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hnmunology Today, vol. 3, Wb. 10, 1982 Med. 148,424 43 .Janeway, C. A., Jr, Sakato, N. and Eisen, H. N. (1975) Proc. Nat[ Acad. &:i. U.S.A. 72, 2357 44 Sakato, N., Janeway, C. A., ,Jr and Eisen, H. N. (19761 (,'old &ring Harbor,~'ymp. Qua.L Biol. 41,719 45 Jorgensen, T., Boegen, B. and Hannestad, K. 11981) in Imrnur.)globulin Idio(ype,~ (Janeway, C., Sercarz, E. and Wigzcll, H., eds), p. 573, Academic Press, New York

(,.-,.,,

46 Kawahara, D.J., Marrack, P. and Kappler, J. (1982) t+d. Proc. 41,366 47 Cerny, J. and Caulfield, M.J. (1981) J. lmmu,oL 126, 2262 48 Eichmahn, K., Falk, I. and Rajewsky, K. (1978) Eur. J. ImmuTtol. 8,853 49 Bottomly, K. (19811 in I,~muuoglobulm ldmtypes (Janeway, C., Sercarz, E. and Wigzell, H., eds), p. 517, Academic Press, New York

)

The characterization of cell receptors for IgG Extensive study of the b i n d i n g of lgG subclasses to intact homologous cclls (reviewed by Leslie and Alexander I) has resulted in two general observations. First, individual cell populations, particularly macrophages, may express more t h a n one type of receptor activity, which may differ in IgG subclass specificity, b i n d i n g affinity and the n u m b e r of b i n d i n g sites per cell (Table I). Second, similar receptor activities may be expressed by different ceil populations. Thus, h u m a n monoeytes a, neutrophils 7 a n d K-cells s all express receptors fk)r h u m a n lgGl a n d IgG3, guinea pig m a c r o p h a g c s a n d neutrophils share b i n d i n g specificity for lgG2 ( C o u p l a n d and Leslie, s u b m i t t e d for publication) and m u r i n e lympbocytes display IgG binding characteristics similar to the I g G l / I g G 2 b b i n d i n g activity expressed by m a c r o p h a g e s '~-t0. T w o questions arise from these observations. F'irstly, are the diverse activities expressed by individual cell populations associated with distinct receptors? And, sccondly, are the receptors w h i c h give rise to similarity in the b i n d i n g activities of different cell types structurally related? In tackling these questions, most attention has been focused on murine receptors, primarily because the availability of large

n u m b e r s of cells from m o u s e macrophage-like and lymphoid cell lines can provide sufficient receptor material for structural a n d functional analysis (111~ cells yield only 1tag of the required glycoproteins 1,121. T h e study of m u r i n e receptors has also been greatly assisted by the generation of a rat monoclonal antibody (2.4G21 which reacts specifically with one of the IgG binding sites on mouse macrophages~L Functional studies of the receptors on intact mouse cells have proved u n a m b i g u o u s . T h e existence of two receptors on mouse m a c r o p h a g c s (FcRI w h i c h binds m o n o m e r i c and a g g r e g a t e d lgCZ2a with high affinity and the I g G 1 / I g G 2 b - b i n d i n g receptor, FcRII) has been established on the basis of their differencc in susceptibility to tryptic digestion 2 (Table I1), ind e p e n d e n t variation in their expression by different macrophage-like cell lines ~* a n d the d e m o n s t r a t i o n that 2.4G2 reacts selectively with FcRII I~. Similarly the structure of t h e receptors on mouse m a c r o p h a g c s a n d l y m p h o c y t e s has been intizrred from the observation that a n t i b o d y 2.4(;2 will block the a t t a c h m e n t of c o m p l e x e d IgG to B-cell lines 13, an activity w h i c h is not affected by the I I - 2 h a p l o t y p e of the mice but which is highly species specific.

'FABLE 1. Binding of lgG subclasses to monocyte and rnacropbage receptors Species

Cell typc

Specificity ol receptor h~r homologous lgG

Association constant Ka

Number of receptor sites per cell (x 1041

Mouse

Peritoneal macrophages

IgG2a (FcP, I)

1.3 x 10~a

lgGl/lgG2b(FcRlI)

IgG2: 6.8-9.0x I06a

lgG 1 IgG2 lgG

6.1 x 105b 14.6 x 117b 7.6 9.0 x 105c

IgG 1/ lgG3

lgGl:l.07 x 10*b IGG3:7.8 x l0 rb 4.4 x 107b

110 (resident) 440 (stimulalcd) 5(1 (resident) 290 (stimulated) 1,330 (stimulated) 2,500 (stimulated) 1,210 (resident) 2,160 (stimulated) 31-34

Guinea Pig Rabbit Man

Peritoneal macrophages Alveolar macrophagcs Blood monoeytes

IgG4

Reference

21

a Estimated at 4°G b Estimated at 20°C c Estimated at 37°C ElscvicrBiomcdicalPrcss1982

0167~1919/82/00000000/S1,00

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Immunology Today, vol. 3, No. 10, 1982

T h e characterization of the isolated receptors has been more equivocal. While some studies have identified two distinct IgG-binding glycoproteins on m a c r o p h a g e s t2,1~,1(', others have either failed to d e m o n s t r a t e the existence of separable receptors ~v, or have recorded a loss in receptor specificity following isolation from the cell m e m b r a n e TM. T h e latter observations have led to speculation that the degree of aggregation or the m e m b r a n e e n v i r o n m e n t of the receptor on the intact cell may be crucial in conferring subclass specificity. F u r t h e r m o r e , estimates of the size of the receptor(s) have varied greatly from a tool. wt of 35,000 to 120,000 I. M a n y of these discrepancies may be a t t r i b u t e d to differences in methodology; differences in the techniques used for receptor release from the m e m b r a n e s ; differences in the form and a m o u n t of insolublized ligand e m p l o y e d to extract the receptors from cell lysates; a n d differences in the analytical p r o c e d u r e s used in their characterization. In a series of experiments, S h e l d o n C o o p e r a n d coworkers have used ' S e p h a r o s e ' - b o u n d aggregated mouse IgG2a, IgG2b or IgGl in small a m o u n t s to isolate, by affinity c h r o m a t o g r a p h y , the receptors from thiogycollate-elicited peritoneal m a c r o p h a g e s ( T E M ) 12 and, more recently, from the m a c r o p h a g e like cell lines J774 and P388D I(' and a B-cell l y m p h o i d leukaemia Io. In the m a c r o p h a g e studies, the use of low quantities of IgG subclasses has proved crucial in avoiding co-isolation of both receptors on c o l u m n s w h e r e a second IgG .subclass m a y be present as a m i n o r c o n t a m i n a n t ~7. For the cell-line studies, two recovery p r o c e d u r e s were used: acid elution and rapid

neutralization was e m p l o y e d to yield receptor preparations which retained biological activity a n d could be retested for subclass specificity; a n d elution with SDS to recover receptors for structural analysis by a c o m b i n a t i o n of isoelectric focusing a n d SDS-polyacrylamide gel electrophoresis. In addition, affinity c h r o m a t o g r a p h y on insolublized 2.4G2 was used in the m a c r o p h a g e ceil-line study. T h e investigations of T E M receptors ~2 and receptors on m a c r o p h a g e cell-lines 1(' were consistent in that different receptors were isolated using lgG2asepharose a n d IgG1- or l g G 2 b - s e p h a r o s e columns, a n d in that trypsin p r e t r e a t m e n t of the intact cells resulted in the selective failure to recover' the IgG2a receptor, FcR1. However, p h y s i c o - c h e m i c a l differences between the receptors from the two cell sources were recorded, the cell-line receptors being slightly larger and more acidic ('Fable II). T h e molecular weight differences noted for F c R I I were similar to those previously observed by M e l l m a n a n d Unkeless *~ (Table ll) who suggested that modification of the T E M receptor by lysosomal proteases, either i~ .silz~or during isolation, m a y have been i m p o r t a n t in determining the size of the isolated glycoprotein. T h e basis of the differences in pl b e t w e e n T E M a n d the cell-line receptors is less certain a n d may arise either from methodological differences b e t w e e n the two studies in p H determination, or t~om differences in glycosylation of the receptors in the two cell p o p u l a t i o n s (cf. Ref. 18). Using 2.4G.2-sepharose fbr affinity c h r o m a tography, I,ane a n d C o o p e r ~(' recovered two proteins, the 65,000 mol. wt F c R I I and a m i n o r c o m p o n e n t of

TABLE II. Characterization of murine lgG receptors Cell type or line

Ligand used in affinity chromatography

Receptor isolated

Molecular weight

lso-electric Trypsin point, pl sensitivity

Specificity of isolated receptor

Retie> ence

T.E.M. a

aggregate IgG2a aggregate tgG2b or IgGl rat monoclonal 2.4G2 rat monoclonal 2.4G2 rat monoclonal 2.4G2 aggregate tgG2a

FcRI FcRII

67,000 52,000

6.0 6.6 6.6-7.5

Yes No

n.t. n.t.

12 12

FcRII

47,000

--

No

n.~.

18

FcR[I

60,000(+47,000)

--

n.t.

18

FcRII

60,000+47,000

4.8-5.8

~47,0t)0d

18

FcRI

70,000+60,000

3.8-4.7

Yes

aggregate or lgG1

FcRI[

65,000

4.7-5.5

No

rat monoclonal 2.4G2 aggregate [gG2a, IgG2b or lgGl rat monoclonal 2.4G2

FcRII

65,000 (+47,000)

5.5

No

IgG1, lgG2b+ IgG2a IgG2a>>lgG1 IgG2b IgG2b>IgG2a= [gG1 n.t.

16 16 16

'l"cRll-like'

c. 60,000

4.5-5.5

No

n.t.

"i9

'FcRll-like'

70,000

--

--

--

18

T.E.M. a P388b J774 b P388D and J774 b

L1210c WEHI231 c

a T.E.M. are thioglycollate elicited peritoneal macrophages bj774 and P388(D) are macrophage-like cell lines c L1210 and WEH1231 are B-cell lymphoid leukaemias d reduction in molecular weight without loss of activity.

16

267

Immunology Today, voI. 3, No. I0, 1982

47,000 mol. wt in variable yield. In this respect their findings were identical to those of M e l l m a n and Unkeless TM. The biological activity of the isolated receptors was tested by rebinding to the insolublized lgG subclasses and 2.4G2. FcRI retained its original IgGa-binding specificity, though interaction was also observed with 2.4G2. This may have been due to interaction of the receptor with the Fc portion of 2.4G213, but may also reflect antigenic alteration of FcRI following acid denaturation. FcRII displayed some loss of specificity, showing significant binding to aggregated IgG2a. The authors suggest that specificity loss may be associated with removal of the receptor from its m e m b r a n e environment, citing the reported susceptibility of the receptor to phosphofipase C 15 as an indication of the importance of boundary lipids in conferring biological specificity. The structural studies on murine B-cell receptors are conflicting. Using insolublized 2.4G2 as absorbent, M e l l m a n and Unkeless TM recovered a 70,000 mol. wt component from the B-cell line W E H 1231 displaying little similarity with the FcRII receptor on macrophages (Table II). O n the other hand, Lane et al. 19, using scpharose-bound oligomeric h u m a n , rabbit or mouse IgG recovered from the B-cell leukaemia, L1210, a protein almost identical in molecular weight and pl to FcRII. T h e distinguishing features of the latter protein were that on the intact cell surface it displayed a broader subclass specificity t h a n FcRII by reacting with monomeric IgG2a, and that it was recovered from the cell lysate with aggregated IgG2asepharose. Regrettably, in this study the authors did not examine its reactivity with 2.4G2. In summary, recent studies ~-~',1~,~2 have established the existence of two structurally distinct receptors on macrophages and macrophage-like cell lines, though apparent physico chemical differences between the receptors on normal macrophages and the cell-lines

remain to be evaluated. Degrees of similarity, both antigenic ~s and physico-chemical% have also been demonstrated between FcRII on macrophages and the IgG receptor on B-cells, though there are still discrepancies in the specificity of these receptors to be explained. The major question that remains is whether the two receptor types are individual gene products which are subsequently modified chemically, by glycosylation or cleavage, and by their m e m b r a n e environment, or whether multiple genes are involved in encoding two families of receptors. R. (], ~ . L E S L I E

Department of hnmunofogy~ Queen's Medical Cenlre, ,Nottingham NG7 2UH, U.K.

References 1 I,eslie, R. G. Q. and Alexander, M. D. (1979) (,)err. Top. Mler, biol. lmmu,ol. 88, 25 2 Unkeless,J. C. and Eisen, H. N. (1975)J. Exp. Med. 142, 1520 3 Leslie, R. G. Q. and Cohen, S. (I 976) Eur. J. lmmunoL 6,848 4 Leslie, R. G. Q. and Cohen, S. 11974) hnmanology 27, 577 5 Arend, W. P. and Mannik, M. (1973).7. lmmuno[. 110, 1455 6 Alexander, M. D., Andrews, J. A., Leslie, R. G. Q. and Wood, N.J. 11978)Immunology35, 125 7 Messner, R. P. andJelinek,J. (1971))J. Clin. lnve~t. 49, 2165 8 Spiegelberg, H. L., Perlmann, H. and Perlmann, P. 11976) J. hnmunoI. 116, 1464 9 Basten, A., Miller, J. F. A. P., Sprent, .J. and Pye, J. 11972) j . Fvp. Med. 135,610 10 Anderson, C. L. and Grey, H. M. (1974)J. Exp. Med. 139, 1175 11 Loube, S. R., McNabb, T. C. and Dorrington, K. J. (1978) J. lmmumd. 120, 709 12 Lane, B. C., Kan-Mitchell, J., Mitchell, M. S. and Cooper, S. M. (1980)J. Ex/';.Med. 152, 1147 13 Unkeless,J. C. 11979),7, Exp. Med. 150, 580 14 Unkeless,J. C. (1977)J. Exp. Med. 145, 931 15 Anderson, C. L. and Grey, H. M. (1978)J. ImmunoL 121,648 16 Lane, B. C. and Cooper, S. M. (1982).7. lrnmunol. 128, 1819 17 Schneider, R. ,]., Atkinson, J. P., Krause, V. and Kulczycki, A. (1981) J. lmmuno/. 126,735 18 Mellman, I. S. and Unkeless, J. C. (1980) j . Exp. Med. 152, 1048 19 Lane, B; C., Bricker, M. D. and Cooper, S. M. (1982) ,7. hmnunol. 128, 1825

The symbiosis of immunocompetent and endothelial cells Immunologists frequently envision blood vessels as passive conduits through which cclls, antibody and complement components flow. Yet it is evident now that there is a dynamic interplay between immunocompetent cells and vascular endothelium that may be important in the initiation, progress and resolution of a wide range of immune responses. The extent of the interaction may dcpend on the type of blood vessel involved and on the physiological state of the endothelial cells. Endothelial cells can be altered morphologically, physiologically and antigenically by their interaction with lymphocytes and monocytes. For example, it has long been known that the characteristic 'high endothelium' of the postcapillary venules in the lymph nodes of some species is dependent on T-lymphocyte traffic; it regresses when T lymphocytes are con-

genitally absent or experimentally depleted and regenerates when rF lymphocytes are restored to these animals 1,2. W h e n T-lymphocyte traffic is increased by antigen deposition in the lymph node, capillaries proliferate and enzyme activity increases in the endothelial cells 3. Such stimulated lymph node endothelial ceils produce a sulfated glycolipid that may contribute to local lymphocyte accumulation 4. Similarly, capillary proliferation is stimulated in the skin by such T-lymphocyte-dependent responses as graft v. host and delayed hypersensitivity reactions s.~'. Both of these reactions involve helper T lymphocytes which secrete a variety of lymphokines to regulate the activity of i m m u n o c o m p e t e n t cells. In addition, lymphokine-containing s u p e r n a t a n t s from concanavalin-A-activated lymphocytes can influence the activity of endothelial cells in culture, as demonstrated Elsevier Biomedical Press 1982 01674919/82/00004)000/$1.00