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immunologytoday, October1980

3 Bonnard, G.D., Glauser, A., Chappuis, M., Lemos, L., Gauthier, E., Barrelet, V. and Jeannet, M. (1974) in Immunology in Obstetricsand Gynnecotogy(Centaro, A. and Caretti, N., eds), pp. 147-155, Excerpta Medica, Amsterdam 4 Ceppellini, R., Bonnard, G.D., Coppo, F., Miggiano, V.C., Pospisil, M., Curtoni, E.S. and Pellegrino, M. (1971) Transplant. Proc.3, 58-63 5 Bonnard, G.D. and Lemos, L. (1972) Transplant. Proc. 4, 177-180 6 Lawler, S.D., Ukaejiotb, E.O. and Reeves, B,R. (1975) Lancet ii, 1185-1187 7 Birkeland, S.A. and Kristoffersen, K. (1980) &and. J. hnmunol. 11,311-319 8 Finn, R., St. Hill, C.A., Davis, C., Hipkin, L.J. and Harvey, M. (1977)Lancetii, 1200-1202 9 Olding, L.B., Murgita, R.A. and Wigzell, H. (1977) J. Irnmunol. 119, 1109-1114 10 Olding, L.B., Benirschke, K., Oldstone, M.B.A. (1974) Clin. Immunol. Immunopatkol. 3, 79-89 11 Adinolfi, M. (1976) Lanceti, 97 12 Oldstone, M.B.A., Tishon, A. and Moretta, L. (1977) Nature (London) 269,333-335

13 Chaudhuri, J.P. and Zang, K.D. (1976) tlum. Genet. 34, 307-310 14 Hayward, A.R. and Lydyard, P.M. (1978) Chn. Exp. Immunot. 34, 374-378 15 Moriya, N., Nagaoki, T., Okuda, N., and Taniguchi, N. (1979) J. Irnmunol. 123, 1795-1798 16 Durandy, A., Fischer, A. and Griscelli, C. (1979) J. lmmunol. 123, 2644-2650 17 Wolf, R.L., Lomnitzer, R. and Rabson, A.R. (1977) Clin. Exp. lmmunol. 27,464-468 18 Tiilikainen, A., Schr6der, J. and de la Chapelle, A. (1974) Transplantation 17,355-360 19 Toivanen, P., Asantila, T., Granberg, C., Leino, A. and Hirvonen, T. (1978) Immunol. Rev. 42,185-201 20 Herva, E. (1976) Lanceti, 919-920 21 Granberg, C. (1980) Ceil. Immunol. 53, 10-18 22 Pavia, C.S. and Stites, D.P. (1979)J. Immunol. 123, 2194-2200 23 Pavia, C.S. and Stites, D.P. (1979) Cell. Immunol. 42, 48-60 24 Droege, W. (1976) Eur. J. Immunol. 6, 279-287 25 Du Pasquier, L. and Bernard, C.C.A. (1980) Differentiation 16, 1-7 26 Chaouat, G. and Voisin, G.A. (1980) Immunology39,239-248

Macrophage handling of soluble immune complexes t

R.G.Q.

Leslie

Department of Immunology, University Hospital, Queen's Medical Centre, Nottingham, U.K.

Two in-vivo observations underline the importance of stu@ing macrophage interactions with soluble immune complexes. The first is the clearance of soluble complexes from the circulation after experimental administration of free or compIexed antigen, during an acute infection, or even after a heavy meal. Clearance is mediated by the mononuelear phagocyte system, particularly by the Kupffer cells of the liver, and its rate is dependent on both the macrophage-binding activity of the compIexed antibodies and complex size. The second is the association of circulating immune complexes with diseases such as rheumatoid arthritis and systemic lupus erythematosus, where damage to specific organs and tissues is thought to be caused by complexes deposited at these sites. The basis of the persistence of soluble complexes in the blood, and the part played by the circulating, as opposed to the deposited, complexes in pathogenesis are aspects of these diseases in which lhe activity and responsiveness of mononuclear phagocytes may be extremely important. Studies in vitro provide a valuable backgroundjbr investigating the activities in vivo of soluble complexes by defining (i) the way in which macrophages recognize cytophilic antibodies and lhe basis of binding enhancement which follows anlibody combination with antigen; ( ii) the kinetics of complex uptake and destruction by phagocytes and the biochemical mechanisms involved; and ( iii) the regulatory effecl that soluble immune complexes may have on macrophage activities such as oxidative metabolism and lysosomal enzyme release. In this review, Graham Leslie outlines the progress that has been made in characterizing lhese evenls.

Macrophage recognition of antibody and immune complexes A significant advance in our u n d e r s t a n d i n g of soluble i m m u n e c o m p l e x uptake by m a c r o p h a g e s has been achieved t h r o u g h quantitative c o m p a r i s o n of the b i n d i n g of free i m m u n o g l o b u l i n s with that of complexes or aggregates of defined size 1-3. T h e interaction of m o n o m e r i c i m m u n o g l o b u l i n with macrophages was first characterized, in terms of an association constant and the n u m b e r of receptor sites per cell, © Elsevier/North-Holland Biomedical Press 1980

by A r e n d and M a n n i k 4, who m e a s u r e d the equilib r i u m binding of rabbit IgG to h o m o l o g o u s alveolar m a c r o p h a g e s and analysed the data using the Scatc h a r d plot (Fig. 1). T h i s a p p r o a c h was s u b s e q u e n t l y a p p l i e d to h o m o logous interactions in guinea-pigs, mice and m a n (for review see Ref. 5). C o n s i d e r a b l e interspecies variation is a p p a r e n t , both in the affinity of I g G for the m a c r o p h a g e s and in the n u m b e r of receptor sites per cell (Table I). In guinea-pigs, mice and, possibly,

79

immunology today, October 1980

TABLE I. Association constants and numbers of receptor sites for IgG on mononuclear phagocytes in guinea-pigs, rabbits, mice and man. Species

Guinea-pig Rabbit Mouse Man

IgG

Macrophage

Subclass

type

IgG1 IgG2 IgG IgG2a IgG2b IgG1

Peritoneal Peritoneal Peritoneal Peritoneal Peritoneal Peripheral blood Monocytes Peripheral blood Monocytes Peripheral blood Monocytes

IgG3 IgG4

Incubation temperature (°C)

Association constant (K~) (x 10-21 mol")

Sites per cell (x 10-2)

Ref.

20 20 20 4 4 20

6.1 ! 1.5 14.6 + 4.5 5.9 + 1.6 1300 90 1070 ± 390

13.3 + 1.8 25.0 +- 4.0 46 ± 21 4.4 2.9 0.31 -+ 0.16

21 22 23 6 6 24

20

780 ± 460

0.34 + 0.11

24

20

440 ± 21

0.21 + 0.07

24

h u m a n s , there a p p e a r to be t w o types of receptors distinguishable by their subclass specificity, their affinity for i m m u n o g l o b u l i n and their a b u n d a n c e on the cell surface. In mice, the existence of two receptors has been confirmed on the basis of differitlg susceptibility to proteotysis 6 and i n d e p e n d e n t variation in their expression on m a c r o p h a g e - l i k e cell lines 7. T h e m u r i n e receptors have been further distinguished as m o n o m e r - b i n d i n g and c o m p l e x - b i n d i n g 8, b u t this distinction is s o m e w h a t arbitrary since careful investigation has shown that the latter receptor will b i n d unc o m p l e x e d i m m u n o g l o b u l i n , albeit with lower affinity t h a n the ' m o n o m e r ' receptor 6. T h e binding activity of antibodies c o m p l e x e d with polyvalent antigens has been analysed by the m e a n s e m p l o y e d for m o n o m e r i c i m m u n o g l o b u l i n s . T h e att a c h m e n t of small complexes, c o n t a i n i n g two, three or four a n t i b o d y molecules, to mouse and g u i n e a - p i g macrophages~, 3 was e x a m i n e d at 4 ° C , to avoid ingestion, and each c o m p l e x was observed to b i n d with a characteristic, uniform avidity which increased progressively with c o m p l e x size (Fig. 2, T a b l e II). T h e basis of e n h a n c e d i m m u n e c o m p l e x binding has been investigated by c o m p a r i n g the free energy changes associated with m o n o m e r a n d o l i g o m e r binding. A progressive increase in the avidity of complex b i n d i n g with size indicates that a n t i g e n - l i n k e d

a n t i b o d y molecules are acting co-operatively in att a c h i n g to the m a c r o p h a g e m e m b r a n e resulting, ideally, in a s u m m a t i o n of the b i n d i n g energies of the constituent antibodies. In the absence of conformational change, the value w o u l d be equal to the s u m of the binding energies of an e q u i v a l e n t n u m b e r of free i m m u n o g l o b u l i n s (Fig. 3a), whereas if structural alteration were involved, l e a d i n g to an increase in the intrinsic affinity of the antibody, the free energy change for c o m p l e x a t t a c h m e n t w o u l d be greater t h a n this s u m (Fig. 3b). In fact, the b i n d i n g energy of the complexes is less t h a n that e x p e c t e d from the activity of the free m o n o m e r s ( T a b l e II) indicating that enh a n c e m e n t can be e x p l a i n e d w i t h o u t resorting to the hypothesis of c o n f o r m a t i o n a l change, and that crosslinking of a n t i b o d y molecules by antigen places a strain on the i m m u n o g l o b u l i n - r e c e p t o r interaction. O n the basis of similar observations, Segal and H u r w i t z 3 p r o p o s e d that the strain imposed by antigen on the interaction of rabbit I g G antibodies with m u r i n e celt receptors m i g h t arise from (i) re-orientation of antibodies in the i m m u n e c o m p l e x to ensure a t t a c h m e n t to the cell surface; (ii) redistribution of the i m m u n o g l o b u l i n receptors on the cell surface, resulting in steric h i n d r a n c e ; (iii) restriction on the r a n d o m m o v e m e n t of the receptors in the plane of the m e m .brahe, leading to a drop in e n t r o p y for the system; or

TABLE II. Binding of immune complexes of IgG2 and DNP~gBSAto macrophages Ab:Ag ratio in incubation mixture

3.0:1 0.9:1 0.3:1 1.0:0

Mean composition of complex

Ab3. 8 Agi. 4 Ab2. 9 Ag2. 3 Ab2. 0 Ag2. 2

Ab~ Ago

- a 6' = R T n K a

Apparent association constant (Ka) (1 mol< x 10 -7) 59.0 ± 11.2 33.4 + 3.4 15.4 -+ 2.3 0.206 ± 0.045

where R = 2 cais/degrees and T= temperature (K).

AG per mole IgG2 = - AG per mole complex Average number of antibodies per complex

Free energy change - AGa (kcal)

-

per mole complex

per mole IgG2

11.2 10.9 10.4

2.95 b 3.76L 5.20u 8.05

-

immunology today, October 1980

80

Macrophage c a t a b o l i s m of s o l u b l e i m m u n e complexes

0.010

,-

0.005

•

0

•

0.i

The breakdown of soluble immune complexes to low-molecular-weight catabolites by macrophages involves a chain of at least five events (Fig. 4): attackmerit of the complexes, via antibody, to the phagocyte membrane receptors; ingestion of the m e m b r a n e - b o u n d complexes in pinocytic vesicles; fusion of the pinosomes with lysosomal granules; digestion of the complexes within the secondary lysosomes; and excretion of the catabolic products. An early in-vilro study of immune complex degradation by guinea-pig macrophages demonstrated that the overall process was dependent on complex sizd;, but no attempt was made then to determine which stages were size-discriminating or to identify the rate-determining step. With the recent development of assays which measure attachment, ingestion and intracellular destruction of complexes, as separate events, these questions are now being tackled. Immune complex attachment to macrophages in vilro has been examined in terms of both binding equilibria (see above) and the kinetics of association and dissociation% ~°. The discrimination on the basis of complex size, which is apparent from the equilibrium studies, has been correlated with differences in the rate of dissociation from the phagocyte m e m b r a n e rather than the rate of uptake, which was relatively invariant tot immunoglobulin aggregates over a six-

•

0.2 0.3 r (M, x 108)

0.4

Fig. 1. Scatchard plot for the binding of gulnea-plg IgG2 to oil-stimulated peritoneal macrophages at 4°C. r / c = - K a r + K a n , where r is the concentration of bound IgG, c is the concentration of free IgG, n is the total concentration of immunoglobulin receptors in the system and Ka is the association constant for IgG binding to the receptors.

(iv) negative co-operativity between the receptors arising from induced conformational change in receptors adjacent to a site of immunoglobulin attachment.

Ab : Ag •

"~

Ab : Ag 0"3:1

Ab : Ag 0"9:1

• 3-0:1

2.0

2.0

2.0

1.6.

1.6 I

1.6

1.2

1.2

O.8

0.8

0.8

0,4

0.4

0.4

m

1.2

•

l 0

0.2

0.4

0.6

I

0

0.2

0.4

0.6

0

0.2

0.4

0.6

r(M, x 108) Fig. 2. Scatchard plots for the binding of IgG2-containing complexes, formed at three different Ab : Ag ratios, to guinea-pig peritoneal macrophages at 4°C. from Ref. 3. Reproduced with the permission of Verlag Chemie.

81

immunology today, October 7980

fold size range 2. Since attachnaent is reported to be rate-determining for the catabolism of i m m u n e aggregates at low concentrations m, invariance in this rate appears to conflict with the bz-vivo observations on the size dependence of complex clearance 2. However, it has been proposed that complex attachment takes place in two stages; a relatively rapid interaction, involving perhaps a single antibody molecule in the complex, followed by a slower stage in which multipoint binding occurs 2. On this basis, Knutson et al. 2 concluded that discrimination may be associated with the preferential ability of the larger complexes to undergo the comparatively irreversible secondary binding, providing sufficient permanency of attachment for subsequent ingestion to take place. Equilibrium studies have also demonstrated that the susceptibility of complexes to inhibition by monomeric IgG is inversely related to complex size 3, indicating that phagocytic discrimination between complexes may be enhanced in physiological concentrations of free immunoglobulin. The effect of monomer IgG on the binding kinetics of complexes still requires investigation.

I

II

S IMPLE COOPERATION

-AGm

-ZXGc ~ 2 (-AGm)

CONFORMATIONALCHANGE+ COOPERATION

-AGm

-/XGc>2 (-/XGm)

Fig. 3. Schematic representation of two hypothetical mechanisms for the enhanced attachment of IgG-containing complexes to cell receptors.

-- AGin: molar free energy change for binding of IgG monomer - gxGc:molar free energy change for binding of complex

III

..

#

P

Lp LP

-:-:-:-:.>>:.:.:.:.:.:.:.:.:.:.:.>>:.:+>>:.:.:'>:':.:':'>i

Fig. 4. Schematic representation of the processes involved in macrophage degradation of soluble immune complexes.

The arabic numerals refer to the individual steps in the process: (1) attachment, (2) ingestion, (3) secondary lysosome formation, (4) complex digestion and (5) catabolite excretion. The roman numerals between vertical lines denote the stages investigated in different assays.

immu~zotogy today, October 1980

82

90'at4oc

~

O- 60' at

~.~l

wash

4,20°r3l°C ~

=E

DNPBSA-IgG1or IgG2(~) + Macrophages(2-4x 106)

. . ~ ~ 1 . 1251(Fab')2anti-DNP(A)

Count 125I Fig. 5. Assay scheme for measuring the rate of ingestion of membrane-bound soluble immune complexes by macrophages.

The kinetics of complex ingestion have been examined ~1 by attaching soluble complexes to macrophages at 4°C, incubating the complex-laden cells at elevated temperatures for various times and detecting complexed antigen remaining at the cell surface with ~2SI-labelled F(ab')2 fragments of the appropriate antibody (Fig. 5). Ingestion was found to obey first order kinetics at 20 and 37°C (Fig. 6) and to proceed at a rate (12.5%/rain at 37°C) that was fourfold faster t h a n the rate of r a n d o m m e m b r a n e t u r n o v e r (3%/min) which accompanies pinocytosis by resting 4o 100

2.0

5O

o "~

-~

_'2

20

~--

g i0

•

'

1.0

5 li0

I

I

I

I

I

20

30

40

50

60

I

]ncubatf0nTime (minutes) Fig. 6. Kinetic plots of the clearance of m e m b r a n e - b o u n d complexes by macrophages at 4, 20 and 37°C. From Ref. 11. Reproduced with the permission of Verlag

Chemic.

macrophages ~2. Complex ingestion may thus be regarded as a selective process, though it is as yet uncertain whether the selection is a result of complex concentration at the sites of forming pinosomes or whether the complexes themselves induce pinosome formation de novo. Variation of complex size, on the other hand, was not found to influence the ingestion rate 1~, indicating either that the cross-linking of as few as two Fc receptors on the phagocyte m e m b r a n e provides a signal for irreversible endocytosis by an essentially indiscriminate mechanism or, more probably, that rapid reorganization of the m e m b r a n e - b o u n d complexes into large immune aggregates precedes endocytosis. Intracellular destruction of immune complexes by macrophages has been examined as an overall process by measuring the release of trichloroacetic-acidsoluble catabolites atier loading the cells with radiolabelled complexes at 4 or 20°C (Fig. 7). Kinetically, catabolism was estimated from the decline, with time, in the proportion of radiolabel that remained undigested 11 and was found to be a temperature-dependent pseudo-first order reaction (Fig. 8), with a rate that was 20- to 60-fold slower ( 0 . 3 - 0 . 6 % / m i n at 37°C) than the rate of ingestion. No variation was observed when complexes of different size were employed in the assay but the rates of degradation of antigen and antibody in the same complex were found to differ markedly ~1, indicating that the susceptibility of the target proteins to hydrolysis by lysosomal enzymes, rather than the kinetics of secondary lysosome formation or catabolite excretion, was the rate determining factor. The kinetic studies, alone, have provided little inforrnation on the individual intracellular events involved in complex catabolism. Preliminary characterization

immunology today, October 1980

83

~, ~

"

~

~

1251

/~ @ii!~

i hr ,20°C, or 4Oc~ I. 5 hr, wash

0-120' at 37°C

TCA precipitablecpm

TeA soluble

~

cpm

IgG2-ONP19BSA( ~/ )

+ Macrophages Fig. 7. Assay s c h e m e for measuring the catabolism of m e m b r a n e - b o u n d soluble i m m u n e complexes by macrophages.

of these events has been achieved, however, by observing the effect of metabolic inhibitors [potassium cyanide (KCN) and 2-deoxyglucose (2dG)], serine esterase inhibitors [tosylphenylananyl chloromethyl ketone (TPCK) and tosyllysyl chloromethyl ketone (TLCK)], disruptors of cellular orgar/ization [colchicine(Coi) and cytochalasin B, (Cyto B)] and a membrane perturber [lidocaine (Lid)] upon the rates of complex ingestion and catabolite excretionllJ3,14. Selective action of the individual inhibitors on different stages of complex handling was observed, indicating that each stage is controlled by distinct biochemical mechanisms. Ingestion of membrane-bound complexes was partially blocked (37~ inhibition) by an inhibitor of respiration (KCN), was unaffected by stopping glycolysis with 2dG, and was substantially reduced (73~0) by a combination of both agents 1I, suggesting that the endocytic process involved was fluid-phase pinocytosis ~5. Support for this view was provided by the observation that complex internalization is also reduced to 10~o of the original rate by microfilament disruption with Cyto B 14, whilst remaining unaffected by Col or Lid. Complete inhibition was not observed in either study, however, indicating that an alternative process, adsorptive micropinocytosis, may also play a part in complex ingestion. The esterase inhibitor, TPCK, which has been reported to prevent phagocytosis ~6, was also effective in blocking complex endocytosis ~4 but, since the inhibitor caused cell death, its action cannot be regarded as selective. Complex catabolism was blocked by a Combination of the metabolic inhibitors, but differed from ingestion in showing greater sensitivity to the action of 2dG than KCN 12. Selective inhibition of complex digestion was also recorded with 5 mmo1-1 Lid ~3 and the esterase inhibitor T L C K 14, whereas Cyto B and Col were only slightly inhibitory. Confirmation that Cyto B acted primarily on the ingestion step, and identification of the intracellular events blocked by. Lid and TLCK, were obtained by preincubating the complex-laden macrophages for different times at 37°C before adding the inhibitors. The partial blockade of digestion observed with Cyto

B (c. 24%) was completely abolished by 15 min preincubation (Fig. 9), indicating that its sole effect was on the rapid ingestion of complexes at 37°C and not on subsequent events. The action of Lid was also affected to a lesser extent, and increasing the preincubation period to 45 min led to a further reduction in its inhibitory capacity, suggesting that the local anaesthetic acts upon a relatively early intracellular event, presumably by preventing pinosome-lysosome fusion rather than proteolysis within the secondary lysosomes or catabolite excretion. Inhibition of complex degradation by TLCK, on the other hand, was unaffected by preincubation and its inhibitory action upon intralysosomal digestion of the complexes, rather than upon excretion, was confirmed by the absence of catabolite accumulation within the inhibited cells I4. Macrophage activation by soluble i m m u n e complexes The destruction of soluble immune complexes by macrophages, though selective at the levels of uptake 2.0

100-

.g -X E

8 -1.8

'~ 8

5O 1.6 t 0

I

30

J

I

I

60

90

120

I

Incubation Time Imins) Fig. 8. Kinetic plots of the digestion of soluble i m m u n e complexes b y m a c r o p h a g e s at 4 (~ . . . . . A), 20 (It . . . . . II) and 37°C (O 0). The log phase before onset of digestion is most pronounced when initial complex binding is performed at 4°C, and reflects the time required for complex transport from the external membrane to secondary lysosomes. From Ref. 11. Reproduced with the permission of Verlag Chemie.

immunology today, October 198,0

84 100

-

•__

_

-

/ 80

/

-

/.x

/

/ /

,/

/ /

~ E

60

-

40

-

/

/f

O

/ /

/

o

•

Cytochalasin B

/

0

/

20

-

X

0

-

I 0

/

• TLCK x

kid0caine HCL

I ,I 15 45 Preincubati0n time (mins) Fig. 9. The effect of prelncubating complex-laden macrophages at 37°C on the capacity of Cyto B, Lid and TLCK to inhibit complex digestion (see text).

and ingestion, is not necessarily a process that involves specific triggering, since phagocytes constantly pinocytose and process their fluid environment in the absence of stimuli. Activation is most readily measured in terms of functions which are not normally expressed by the cells, such as directed movement, the release of lysosomal enzymes and neutral proteases or the production of cytotoxic oxygen compounds. Phagocytic stimuli, surface-bound IgG, C3b and activated T-cell products have been shown to promote these activities but, until recently, there has been no clear evidence that soluble i m m u n e complexes are stimulatory. Following reports that polymorphonuclear leukocytes respond to soluble complexes with a burst of oxidative metabolism I7 and the release of lysosomal hydrolases ~8, and that h u m a n monocytes respond to surface-bound IgG by generating superoxide and singlet oxygen 1'), Connell and co-workers 2° have looked a t singlet oxygen production by guinea-pig macrophages upon incubation with soluble complexes of defined size. They demonstrated that complexes containing two to tour antibody molecules produced a rapid two-phase response, comprising a brief burst of high chemiluminescent activity, due to singlet oxygen decay, which was inhibitable by superoxide dismutase but not catalase, and a persistent lower level of response susceptible to both enzymes. These observations indicate that soluble complexes are capable of stimulating oxidative metabolism in macrophages both upon attachment to the cell surface and following ingestion and transport to secondary lysosomes. Summary and conclusions In-vitro study of macrophage interactions with free immunoglobulins and soluble i m m u n e complexes has

provided some understanding of the binding enhancement which follows antibody combination with antigen and of the discimination between complexes of different size, as well as furnishing detailed information on the kinetics of complex uptake, ingestion and catabolism. The assays developed to measure these events should prove invaluable in assessing phagocyte function in diseases characterized by a persistence of circulating immune complexes. The inclusion of selective inhibitors in the kinetic studies of ingestion and catabolism has provided information about biochemical mechanisms involved in complex endocytosis, transport to secondary lysosomes and intralysosomal digestion, and should provide a means of investigating these events individually. The ability of soluble complexes to trigger macrophage elaboration of cytotoxic compounds has recently been demonstrated. This finding, and the observation that soluble complexes promote lysosomal enzyme release from neutrophils, indicates the necessity for investigating the pathogenetic role of circulating, as well as deposited, complexes in disease.

References

1. Segal, D.M. and Hurwitz, E. (1977)J. lmrnanoL 118, 1338 2 Knutson, D.W., Kijlstra, A. and V. Es., L.A. (1977) J. Exp. Med. 145, 1368 3 Leslie, R.G.Q.. (1980a) Eur. J. [mmanol./0, 317 4 Arend, W.P. and Mannik, M. (1973)J. ImmunoL 110, 1455 5 Leslie, R.G.OQ. and Alexander, M.D. (1979) Curt. Top. Microbiol. Imraunol. 88, 25 6 Unkeless,J.C. and Eisen, H.N. (1975)J. Exp. Med. 142, 1520 7 Unkeless,J.C. (1977)J. Exp. Med. 145,931 8 Silverstein, S.C., Steinmann, R.M. and Cohn, Z.A. (1977) Annu. Rev. Biochem. 46, 669 9 Shinomiya,T. and Koyama,K. (I 976) Immunology 30,267 10 Knutson, D.W., Kijlstra, A. and V. Es., L.A. (I979) J. ImmunoL 123, 2040 11 Leslie,R.G.Q (1980b) Eur..7" Irnmuno/. 10, 323 12 Muller, W.A., Steinmann, R.M. and Cohn, Z.A. (1980) in Mononuc/ear P/zagocytes - Functional Aspects (van Furth, R., ed.), Part 1, p.595, Martinus NijhoffMedical Division,The Hague 13 Kijlstra, A., V. Dorp, W., Daha, M.R. and Leslie, R.G.Q. (1980) Immunology (in press) 14 Leslie,R.G.Q. (1980) Eur. J. lrumunoL (in press) 15 Steinman, R.M., Silver, J.M. and Cohn, Z.A. (1974) J. Cell Bio. 63,949 16 Nagai, K., Nakamura, T. and Koyama, J. (1978) FEBS Lea. 92, 299 17 Henson, P.M. and Oades, Z.G. (1975)J. Clin. Invest. 56, 1053 18 Morrison, A.D., Pruzanski, W. and Ranadive, N.S. (1978) Scand. J. Rheumatol. 7,241 19 Johnston, R.B., Lehmeyer, J.E. and Guthrie, L.A. (1976) J. Exp. Med. 143, 1567 20 Connell, P.A., Seehra, M.S., Leslie, R.G.Q. and Reeves, W.G. (submitted for publication) 21 Leslie,R.G.Q. and Cohen, S. (1976) Eur. J. lmmunol. 6, 848 22 Leslie, R.G.Q. and Cohen, S. (1974) Immurlology 27, 577 23 Ganczakowski, M. and Leslie, R.G.Q. (1979) hnrnunology 36, 487 24 Alexander, M.D., Andrews, J.l.., Leslie, R.G.Q> and Wood, N.J. (1978)Immunology35,125