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100 9 Greenberg, A. H. and Greene, M. (1976) Nature (London) 264, 356-359 10 Rygaard, J. and Povlsen, C.O. (1975) Transplant. Rev. 28, 43-60 11 Shwartz, R. S. (1975) N. Engl.J. Med. 293, 181-184 12 Roder, J.C. in Natural Cell Mediated Immunity Against Tumors (Herberman, R. B., ed.) Academic Press, New York (in press) 13 Roder, J. C., Rosen, A., Fenyo, E. M. and Troy, F. A. (1979) Proc.Natl. Acad. Sci. U.S.A. 76, 1405-1409 14 Collins, J. L., Patek, P. Q. and Cohn, M. J. Immunol. (in press) 15 Patek, P. Q., Collins, J. L. and Cohn, M. (1978) Nature (London) 276, 510 16 Kiessling, R., Petranyi, G., Klein, G. and Wigzell, H. (1975) Int. J. Cancer15, 933 17 Sendo, F., Aoki, T., Boyse, E. A. and Buofo, C. (1975)J. Natl. CancerInst. 55,603 18 Harmon, R. C., Clark, E., O'Tooke, C. and Wicker, L. (1977) Immunogenetics4, 601 19 Klein, G. O., Klein, G., Kiessling, R. and Karre, K. (1978) Immunogenetics6, 561-569 20 Petranyi, G., Kiessling, R., Povey, S., Klein, G., Herzenberg, L. and Wigzell, H. (1976) Immunogenetics3, 15 21 Riccardi, C., Puccetti, P., Santoni, A. and Herberman, R. B. (1979) J. Natl. CancerInst. 63, 1041-1045 22 Kiessling, R., Petranyi, G., Klein, G. and Wigzell, H. (1976) Int. J. Cancer17, 1 23 Cantor, H., Kasai, M., Shen, F., Lederc, J. and Glimcher, L. (1979) Irnmunol.Rev. 44, 3-12 24 Warner, N. L., Woodruff, M. and Burton, R. C. (1977) /nt..7" Cancer20, 146 25 Gershwin, M. E., Ikeda, R. M., Kawakami, T. G. and Owens, R. B. (1977)J. Natl. Cancerlnst. 1455-1460 26 Hailer, O., Hansson, M., Kiessling, R. and Wigzell, H. (1977) Nature (London) 270, 609-611 27 Riesenfeld, I., Orn, A., Gidlund, M., Axberg, I., Alto, G. and Wigzell, H. Int. J. Cancer(in press) 28 Haller, O., Kiessling, R., Orn, A. and Wigzell, H. (1977) J. Exp. Med. 145, 1411 29 Brodt, P. and Gordon, J. (submitted for publication) 30 Brodt, P. and Gordon, J. (t978)ff. hnmunol. 121,359-362

31 Gorczynski, R. M. (1974)J. Immuno[. 112, 533 32 Roder, J. C. and Duwe, A. (1979) Nature (London) 278,451-453 33 Karre, K., Klein, G. O., Kiessling, R., Klein, G. and Roder, J. C. (1980) Nature (London) 284,624-626 34 Talmadge, J. E., Meyers, K. M., Prieur, D.J. and Starkey, J. R. (1980) Nature(London) 284,622-624 35 Hanna, N. and Fidler, I. J. ft. Natl. CancerInst. (in press) 36 Trinchieri, G., Santoli, D. and Knowles, B. (1977) 3/ature (London) 270, 611 37 Gidlund, M., Orn, A., Wigzell, H., Senik, A. and Gresser, I. (1977) Nature (London) 273, 759 38 Brunda, M, Herberman, R. B. and Holden, H. T. (submitted for publication) 39 Lain, K. M. and Linna, T. J. (1977) in Advances in Comparative Leukemia Research (Bentvelzen, P., Hilgers, J. and Yohn, D. S., eds.), Elsevier/North-Holland Biomedical Press, Amsterdam 40 Roder, J. C., Haliotis, T., Klein, M., Korec, S., Jett, J., Ortaldo, J., Herberman, R. B., Kutz, P. and Fauci, A. (1980) Nature (London) 284, 553-555 41 Haliotis, T., Roder, J., Klein, M., Ortaldo, J., Fauci, A. and Herberman, R. B. J. Exp. Med. (in press) 42 Klein, M., Roder, J., Haliotis, T., Korec, S., Jett, J., Herberman, R. B., Katz, P. and Fauci, A. J. Exp. Med. (in press) 43 Griscelli, C., Durandy, A., Guy-Grand, D., Daguillard, F., Herzog, C. and Prunieras, M. (1978) Am. J. Med. 65,691-702 44 Blume, R. S. and Wolff, S. M. (1972) Medicine 51,247 45 Lipinski, M., Virelizier, J. L., Tursz, T. and Griscelli, C. Eur. J. Immunol. (in press) 46 Lipinski, M., Tursz, T., Kreis, H., Finle, Y. and Amiel, J. L. (1980) Transplantation29, 214 47 Kersey, I. H., Spector, B. D. and Good, R. A. (1973) Int. J. Cancer12, 333 48 Kasai, M., Iwamori, M., Nagai, Y., Okumura, K. and Tada, T. Eur. J. Immunol. (in press) 49 Gorelick, E., Fogel, M., Feldman, M. and Segal, S. (1979) J. Natl. CancerInst. 63, 1397-i404 50 Klein, G. (1979) Proc.Natl. Acad. Sci. U.S.A. 76, 2442-2446 51 Haliotis, T. and Roder, J. in GeneticControlof Natural Resistance (Skamene, E., ed.), Academic Press, New York (in press)

Biliglobulin: a new look at IgA j. G. Hall and E. Andrew Block X, Institute of Cancer Research, Department of Turnout Immunology, Clifton Avenue, Sutton, Surrey, UK.

The teachings of Hippocrates and Galen ensured that physicians have always had a lively appreciation of the importance of bile, but somehow immunologists never got the message and hitherto they have paid it scant attention. Here Joe Hall and Liz Andrew discuss recent discoveries in Belgium and Britain which have shown that bile is a rich source of the secretory immunoglobulin IgA because the hepatocytes rapidly and actively transport this immunoglobulin from blood to bile. Not only are thesefindings important for understanding diseases involving the liver, the gut, immune complexes and tolerance to dietary antigens, but, in addition, they have provided research workers with an easy way of investigating secretory antibodies in small laboratory animals.

The beginning T h e ravages of enteric infections were a potent stimulus to the pioneer i m m u n o l o g i s t s of the final q u a r t e r of the n i n e t e e n t h century, who were well aware that b o t h the theory and practice of their science should be applicable to the gut. D u r i n g the 1920s specific agglutinins were d e m o n s t r a t e d in the © Elsevier/North-Holland Biomedical Press 1980

stools of patients with bacillary dysentery, and Besredka (a student and colleague of Elie M e t c h nikoff) showed that this disease could be p r e v e n t e d or m i n i m i z e d by an oral vaccine. T h e s e results were confirmed and e x t e n d e d by e x p e r i m e n t s in l a b o r a t o r y animals and livestock so that by the 1950s there was a substantial body of evidence that a special class of

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protective antibodies was present in the mucous secretions of the alimentary, respiratory and genital tracts ~. BILE CANALICULUS

Molecular aspects and transport mechanisms IgA, the principal immunoglobulin in the mucous secretions and colostrum of man, mice and rats, is a minor component of blood serum immunoglobulins (which are mainly IgGs) and it is electrophoretically and antigenically distinct from them. IgA was found often to be about twice the size of IgG and there was some confusion until it was realized that much IgA occurs in a dimeric form, the two pairs of heavy chains being linked by a joining or 'J' chain. The elucidation of these facts is a long story that owes much to the work of the late Joseph Heremans whose review 2 is still paramount. However, even dimerization was insufficient to account satisfactorily for the size of the IgA found in secretions and Tomasi and his colleagues 3 defined an additional 'piece', now conventionally called 'secretory component' (SC) with which the dimerized IgA was obliged to combine before it could undergo secretion. At first, SC was sometimes envisaged as part of the humoral antibody system but the development of immunohistochemical techniques showed this not to be so. SC is not produced by the plasma cells that secrete IgA but is properly regarded as a m e m b r a n e receptor on the epithelial cells that ultimately transport IgA to their external surface. The mechanism of this transport by epithelial cells is now well documented 4& Dimeric IgA, produced usually by subrnucosal plasma cells, unites with SC on the surface of, for example, enterocytes, and the whole (IgA)2-SC complex (often designated SIgA) is transported across the cell in endocytic vesicles6,7 and discharged into the lumen of the gut. It is most important to note that monorneric IgA does not unite with SC and cannot be actively secreted.

Local v. systemic production of IgA In some early experiments, in which radiolabelled SIgA was injected intravenously, it was reported that the injected material was actively transported into the secretions. Therefore, for a time it was believed that the IgA in the secretions was derived directly from the blood either by an active and efficient extraction mechanism in mucous epithelia or by selective transudation. Later, when an overwhelming amount of i m m u n o h i s t o c h e m i c a l evidence showed IgA production to be concentrated in the submucosal plasma cells, it was concluded that most of the IgA in secretions was produced locally. Certainly, local production is important, particularly in the gut. Experiments in which the segmental i m m u n i z a t i o n of surgically prepared loops of gut have led to the local accumulation of SIgA antibody of defined specificity and protective value s,9 leave no doubt that the local production of IgA can occur on a decisive scale.

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Fig. 1 Diagramof the pathwaysby which IgA reaches the gut. The submueosalplasma cells secrete IgA, dimerizedbyJ chain, into the extravaseular tissue space of the gut. Some of this dimeric IgA unites immediately with secretory component (SC) on the basal aspect of the enteroeytes;the IgA-SC complex is transported across the cytoplasm in endocytie vesiclesand discharged into the lumen of the gut. The remaining IgA is collected into the lymphatic lacteals and conveyed, via the intestinal and thoracic ducts, to the great veins. The IgA then circulates in the blood until it reaches the portal vein where it quickly passes through the fenestrated endothelium of the portal sinusoidsand unites with SC on the hepatocyte membrane. Endocytic vesicles carry the IgA-SC complex across the hepatocyte cytoplasm and discharge it into the biliary system which conveysit to the duodenum. However, this does not mean that it is the only way by which secretions may be provided with antibody. M a n y glandular structures whose secretions contain IgA often lack a significant population of plasma cells: they can get IgA only from the blood. It is important to remember that in an individual animal the IgA in the secretions may be derived from local production, by transport from the blood or by a combination of both. Also, the relative contribution of these two sources may vary with age and hormonal changes associated with pregnancy and lactation. What is crucial, as can be seen from Fig. 1, is that the fgA supplied by blood must be free of SC, otherwise its ability to combine with the SC on the epithelia will be

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blocked. Thus experiments in which SIgA is injected intravenously are b o u n d to be somewhat unphysiological and difficult to interpret. T h e m i g r a t i o n of l y m p h o i d cells Reference has been made already to the submucosal plasma cells that synthesize IgA but these are, for the most part, ephemeral cells with a half-life of a few days. Most mammals are endowed at birth with a submucosal lymphoid apparatus which is meagre compared with what develops later. It seems now that there soon develops a subset of circulating lymphocytes which recirculate from blood to lymph by extravasating in the mesenteric nodes and the lamina propria of the gut. When they encounter an antigen some of them transform into immunoblasts, and although some are sessile many are swept into the lymph stream and discharged into the venous blood. However, as soon as the arterial blood conveys them back to mucosal sites, they immediately extravasate into the submucosae and transform into the true plasma cells which secrete the IgA antibody into the interstitial fluid. The fluid delivers it to the enterocytes on the one hand, and the mesenteric lymph on the other. This outline is necessarily superficial and glosses over some rather obtrusive differences between species, but it has been reviewed elsewhere 1° and is a necessary explanation of how a local antigenic stimulus, say in a small segment of bowel, can lead to the production of specific secretory antibodies in remote locations such as lactating m a m m a r y glands. T h e l i v e r and bile: essential factors in the e c o n o m y of the IgA system So far, we have confined ourselves to a brief, qualitative description of the secretory immune system as it was generally understood up to about 1977. We must turn now to a quantitative treatment, for it was this approach which led to the important finding that in m a n y animals the liver is a crucial component of the secretory i m m u n e system. Heremans and his colleagues had shown that in many species the mesenteric lymph contains IgA at a concentration much higher than in the blood. Thus the mesenteric or thoracic-duct lymph of rats contains 0.6 mg m1-1 IgA while the blood contains only 0.1 mg ml-~ - i.e., lymph from the gut, although relatively dilute in terms of total protein and conventional Igs contains six times as much IgA as does the blood. A simple calculation involving flow rates and volumes shows that the lymph delivers daily to the blood enough IgA to replenish it fifty times over 11. The reason for the evident disappearance of this IgA is that rat bile is a rich source of IgA 12 because this immunoglobulin is transported rapidly and actively from blood to bilC~, 13, The scale of this transport is quite enough to reconcile the above discrepancy

Fig. 2. Electron micrograph, by David Robertson and Michael Birbeck, of a negatively stained IgA antibody molecule from rat bile, attached to a flagellum prepared from Salmonella paratyphi A-H (x420,000)

A suspension of these organisms had been injected into the Peyer's patches of the rat sevendays before the bile was collected. The scale bar alongside the IgA molecule represents 26.5 nm which corresponds well with the accompanyingdiagram of an IgA molecule (inset). This diagram is based on models proposed in Hereman's review2. between levels of IgA in intestinal lymph and blood, and it is made possible because the hepatocytes, which in lolo amount to a significant proportion of the animal's body weight, are the ceils which actively carry it out. The transport process has been visualized by electron microscopic autoradiography 14 and it seems to be analogous in every way to that of the enterocytes. The hepatocytes display SC on their surfaces and transport the IgA which unites with it in endocytic vesicleslS; after all, hepatocytes are derived embryologicatly from the gut and may be regarded, in this context, as a specialized epithelial cell. However, in spite of these undeniable similarities between hepatocytes and enterocytes the experimentalist must remember at least one important difference. Because the endothelium of the portal venous sinusoids is discontinuous and exhibits many fenestrations, an intravenous test dose of, for example [125I]IGA, is able to come into virtually instantaneous contact with the very active sinusoidat face* of the hepatocyte membrane, which will convey the IgA to the bile long before it has a chance to diffuse through the conventional capillaries in the gut a n d reach the enterocytes (Fig. 1).

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T h e transport of polyclonal IgA with defined antibody activity Many of the above experiments depended on the availability in man, mice and rats of plasma-cell tumours that produced monoclonal IgA in sufficient quantities to make its isolation and purification a practical proposition. Although such materials have been invaluable it is the job of a physiologist to show that the natural product of non-malignant, antibodyproducing plasma cells behaves in the same way. This can now be done fairly easily in animals. By injection of suspensions of antigens into the Peyer's patches of the small intestine of rodents it is possible to stimulate preferentially the gut-associated lymphoid tissue (GALT) so that antibodies are produced. Under these circumstances much dimeric IgA antibody is synthesized, conveyed via the lymph to the blood and then, by the mechanism described above, secreted into the bilC 6. Thus by collecting bile from appropriately immunized animals specific SIgA antibodies, virtually uncontaminated by other Ig classes, may be easily obtained. Fig. 2 shows an electron micrograph of antiflagellin antibody obtained from the bile of a rat whose Peyer's patches had been injected with killed Salmonella t.yphi A-Horganisms seven days previously. Similarly, it is possible to transfer IgA antibodies passively. Mesenteric or thoracic-duct lymph from actively immunized donors contains dimeric IgA antibody which has not combined with SC. When such antibodies are injected intravenously they appear rapidly and in high titre in the bile (and thus the gut) of unimmunized recipients. When biliary antibody is injected it remains in the recipients' serum since its receptors for the SC on the hepatocytes are blocked by the SC which it previously acquired in the donors' hepato-biliary system ~7.

Monomer, polymer and blood levels Although IgA plasma-cell tumours in animals and man often secrete a mixture of monomer, dimer and higher polymers this is not true necessarily of the nonmalignant IgA plasma cells. Normal mice and rats produce little monomeric IgA, and because the dimeric (or polymeric) forms oflgA are so rapidly and efficiently cleared from the blood by the liver, the amount of IgA actually in the blood at a given moment is small. In this context man is an unusual animal in that human sera contain appreciable amounts of IgA monomer and this phenomenon introduces complications. A 'high' serum IgA level may reflect an overproduction of monomer with, perhaps, a genuine defect in the production of dimer or, alternatively, it could result from hepato-biliary dysfunction causing a defect in the export of dimeric IgA and its consequent accumulation in the blood. Conversely a 'low' serum IgA may be the result of the predominant production of polymeric forms which are rapidly and abundantly

secreted. The picture is further complicated by the fact that the immunodiffusion methods often used to measure serum IgA depend critically on the molecular size of the reactants; hence unless the range of polymeric forms is known, and a corresponding standard used, the results may be grossly misleading. It is not hard to see why the diagnosis of selective IgA deficiency must rest on measurements of IgA in the secretions and counts of IgA-secreting plasma cells in mucosal biopsies.

Biological properties

of IgA

Most enteropathic bacteria and viruses have to adhere closely to the epithelial cells before they can cause mischief and it is through the steric hindrance of this close contact that the protective properties of specific IgA antibodies are manifested. In addition, there is evidence that such antibodies can induce the loss from bacteria of the genetic elements responsible for directing the synthesis of virulence determinants TM. In these types of activity the structure of SIgA is important. The SC probably helps to anchor the molecule into the boundary layer of mucus that overlies epithelia, and the whole structure seems more resistant to gastrointestinal proteases than are other Igs. However, it should be recognized that in the absence of IgA, IgM can take over some of its functions. Also, in some ruminant species the principal immunoglobulin in secretions is IgG, even though IgA is produced as well. It is not perhaps surprising that IgA, which has to operate essentially outside the body in the milieu of the gut and secretions, seems to tack the effector functions of classic humoral antibody: it does not activate complement and is said to be a poor opsonin. None the less, our present view is that the importance of IgA extends far beyond its laudable but essentially unglamorous role in intestinal sanitation. Consider Fig. 1 again. Suppose a dietary or microbial antigen penetrates the mucosa, as indeed such materials constantly dC 9, what could be the consequences? If a high affinity IgA dimer in the submucosa united with this antigen a soluble immune complex could be formed but complement would not be activated. The complex would be carried by the lymph to the blood, and by the blood to the liver, where the whole complex could be consigned quietly to biliary oblivion with no alarming allergic reaction whatsoever. This is not just speculation, for the experiment that shows that the liver can clear complexes in this way has already been done 2°. If, as seems likely, the phenomenon has general applicability it indicates that IgA plays a crucial role in a wider context than was previously supposed. An animal producing only dimeric IgA antibody to a given antigen would, because of hepatic clearance, have little antibody detectable in its serum - it could be dismissed as unresponsive, even tolerant. Indeed it would be tolerant, in the sense that it would

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not over-react to trivial stimuli, b u t would be very far from defenceless. The authors' research is supported by programme and project grants from the Medical Research Council and Cancer Research Campaign. References 1 Pierce, A. E. (1959) Vet. Revs. Annols 5, 17-36 2 Heremans, J. F. (1974) In The Antigens (Sela, M., ed.) Vol. 2, pp. 365-522, Academic Press, New York 3 Tomasi, T. and Bienenstock, J. (1968) Adv. Immunol. 9, 1-96 4 Brandtzaeg, P. and Savilahti, J. (1968) Adv. Exp. Med. Biol. 107, 219-226 5 Brown, W. R. (1978) Gastroenterology75,129-138 6 Porter, P., Noakes, D. E. and Allen, W. D. (1972) Immunology 23, 299-307 7 Nagura, H., Nakane, P. K. and Brown, W. R. (1979) J. lmmunol. 123, 2359-2368 8 Ogra, P. L. and Karzon, D. T. (1969) J. Immunol. 102, 1423-1430 9 Porter, P., Noakes, D. E. and Allen, W. D. (1970) Immunology 18, 909-920

10 Hall, J. G. (1979) BloodCells 5,479-492 11 Orlans, E., Peppard, J., Reynolds, J. and Hall, J. G. (1978)J. Exp, Med. 147, 588-590 12 Lemairre-Coelho, I., Jackson, G. D. F., Vaerman, J-P (1977) Eur. j . Immunol.7, 588-590 13 Jackson, G. D. F., Lemaitre-Coelho, I., Vaerman, J.-P., Bazin, H. and Beckers, A. (1978) Eur. J. Immunot. 8, 123-126 14 Birbeck, M. S. C., Cartwright, P., Hall, J. G., Orlans, E. and Peppard, J. (1979) Immunology37,477-484 15 Mullock, B. M., Hinton, R. M., Dobrota, M., Peppard, J. and Orlans, E. (1979) Biochem. Biophys. Acta 587, 381-391 16 Hall, J., Orlans, E., Reynolds, J., Dean, C., Peppard, J., Gyure, L. and Hobbs, S. (1979) Int. Archs Allerg. Appl. Immunol. 59, 75-84 17 Reynolds, J., Gyure, L., Andrew, E. and Hall, J. G. (1980) Immunology39, 463-467 18 Porter, P., Linggood, M. A., Chidlow, J. (1978) Adv. Exp. Med. Biol. 107, 133-142 19 Hemmings, W. A. (ed.) (1978) Antigen Absorplionby theGut, pp. 1-226, MTP Press, Lancaster 20 Peppard, J., Orlans, E., Payne, A. W. R. and Andrew E. Immunology (in press)

(techniques 1 Assessment of cell-mediated cytotoxicity Benjamin Bonavida and Thomas P. Bradley Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles, California 90024 Several different techniques are used to assess the expression of cellular immunity (reviewed in Ref. l). In this article Benjamin Bonavida and Thomas Bradley discuss ways of measuring one aspect of cellular immunity- the activity of cytotoxic cells on target cells. T h e role of lymphocytes in interactions with target cells which lead to target lysis was not established until the early 1960s. R o s e n a u a n d M o o n 2 were the first to d e m o n s t r a t e the lysis of homologous cells by sensitized lymphocytes in a n in vitro tissue culture system. L y m p h o c y t e s from B A L B / c mice sensitized against L cells from a n allogeneic mouse (C3H), i n d u c e d a striking cytopathic change w h e n coc u l t u r e d with L target-cells. These changes occurred in the absence of a n t i b o d y a n d c o m p l e m e n t . Close contact b e t w e e n the sensitized lymphocytes a n d the target cells was found to be essential for the cytotoxic reaction to occur. I n the t w e n t y years since this demonstration of cell-mediated cytotoxicity (CMC), progress in the direct m e a s u r e m e n t of C M C has been slow, possibly because of the complex n a t u r e of the © Elsevier/North-Holland Biomedical Press 1980

reaction a n d the difficulty in assessing target-cell destruction. In order to make a reasonable assessment of a cytotoxic reaction with a p a r t i c u l a r assay, one should know: (1) the development, differentiation, a n d fate of the cytotoxic cell, (2) the exact n a t u r e of the effector cell involved, (3) whether this effector acts alone or in cooperation with other cells, (4) the biological state of the effector cell before a n d after i n t e r a c t i o n with target-cells, (5) the molecular m e c h a n i s m of the cytotoxic reaction a n d (6) the effect of the target on the cytotoxic celt. F u r t h e r m o r e the assay should provide a m e a s u r e of the absolute frequency of cytotoxic cells present in a mixed population, a m e a s u r e of the affinity a n d avidity of the effector cells to corresponding target-cells a n d a m e a n s to q u a n t i t a t e the cyto-