Immunology Today, vol. 6, No. 5, 1985 149

The study of circulating lymphocytes in vivo: personal view of artifice and artifact

a

Joe Hall

In his book, Advice to a Young Scientist, Peter Medawar ' advised biomedical scientists never to write, 'Mice were injected with serum a l b u m i n . . . ' , on the grounds that few hypodermic needles are large enough for even the smallest mouse to pass through, with or without the lubri- cating effects of serum proteins. His advice seems to have been heeded, for nowadays one does not come across this particular solecism very frequently. Unfortunately, in their efforts to get the grammar right, some immunolo- gists seem to me to have forgotten to think about what they are injecting and where it gets to after they have injected it. Because of this, I have agreed to write some- thing about the whys and wherefores of injecting things into experimental animals, or even people. Much of this has been written before, and better, by others but it seems to have been either ignored or forgotten by many who are working now as immunologists. In addition, some of it is well known only to those who habitually infuse white cells into experimental animals. This comes under the heading of 'unpublished observations' and its inclusion is one reason why I will give few references: it would be mislead- ing to endow certain statements with the spurious authority of a literature citation while others, just as important, cannot be distinguished in the same way. In any case, many of the points I will make represent the application of elementary lymphatic physiology and anatomy and can be gleaned from texts such as Yoffey and Courtice s, now alas somewhat dated.

Much of what I want to say concerns what happens to white cells before and after they are injected into the autologous host or syngeneic recipients but I shall have to consider some simpler things first.

Today, most immunologists have escaped the dubious benefits of a medical or veterinary training; the manipu- lative skills of immunology relate to tissue culture and wet agar rather than to surgical preparations, and new ways of looking at things have emerged. Some people now look at a mouse and see a convenient, self-regulating culture chamber with a leg at each corner. When they give it an injection they seem to imagine that what they inject will slop around inside and get nicely mixed up with every- thing else. It isn't necessarily so. Let us take a few examples, bearing in mind as we do so that antigenic (i.e. macromolecular) material introduced into most tissue spaces depends absolutely on the lymphatic system for its initial transport.

In t ra -pe r i tonea! and subcutaneous inject ions First, let us consider the commonplace i~ect ion of a

solution or suspension of antigen into the peritoneal cavity. I ' m continually puzzled by the belief that much of the antigen given by this route is taken up by intraperi-

Section of Tumour Immunology, Block X, Institute of Cancer Research, Downs Road, Sutton, Surrey, SM2 5PX.

toneal macrophages and conveyed to the mesenteric nodes. This does not happen to any significant extent; the peritoneal cavity is drained by diaphragmatic lymphatics which convey the bulk of the injected material (in or out of macrophages) via the mediastinal nodes, to the thoracic or right lymph duct, and thence to the blood. Some of the particulate material will be retained by the intrathoracic nodes whilst the remainder, together with the soluble material, will gain the blood and thus the spleen. The time taken for this to happen depends largely on the volume injected and on the subsequent activity and respiration rate of the recipient. So, if one wants to time the events of the immune response by the hour, and/or to concentrate on the splenic response, it is better to inject the antigen intravenously (i. v.). Of course, intraperitoneal injections are more convenient, and the induction of immune responses in the intrathoracic lymph nodes is unlikely to always be a disadvantage.

An example o fa favourite subcutaneous (s.c.) route is the injection of antigens into the hind foot pads of labora- tory rodents. Often, the aim of this procedure is to stimu- late the popliteal node (as it does) but frequently much of the injected material bypasses the popliteal node and goes to larger nodes in the inguinal/iliac regions. Again, this need not matter as long as one knows about it, and the knowing is based upon observation. There is a good deal of variation between different strains of rats and mice, and it is instructive to inject lymphography dye instead of antigen, and then perform a dissection to see how the local lymphatics and nodes are arranged in one's own brand of animal .

Minor anatomical variations are no less common in larger animals. In ruminants, too, material injected s.c. into the lower part of the leg goes promptly to the popliteal lymph node but, again, in a large minority of cases, a sub- stantial peripheral lymphatic spirals around the front o f the leg to the inside surface, and leads directly to the inguinal /mammary nodes. This can be an important con- sideration; there are already in the literature important claims which depend for their validity on the assumption that antigens injected into the lower leg go exclusively to the popliteal node. In any general sense this cannot be true. The only way to make sure that antigenic material goes to a particular node is to infuse it directly into one of the afferent lymphatics. This is reasonably easy to do in animals down to the size of rabbits and, given a micro- manipulator and a dissecting microscope, it is by no means impossible in laboratory rodents.

In t ravenous inject ions Detailed anatomical considerations are not needed

where intravenous (i.v.) injections are concerned; material injected into any superficial vein is going to end up in the same place, and this is the preferred route for the adoptive transfer of living white cells. However, before plunging into the subject of how such cells fare, it is

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necessary to distinguish clearly between two contrasting types of experiment.

In the first type the object is not to mimic nature but to demonstrate or measure a particular function contained in a population of cells. A good example of this type of experiment is the enumeration of haemopoeitic colonies in the spleen of lethally irradiated mice after the i.v. injection of cells from syngeneic bone marrow. No one imagines that it is 'normal ' for a mechanically prepared suspension of bone marrow cells to be translated suddenly to the lumen of a superficial vein, or that haemopoeisis occurs normally in the adult spleen. In practice, though, the procedure offers a reproducible and very valuable way of investigating the stem cell potential of bone marrow. Into this category come also those experiments in which, e.g. lymphocytes from primed animals are used to trans- fer immunological 'memory ' to unprimed recipients, or in which functionally athymic animals are reconstituted with T cells. The essential common feature of these experiments is that several days (and sometimes weeks or months) elapse between the injection of the cells and the observation of the experimental result. In the interim, the cells that were injected have ample time to recover from injury, and generally sort themselves out and regroup according to the dictates of a physiological environment. All the experimenter has to do to ensure technical success is to be reasonably gentle in preparing the cells, and to in- ject them slowly enough, and in a small enough volume for the recipient animal to survive. Indeed, it is perhaps the ease, success, ubiquity and general utility of this type of experiment that may have led some people to assume, perhaps unconsciously, that similar procedures are ade- quate for experiments of quite another type.

The other experiments I have in mind are those aimed at observing the migratory behaviour of lymphocytes in vivo. Such experiments usually involve collecting lympho- cytes from blood or lymph, labelling them in vitro with radioisotopes or fluorescent dyes, and then injecting the labelled cell i.v. and studying their distribution in the tissue at various times thereafter. The tempo of the pro- cesses under study is such that the duration of these experiments rarely exceeds 24 h and may be much less. It is the brevity of the interval between the injection of the cells and the observation of the result that causes trouble. The cells under study have litde or no time to recover from the effects of in vitro manipulations, and so their initial behaviour in vivo may be a poor guide to that of normal cells. This problem has been recognized for some time, and some years ago J im Gowans suggested four rules for the study of circulating cells that would help to minimize it.

1. The cells should be those that normally enter the blood.

2. The cells and the recipient should be syngeneic. 3. The transfused cells should be alive and undamaged. 4. The rate of infusion should approximate to the rate at

which the cells normally enter the blood.

Rule 1 means that the cells to be labelled should be col- lected from either blood or lymph. The practice of using suspensions of cells teased from lymph nodes an*d spleens for this type of experiment, seems to me to be quite damn-

Immunology Today, vol. 6, No. 5, 1985

able. There are enough difficulties in interpreting the results of these experiments, without starting offwith cells that may not be in a circulatory mode and which have been exposed to considerable trauma.

Rule 2 seems self-evident but, incidentally, some illu- minating information has come to light by investigating the fate of allogeneic lymphocytes. In a recent article in this journal 2 the late and much lamented Bill Ford pointed out that, at any rate in certain strain combina- tions in rats, allogeneic cells are killed within an hour or so of injection. The killing does not depend upon the pre- sence of antibody or on prior sensitization, and is prob- ably carried out by dendritic interdigitating cells in the paracortices of lymph nodes. Interestingly, almost immediately after they have been injected, the allogeneic cells begin to recirculate normally. Just like syngeneic cells they migrate across the post-capillary venules in lymph nodes but when they enter the parenchyma of the node and encounter the dendritic interdigitating cells they are arrested and ultimately killed. It is most unlikely that this mechanism evolved to deal with allogeneic cells, and it is highly plausible that it is by this means that effete' self lymphocytes are recognized and taken out of circulation. However, a syngeneic lymphocyte that has been manipu- lated in vitro and labelled with 51Cr may well be deemed effete, and I have litde doubt that many syngeneic, labelled lymphocytes that end up in lymph nodes do so because they are recognized as being damaged. Thus, in the experimental situation, a labelled lymphocyte may be in a lymph node for one of two reasons. It may be there for truly physiological reasons, i.e. because it is migrating normally, or because it has been recognized as being damaged, and has been sequestered. It is impossible to distinguish between these two possibilities merely by assaying the total radioactivity in individual lymph nodes. In this type of experiment clear results can be obtained only by counting the radioactivity in the cells which continue to circulate normally in lymph and blood. These may be a selected population but at least they are functionally intact.

The removal of effete lymphocytes by dendritic cells casts a revealing and not entirely flattering light on some aspects of modern immunology. The phagocytosis of effete nucleated cells and red cells by dendritic (veiled) cells can be observed in routine electron micrographs of the lymphoid tissues of, for example, normal foetal and post-natal sheep. Yet because dendritic ceils will not phagocytose in artificial in-vitro systems containing serum (an unnatural medium) or plasma (replete with polyionic or metal-chelating anticoagulants) their status as macro- phages has been denied, and considerable confusion has resulted. In fact, it is very easy to show that under physio- logical conditions in vivo these cells will enthusiastically phagocytose immune complexes (as well as effete cells).

I have included the digression above because it is one of the clearest demonstrations that standard in-vitro condi- tions can inhibit the primary functions of leucocytes. Cells kept under such conditions may well be damaged, albeit temporarily, and this consideration leads us naturally on to Gowans Rule 3.

My own experience suggests that it is virtually im- possible to collect and label lymphocytes without

Immunology Today, vol. 6, No. 5, 1985

damaging them to some extent. The shorter the collection period (of lymph for example) the better. Unfortunately, shorter collections mean fewer cells, which must be given correspondingly more label to make the experiment feasible, and so more radiation and/or biochemical damage accrues. In other words, everything one does to the cells is bad for them. The damage can be minimized by being careful and considerate but, in spite of one's best efforts, the labelled cells are always cleared from the blood of the recipient much more rapidly than can be reason- ably accounted for in terms of physiological extravasa- tion. In certain situation cells can be labelled in vivo by in- stilling fluorochromes or radioisotopes into lymphoid organs by direct injection into the substance of the tissue or, better, by perfusion via the regional blood or lympha- tic vessels. Such techniques certainly minimize the initial damage to the cells and have much to offer but they are not universally applicable.

Rule 4 is a counsel of perfection and brings a fresh set of difficulties. I f labelled cells are to be infused intravenously (i. v.) at a physiological rate, they usually have to be stored in the reservoir of the perfusion apparatus and this is often just as bad for them as being injected too rapidly as a bolus. In theory, it is possible to design an apparatus .that continuously collects, labels and washes the cells and then delivers them at a physiological rate, but such equipment is out of reach of all but the wealthiest and most sophisti- cated laboratories. The crucial point is that lymphocytes, and particularly the large lymphoid immunoblasts, are extremely delicate cells which under natural conditions in vivo spend very short periods of time as free-floating cells. This may seem a surprising statement to the many immunologists who work with lymphocytes in vitro. After all, the transformation in vitro of lymphocytes into blast cells, in response to a bewildering variety of stimuli, is one of the corner stones of modern immunology, and it is all done in simple suspension cultures. Nonetheless, the fact remains that the conditions of these cultures are totally inimical to the survival of lymph-borne immunoblasts that are generated by bona fide immune responses in vivo. When such cells are placed in suspension cultures, either in the usual media containing 10 % foetal calf serum or in their own lymph, they die. Their half life in vitro is only some 6-8 h and 25 years of juggling with the culture conditions has not so far significantly extended this time. In vivo, on the other hand, most of them do not die. After being injected i.v. they extravasate in various lymphoid tissues and, if they are B cells, they provide themselves with highly organized, concentric lamellae of endo- plasmic reticulum and, as mature, antibody-producing plasma cells they have a half-life of several days. Apparent ly they can do this only when supported by the reticulum of lymphoid or haemopoeitic tissue. Whether this support is purely mechanical, or involves more specific cell contacts or growth factors remains to be determined. What is certain is that, in vivo, the time that elapses between the discharge of an immunoblast from stimulated lymphoid tissue into the lymph, and its later extravasation into the lymphoid tissue where it will mature, is a matter of minutes, or even seconds. Is it so surprising that cells which under natural conditions spend so small a part of their lives as free-floating cells, should be

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so badly equipped to survive in suspension cultures? When such cells spend hours in collecting bottles, and are then subjected to incubation with isotopes and the t rauma of repeated washings by centrifugation, they are likely to be in a rather unhealthy state. Being squirted through a needle or a cannula into a superficial vein must be almost the last straw, and it is surprising that enough survive to make experiments possible. Yet it is at this stage of the game that the cells have to face their greatest ordeal; they have to run the gauntlet of the pulmonary capillary bed, the graveyard of many a cell and not a few experiments.

The lungs as a t rap for in t ravenous ly injected white cells

When a suspension of white cells is injected into a superficial vein the first capillary bed which the cells must pass through is that of the lungs. Many of them do not succeed in passing; some of them are immobilized in the capillaries only temporarily, others are destroyed. This problem was first recognized during attempts to treat patients suffering from agranulocytosis with i.v. trans- fusions of white cells. Of necessity, the cells which were infused were allogeneic but the same trapping of cells in the lungs was noticed when radio-labelled, syngeneic lymphocytes were infused into experimental rats and sheep. When the lungs were removed from the animals 20 rain or so after the cells had been injected, they were found to contain up to 40% of the injected dose of radio- activity. This trapping seemed to be peculiar to the lungs, for when the cells were injected intra-arterially, instead of i.v., the results were unaltered. That is to say, the injected cells successfully negotiated all the somatic capillary beds to which the arterial blood carried them but, upon arrival at the lungs, they were immediately arrested. Similar results were obtained when labelled cells were infused slowly into the hepatic-portal vein. What is the reason for these results? What is it about the lungs that makes them such a trap for infused white cells? The most obvious explanation is that the pulmonary circulation is driven by the right ventricle at pressures which are modest com- pared with those generated by the left ventricle for the systemic circulation. As a result the net perfusion pres- sures across the pulmonary capillaries are equivalent to a few millimetres of mercury only. These pressures are evidently insufficient to 'blow through' moribund cells which tend to aggregate and form emboli. In addition, the expiratory reduction in lung volume may cause transient narrowing of the capillaries which may help to trap cells which are too sick to adjust their shape and cross section with sufficient rapidity. Many of the cells trapped in this way soon escape, reach the systemic circulation and extravasate so that radioactivity starts to accumulate in the appropriate lymphoid tissues. Lungs removed from experimental animals 2 or 3 h after the labelled cells have been injected contain only a few per cent of the injected dose of radioactivity. However, it is wrong to assume that all of the cells which were trapped initially, ultimately escaped unharmed. The reduction of radioactivity in the lungs can be accounted for to some extent by the catabo- lism of the trapped cells and the excretion of their label. When rats are given macrophage poisons before the start of the experiment, the local lung macrophages become

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less able to degrade the trapped cells and, under these conditions, high levels of radioactivity can persist in the lungs for many hours.

The proof of this rather unpalatable pudding is that, in these sorts of experiments, the amount of radioactivity remaining in the animal at, say, 20 h after the injection is often no more than half of what was injected. This fact is often ignored in the Results section of formal papers and the amount of activity in a given organ is expressed as 'percentage of recoverable activity'. This last figure refers, of course, to the amount of radioactivity that remained in the major organs when the experiment was terminated. It is clear that one must be rather careful about attributing physiological significance to the results of such experiments. Very big effects probably do give some indication of physiological reality but it is hard not to worry about a situation where half of the experimental material has been destroyed.

Where labels like [3H]thymidine have been used, the destruction of the cells may lead to reutilization of the label. This can be avoided by using x25IdUr instead but this has its own sources of error. In many experiments, the object of the study is to demonstrate the extravasation of immunoblasts into the gut and peripheral exocrine glands like the salivary and lachrymal glands. Unfortunately, these glands, together with parts of the glandular stomach, are capable of incorporating iodine into their secretions. Thus, some of the 125-iodine released from dying immunoblasts can become associated with these secretory organs, and their content of radioactivity may be equated erroneously with the presence of intact

Immunology Today, voL 6, No. 5, 1985

immunoblasts. Simple biochemical analyses (or auto- radiographic studies, in small animals) can resolve the situation, and they reveal frequently that only 20 % or so of the radioactivity is actually associated with nuclear DNA.

In this article I have tried to show how seemingly trivial technical details may have important consequences because they relate to those aspects of anatomy and physiology that help to integrate the immune responses of higher vertebrates. Also, I have emphasized that some of the inherent (as opposed to adaptive) recognition systems that discriminate between ' se l f and 'non-self probably evolved to discriminate between 'self' and 'damaged- sell'. Because of this, they impinge directly on experi- ments aimed at investigating quite different aspects of immunology. The difficulties introduced by such phenomena cannot always be avoided but they can be recognized and taken into account in both the design and interpretation of experiments. Like the bunkers in a golf course they contribute a good deal to the fun; they are a challenge to the participants, and a source of endless amusement to the informed observer. IT]

Acknowledgement The author's research is supported by a programme grant awarded

by the joint committee of the Cancer Research Campaign and the Medical Research Council.

References 1 Medawar, P. B. (1979) Advice to a Young Scientist, Harper & Row Ltd 2 Ford, W. L. (1984) ImmunoL Today 5, 227 3 Yoffey, J. M. and Courfice, F. C. (1970) Lymphatics, Lymph and the

Lymphomyeloid Complex, Academic Press, NY