeptide

Animal Rabbit liver Avian liver Helix pomatm

Gal Glc NAc Gal NAc

(garden snail) Limulus polyphemus

NANA

(horseshoe crab) Electrophorus electrzcus

13-L>Galactoside

(electric eel) Rat Kupffer cells Mouse lymphocyte (thymus) (spleen) Mouse peritoneal macrophages

Gal 13-1~-Galactoside,D-GalNAc a-I>mannoside Glc, Gal, Glc NAc, Gal NAc

Gal Nac: N-acetyl galactosamine; Glc NAc: N-acetyl glucosamine; Gal: galactose; Man: mannose; Glc: glucose; NANA: Nacetyl neuraminic acid.

47

zmmunology today, August 7980

TABLE II. Bindingof organismsto mouse peritoneal macrophage monolayersand inhibition by glucoseor galactose. Organism Streptococcus virzdans Streplococcuspyogenes Streptococcuspneumoniae B. anthracoides Staphylococcus albus Staphylococcus oureus Escherichia coli Pseudomonas aerugmosa

(mucoid)

Per cent (S.E.) Inhibitory sugars binding Glucose Galactose 33,7 19.5

(1.0) (0.8)

+ +

-

23.0 21,1 19.5 21.0

(0.8) (1,0) (0.9) (1.0)

+ + +

+ + +

19.9

(1.0)

+

+

20.9

(1.0)

+

+

28.9

(1.9)

+

+

0

P, aeruginosa

(non-mucoid) *Corynebacteriumparoum

(10390)

* Data from Ogmundsd6ttir, H. M. and Weir, D. M. (1976) Clin. Exp. lmmunol. 26, 334.

comes a cohesive muhicellular sessile slug. Cohesiveness is due to developmentally controlled tectins called discoidins with specificity for N-acetylgalactosamine. Lectin-like activity has been described in invertebrates such as lobsters, snails, horseshoe crabs, and bivalve clams 7. It is likely that both the opsonins which promote phagocytosis and the red-cell agglutinating activity in invertebrate haemolymph depend on recognition of carbohydrates such as N-acetylglucosamine, N-acetytgalactosamine and N-acetylneuraminic acid. The electric organ of the electric eel contains about 400mgkg -1of a lectin with ]3-O-galactoside specificity s. In more highly developed species cell interaction involving carbohydrate recognition has been noted in embryo neural retinal cells 9. The recognition molecules involved are cell-type specific and not species specific - thus chick and mouse neural retinal cells after disaggregation will reaggregate together irrespective of their species of origin. Species-specific recognition by surface carbohydrates has been implicated in sperm-egg interactions in the ascidian Ciona intestinalis, with fucose playing a key role in the binding reaction and in fertilization 1°. It has been suggested that the process of haemostasis may involve the expression of lectin molecules in activated platelets, leading to platelet and red-cell aggregation and clot formation 1~. The specificity of such lectins may be directed at amino sugars and some basic amino acids 12. Lectins appear to be components of intracellular membranes as welt as external membranes and a role in regulation and segregation has been proposed for lysosomal enzymes which are glycosylated 13,14. An external cell-surface receptor (tigatin) has been isolated from thioglycollate-induced mouse peritoneal macrophages which binds acetyl-flD-hexosaminidase A and may be a candidate for a m e m b r a n e receptor that binds and segregates lysosomal enzymes ~5. As far as mammals are concerned one of the best

worked-out examples of carbohydrate recognition by lectin-like molecules is the binding by hepatocytes from a variety of species of asialoglycoproteins such as asialo-orosomucoid or caeruloplasmin. The membrane-bound lectin has been isolated from hepatocytes in homogeneous tbrm and is a glycoprotein consisting of two subunits of mol. wt 48,000 and 40,000 forming aggregates of mol. wt 500,000. 10% of its dry weight is mannose and glucosamine. The integrity of the terminal sialic acids is required for binding which in common with other lectins requires Ca 2+. The asialoglycoproteins with exposed penultimate galactose groups are b o u n d by the hepatocyte m e m b r a n e lectin, taken into the cells, and catabolized. Clearance of circulating asialoglycoproteins is a threshold event and in the case of caeruloplasmin any molecule deficient in only two of the ten sialic acids is selectively removed. It is suggested that hepatic binding of asialoglycoproteins reflects a normal physiological process regulating plasma protein metabolism in vivo 16. In birds, a n d p r e s u m a b l y reptiles, the hepatocyte receptor has a different specificity - in this case for the terminal N-acetylglucosamine residues on Binding (per cent of control)

I

I

I

I

I

stanc

I

I

I

I

acid

Mannose

I

I

•

•

I

I

~amnosc

N acetyl glucosnmine -

Gahctose A

Fucose

Ghcosamine

Oatactonamine Glucose

:

@

:

atue~ontc

acid

Fig. 1. Inhibition of binding of C. parvum strain 10390 to m o u s e peritoneal macrophage monolayers by various carbohydrates at 10 m m o l 1-1 c o n c e n t r a t i o n in D u l b e c c o ' s PBS + B.

Means of at least three replicates * two standard errors.

zmmunology today, August 1980

48 S A L M O N E L L A TYPHIMURIUM (wild type mtd mutants)

hhibition oi binding to mouse PE monolayers by various sugars.

O SOMATIC REPEAT UNIT

acp c'

Man - Rha - G a ~ Genotype Chemotype

STRAIN INHIBITION

OUTER CORE

Glc LI - Gal I -

INNER

''°'I CORE

Ethanolannne

GIc I - Hep II - Hep I

+ smooth

rfaK Rb

Rc

SL 1542

SL 1096

SL 1099

rfaE Re

SL 1102

BY:

GLUCOSE

+

+

GA LACTOSE

+

+

+

GLUCOSAMINE

+

RHAMNOSE

+

+

ND

ND

ND

+

+

+

+

MANNOSE KDO LIPID

A

-

ARABINOSE

Fig. 2. I n h i b i t i o n of b i n d i n g o f S . ty'phimurium w i l d type and mutants to m o u s e p e r i t o n e a l e x u d a t e cell ( P E ) m o n o l a y e r s . A s u g a r w a s r e g a r d e d as i n h i b i t o r y if it r e d u c e d b i n d i n g to 6 0 % of control values w i t h o u t inhibitor. T h e s t r u c t u r e of the l i p o p o l y s a c c h a r i d e of S.typhimurium, wild type a n d m u t a n t s is s h o w n in the s c h e m a t i c f o r m u l a (from F r e i m e r et al. (1978) Acta Path. Microbiol, Scan& Sect. B 86, 53, with permission). + inhibition; - no inhibition. A b b r e v i a t i o n s : G l c glucose; G a l galactose; A b e a b e q u o s e ; Glc N a c N - a c e t y l g l u c o s a m i n e ; M a n m a n n o s e ; R h a r h a m n o s e ; H P Lg l y c e r o - D - m a n n o h e p t o s e ; K D O 2 - k e t o - 3 - d e o x y o c t o n a t e ; N D not done. C o n c e n t r a t i o n of o r g a n i s m s 1-8 x 10~ml -l.

glycoproteins. There is more recent evidence for a similar type of galactose-specific binding by rat Kupffer cells: they have an ability to bind neuraminidase-treated red-blood cells which can be inhibited by N-acetylgalactosamine, D-galactose, lactose (glucose/galactose) and fucose (6-deoxy-L-galactose). The hepatocyte receptor is inhibited in the same way'L In lymphocytes lectin activity has been found in extracts of m e m b r a n e vesicle preparations which agglutinate trypsinized rabbit erythrocytes and neuraminidase-treated rat erythrocytes. There appears to be two types of lectin present in the extracts; one binding complex glycopeptides with terminal [3-galactosides and another binding mannose-rich glycopeptides ~8. Inhibition of binding of mouse anti-Ia and h u m a n D R w sera to the surface of monocytes by mono- and oligosaccharides has led to the view that Ia and D R w surface determinants may be involved in recognition of syngeneic T cells through specific cell-surface carbohydrates on these cells. In addition recognition of target cells by monocytes leading to cytotoxicity might use such a mechanism v),2°.

Binding of bacteria to mouse macrophages O t h e r evidence tot the existence of lectin-like receptors on phagocytes comes from work in the

author's laboratory on mouse peritoneal macrophages. Bacteria are bound to the macrophage membrane by a mechanism that appears to involve recognition of bacterial cell-wall sugars by m e m b r a n e - b o u n d lectin-like receptors of the macrophage. The binding of bacteria by macrophages in monolayers on flying coverslips (90 min at 4°C) can be inhibited by preexposure of the monolayers (15 min at 4°C) to a variety of monosaccharides or their derivatives at a concentration of 10 mmol 1-~. A sugar is regarded as inhibitory if it reduces binding to 60% of control values without inhibitor. Glucose and galactose inhibit the binding of numerous bacterial species (Table II) and Figs 1 and 2 show more detailed results with organisms of known cell-wall composition including mutants with deficiencies in particular sugars. Glucose and galactose or their derivatives appear to be regularly involved in binding and if one or both sugars is not present, the missing sugar becomes non-inhibitory, despite inhibiting the binding of the wild-type organisms. Thus Salmondla lyphimurium core m u t a n t (SL 1102), with no outer core or somatic O repeat units, appears to bind via its inner core components and binding is not inhibited by any of the sugars that inhibit binding of the wild type (Fig. 2). (A similar picture emerges with Klebsiella cerogenes in which galactose fails to inhibit b i n d i n g of the galactose-deficient core m u t a n t

immunologyloday, Augu,~t1980

49

M I O B . ) M a n n o s e , a l t h o u g h a c o m m o n cell-wall sugar, is n o n - i n h i b i t o r y throughout, perhaps because of the p a r t i c u l a r linkages this sugar makes with other sugars 2~. 100

B i n d i n g of t u m o u r cells to m o u s e m a c r o p h a g e s I n a similar assay, mouse peritoneal exudate cells b i n d mouse t u m o u r cells or proliferating mouse e m b r y o fibroblasts at 2 0 ° C over 4 h. Various sugars or derivatives inhibit the b i n d i n g of C 3 H methylchol a n t h r e n e - i n d u c e d fibrosarcoma cells by syngeneic C 3 H a n d allogeneic C F E peritoneal cells (Fig. 3). Glucose a n d galactose or their derivatives seem to be i m p o r t a n t in b i n d i n g in both syngeneic a n d allogeneic c o m b i n a t i o n s while other sugars such as m a n n o s e , L-fucose a n d sialic acid are only slightly inhibitory. W i t h C 3 H mouse e m b r y o fibroblast a n d C 3 H peritoneal cells similar i n h i b i t i o n was found with glucose a n d galactose b u t only w h e n the fibroblasts were in growth phase a n d of low passage n u m b e r (4-5). B i n d i n g to confluent fibroblasts was not i n h i b i t e d by either sugar2L

It- ...............

90

fi-o-Rff~L. . . . . . . . .

i--i-i---i-i

70 50

.

30

.

.

.

10 I

I

I

I

I

I

I

I

i

I

N ~

~o

~

z~

°

z

~

I0O

90

~

z~

Z~

o

~

}i .............

o

;o2~-~ .....

70

N a t u r e of the b i n d i n g site(s) o n m o u s e macrophage membrane T h e ability of mouse peritoneal m a c r o p h a g e s to b i n d Corynebacterium parvum was e x a m i n e d after exposure of the m a c r o p h a g e monolayers to various e n z y m e a n d chemical t r e a t m e n t s 23. T h e possible role of m a c r o p h a g e - b o u n d i m m u n o g l o b u l i n (cytophilic a n t i b o d y ) was excluded by showing that t r y p s i n (0.5 mg ml -~) could remove a n t i b o d y to C. parvura b o u n d in vitro while the ability to b i n d C. parvum was u n c h a n g e d if the cells were allowed to recover from t r y p s i n treatm e n t for 1 h at 37°C in tissue-culture m e d i u m without serum. I m m e d i a t e l y after exposure to trypsin (1 mg ml