Annals of the Royal College of Surgeons of England (I978) vol 6o

HUNTERIAN FESTIVAL, 1978

Inflammation Papers* contributed to a symposium arranged by the Institute of Basic Medical Sciences at the Royal Coliege of Surgeons of England on Ixth February 1978

INTRODUCTION: INFLAMMATION, WITH EMPHASIS ON ITS MEDIATION G P Lewis BPharn PhD Vandervell Professor of Pharmacology, Royal College of Surgeons of England

'This operation of the body tenned in- flammation is to be considered only as a flammation requires our greatest attention disturbed state of parts which require a new for it is one of the most common and most but salutory mode of action to restore them extensive in its effects of any in the anmal to that state wherein a natural mode of action body'-John Hunter, A Treatise on the alone is necessary; from such a view of the Blood, Inflammation and Gun-shot Wounds, subject therefore inflammation in itself is not to be considered as a disease but as a salutory I794. operation consequent either to some violence Hunter was a great experimentalist and was or some disease . . .'. It is difficult to deny this really more of a, researcher than a surgeon. He truth even today, and yet we strive to produce would be delighted to know that there is a anti-nflammatory agents. I think investigators strong element of research in this College, in this field sill tend what Hunter particularly since it has housed within its waUs goes on to point out, '. to. . forget but when the Insdtute of Basic Medical Sciences. The accomplish that salutory purpose asit cannot in the subject of inflammation is one which plays cancer, scrofular, venereal diseases, etc, it does a part in the research programme of each of mischief'. And we might add inflamatory our departments and was one of the special joint diseases such as rheumatoid arthritis to interests of John Hunter. One of his greatest that list. works was a book entitled A Treatise on the So Hunter pointed out to us a long time ago Blood, Inflammation and Gun-shot Wounds, our therapeutic approach should be to that published in 1794, in which he set out to define not acute inflammation itself but the prevent inflammation and provide a description sometimes based on ihis own experiments and 'mischief' that it causes. But before such observations which is not too far from our advances could be made using this approach view of inflammation today. He writes, 'I the concept of chemical transmission to mediate shall call by the name of inflammation what- biological processes was developed. There folrowed three maln lines of reever produces the following local effects, viz. all As we a search was made for the know, rednes'. search-first swelling, pain, vasodilatation, increased vascular permeability, chemical substances which mediate the cardinal and pain are still regarded as the main features signs of inflammation; then attempts were made to synthesise antagonists of these mediaof acute inflammation. In Hunter's day research was directed tors; and thirdly, in order to test these anmainly to description and at attempts to in- tagonists (that is, anti-inflarmnatory agents) terpret the meaning of inflammation in teams models were devised which embodied one or of the body as a whole. In an attemnpt to more of the signs of inflamation. The accompanying diagram (Fig. i) shows determine whether inflammation was a good 'Inthat the simple screening tests like skin redhe for the body, writes, or bad reaction *Some of these papers have been shortened for publication

Inflammation _

Etiology

Release of mediators (extracell.)

Vasodi latation t Penneability Pain

Genetic

Cellular

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I93

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FIG. I Some pathways in_._._...*endothelial volved in inflammation.

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Release of * mediators

dening, rat paw oedema, turpentine pleurisy, and so on are most likely to produce agents which interfere with the chemical mediators of acute inflammation because they limit the search just to those pathways which are marked in heavy type. This is why we have many agents like indomethacin which prevent the signs of acute inflammation in many instances by interfering with the action or formation of mediators like prostaglandins. In inflammatory joint diseases, for instance, although they relieve the symptoms, they do not affect the progress of the underlying disease. In some instances they might even enhance the progress of the disease, possibly because they inhibit, in the words of Hunter, 'salutory mode. of action of inflammation to restore the disturbed parts to their natural state'. Immunologists studied immune complex formation and cellular immune responses long before pharmnacologists thought that these pathways could be pursued as targets for a therapeutic approach. And even then these pathways were not examined in themselves but were incorporated into the models where we still measured the effects of agents on the vascular changes. However, with the array of expert immunologists taking part in this symposium, we can expect to be presented with some interesting possibilities. Dr Peter Dukor has kindly come all the way from Switzerland to tell us about the intriguing subject of 'Immunopharmacological control of inflammation'. Later we are going to hear about some aspects of immune-complex-mediated tissue damage as well as cell-mediated immunity

from Professor John Turk and his colleague Dr Badenoch Jones. What I would like to hear from them is a consideration of the possibility of having models which would allow us to examine the effects of potential therapeutic agents on single immunological pathways. Would it, for instance, ever be possible to develop a drug acting to prevent an immune response and still retain sufficient specificity to avoid over. whelming toxicity? What does it mean for a drug to be active in an Arthus reaction or a Dumonde and Glynn model' in terms of its activity on individual pathways such as comr. plement? To show that the drugs we have available are effective in these models, I think, is not enough because we really want to develop a different kind of antirheumatic drug from the standard ones we use and this caUlls for a new approach. I would like to tell you about an attempt at a new approach which involves several of the pathways outlined in Figure i.

Enzyme activity In an attempt to follow the initial effects of cellular injury we used a model in which we collected lymph directly from hind limbs of rabbits and studied the leakage of cellular contents such as enzymes into the lymph after different kinds of tissue injury2'7. One of the models used by Dr Jasani and me was the homograft reaction'8 9. We started to use this as a model for rheumatoid diseases some years ago because so many of the features of skin graft rejection, both macroscopic and micro-

Inflammation

194

scopic, resemble the changes which occur in inflammatory joints, and we wanted if possible to examine underlying mechanisms in the model. At first we were particularly interested in the biochemical changes which occurred during the reaction. In order to study these changes we collected lymph directly from rabbit hind limbs bearing either autografts (which do not reject) or hornografts (which

do reject). We found that the pattern of intracellular enzymes leaking from the grafts into the lymph was different for autografts and homografts. Figure 2 shows the activities of six intracellular enzymes in the lymph supernatant collected from a rabbit hind limb over a period of I7 days. At day -3 the cannula was implanted into the femoral lymphatic; at day o the skin was grafted on to the lower limb; 2100 and at day 6 the process of rejection started. 1900 The first point we observed in this series of experiments was that there was an increase 1700in the activity of some of the enzymes during 1100 the first 2-4 days after grafting-that is, during o 900 l the healing-in of the grafts. We saw an in-j ~~~~~~~~~~~~1100 during this time of two transaminases, ,crease 1 700 aspartate aminotransferase (GOT) and alanine -1O 900 I ^^>v* >,Ag aminotransferase (GPT), and smaller increases 5 0 j ~ ~ ~ ~ ~ ~ ~ ~ 0 t .o acid phosphatase and cathepsin. It was in I 0 500F k 300 -t usually these four enzymes which increased in 500 LDH ~ ~~:t j [ 100 the pre-rejection period. The main increase, however, occurred just after rejection and at this time there was an increase in all the F00 00 LGT50 e-gnuC. 300 enzyme activities and particularly in lactic 0 dehydrogenase (LDH) and /-glucuronidase 300~~~~~~~~~10

(I?-gluc.).

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-3 -1 0 1 2 3 4 5 6 7 8 9 10 12 14

Days Composition of lymph and lymph flow from hind limb of NZW rabbit bearing 6 skin homografts. Rejection began on day 7. Enzymes and lymph flow expressed as % of control values. Result of one experiment in which the collection of lymph was begun 3 days before grafting and lymph collected several times daily. Clotting occurred on day 7, at the onset of rejection, and on day 9, but once cleared it was possible to continue collections up to the I4th postoperative day. (Reproduced by permission from the Journal of Physiology8.) FIG. 2

Figure 3 illustrates the changes in enzyme lymph collected from a limb bearing autografts. There are two experiments here, one starting 3 days before grafting and continuing to day 6, when the Jlymph clotted, and the other in which cannulation was made 3 days after grafting and continued until day i i. What we see is an increase in the same four enzymes during the ,period 2-4 days after grafting, but no later increase. Since rejection does occur in the case of autografts it seems likely that the main increases which were found in the homograft experiments are related to the specific immunological reaction of rejection. On the other hand -these early increases appear to be related to the non-specific reaction to the trauma of transplantation because it occurs in both homograft and autograft experiments. The question arises, does the appearance of different enzymes in these two, different reactions mean that they might mediate specific effects? The fact that they are enzymes does not in my mind exclude them from being classified as mediators. But a difficulty which

activities in the femoral ~~~~~~~~~~~~500

-C

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I95

creased most of all in the lymph supernatant during rejection. It appears therefore that the L 7 s, *, ",-- ' LDH 0 main increases which we find in supematant 400 lymph originate in the lymphocytes which I 200 undergo necrosis around the graft tissue. v '-' '" F-gluc. No And surely here axe targets for a therapeutic 0O approach-selective reduction of an excess 200 of hydrodlytic and other enzyme ativity "%"~~I % /AA,% (intracellular mediators in Figure i) either by 4~0L 1400 w u I L"''GT 02 C enzyme inhibition or preventing their release. -6 0_ 200 It will be interesting to have the views on this ",IO ~~~~ L-~GPT subject of Dr John Dingle, whose outstanding oLL. work in this area at the Strangeways Labora20 0->~~-, Aci tories, Cambridge, has gained international 1400recognition. Lymphokines 200l 200 " " Ylo PAi L gra gtein -~~~~~~~~400 Another finding from our earlier experiments 200s 0rfter with homografts agreed very nicely with the Aci 3-00 work of Dr Dudley Dumonde and his colleagues at the Kennedy Institute on lympho-3 -1 0 2 3 4 6 7 8 h910.1 1 0 cyte-activation products which they have called 2400 lymphokines. When we examined the lymphatic rabbi hin libbarnhsiuogrfs. Enzme and vascular connections of the graft we found Li ~~~~~~~~~100 that in most high-dose homograft experiments FIG. Comositin a lymphatic connection was never established ro - 3 -1 0 123 4ofIymphand 56 7 8910 ymphflow 12 14 between the donor and the host. This is Days after grafting FIG. 3 Composition of lymph and lymph flow from illustrated in Figure 4, which summarises the rabbit hind limb bearing 6 skin autografts. Enzymes No of animals Autograft and lymph flow expressed as % of control values. 7Homograft m IQ

%

200

/i.

I

k'

%J

Results of 2 experiments, in one of which the collection of lymph was begun 3 days before grafting and in the other on the third day after grafting. Lymph was collected several times daily. (Reproduced by permission from the Joumnal of Physiology8.)

is common to the study of any potential mediators is that of determining whether they are released to perform a function or just incidentaRly as a result of the injury itself. One fact is certain and that is that the nature of the material released depends not only on the extent of the reaction but on the nature of the cells involved in the reaction. A clue to the origin of the enzymes released in the homograft lymph came from experiments in which we examined the enzyme activities of the lymphocytes in the lymph. We found that there was an increase in the enzyme activities of the lymphocytes at the time of rejection. Not an increase in the cell count but an increase in the activities per cell. In adcdition, these cells were particularly rich in LDH and f.-gluc., the two enzymes which in-

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FIG. 4 Presence or absence of lymph drainage in homografts and autograft as judged by the appearance in the lymphatic cannula of Evans blue dye injected into the graft tissue. Each square represents the result of one experiment obtained on the day indicated by the abscissae. The numeral in each square denotes the number of grafts per animal. The open squares above the line give the experiments tin which lymph drainage occurred, the hatched squares below the line those in which it did not occur. (Reproduced by permission from the Journal of Physiology".)

Inflammation

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results obtained from 7 I homograft experiments and contrasts them with those obtainedl from 53 autograft experiments in which rejection does not occur. From day 7 onwards after grafting a lymphatic connection was present in all autografts and it was independent of the number of grafts per animal. Even on day 6 after grafting 7 out of 9 and already on day 5 2 out of 9 autografts had lymphatic connection. On the other hand no lymphatic connection was established in homografts aftet thc 8th postoperative day. Lymphatic con,nections were obtained in only 7 out of the 33 experiments tested between days 6 and 8 an(I only in experiments with a low dose of grafts per animal. Of these 7 experiments with1 lymphatic drainage, in 5 it occurred on day 7 and in i each on day 6 and day 8. An explanation of this finding came when we examined the vascularisation of the grafts. F'igure 5 summa rises results obtained from 53 experiments with autografts and 76 witl honio'grafts. In both groups vascularisatioln between graft and host tissue occurred in about half the experiments on day 4 an(l it was present in all experiments on day 5. From day 6 onwards the autografts and homografts No. of animals 9

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Dyafter grafting

FIG 5 Presence or absence of vascularisation in homografts and autografts as judged by blueing of the skin after intravenous injection of Evans blue. Each square represents the results of one experiment obtained on the day indicated by the abscissae. The numeral in each square denotes the number of grafts per animal. The open squares above the line give the experiments in which vascularity was present, the hatched squares below the line those in which it was absent. (Reproduced by permission from the joumal of Physiology8.)

behaved differently. All the autografts retained their vascularity, while in an increasing number of homografts from day 6 onwards the blood flow ceased as a result of the onset of rejection. According t-o these findings in antimals bearing high-dose homografts a lymphatic connection is not established between the host and(l lhe donior graft unless homnograft rejection is delayed, the explanation being that the vascuhlar shut:-down accompanying the onset of tejection occurs before the lymphatics develop. The reason for mentioning this finding is b)ecauise of its wider implications in the mecha.nism of graft rejection. The sensitisation hiat; leads to rejection of the graft tissue takes platce in tlwo stages: first, cointact between the antigen of the graft tissue and lymphocytes enterin£g the graft from the blood stream; and second, passage of the primed lymphocytes irto, the regional lymph node. To achieve the second stage it is implied that there must be a lymphatic drainage of the graft itself to convey the primed lymphocytes to the regional lymph node. Our experiments show that this is not a prerequisite of homograft rejection. Howvever, a lymphatic drainage of the underlying graft bed does appear toi be present and may take part in a third stage in the sensitisation process-that is, the migration of the primed cell back into the recipient tissue before it is taken by the host's lymphatic system to the regional lymph notde. So, perhaps wve must add to our list of chronic mediators diffusable substances which might bring about ,such cell/cell interactions. Alternatively, it is possible that the diffusable products from lymphocytes which have been called lymphokines" might help to provide an explanation of the effects seen in the regional lymph node during the rejection reaction in a different way. Perhaps lymphocytes activated at the site of the graft produce lymphokines peripherally which diffuse into tle host tissue, are taken up in the lymph as soluble substances, and are transported to the regional lymph node, possibly together with primed lymphocytes to produce the changes observed. Dr Dumonde and his colleagues have already shown that injection of these lymphocyte-activation products into the afferent lymphatics produces effects in the regional lymph node that closely resemble

Inflammation the effects produced in the node by a cellrmediated response in the tissue. Such lymphokines might well mediate someni of the vascular changes as well as celltular events in other chronic inflammatory reactions b[o0o. However, even in a reaction like homograft rejection it seems that some of the changes are brought about by what we normally regard as acute mediators. Dr Beverley Manglhanii an(l I have recently"' been looking foir such mediators in homogenates of grafts taken a. various stages of the reaction and hav1:e attempted to correlate their presence w\it1i c(Jianges in blood flow in the grafts. Ln or(ler to make more precise measurements of blood flow changes than we had previously ve uised a. xenon clearance technique in hoti atitografts and homografts. No blood flow wva.s (letected in either the autografts or the homiiografts until day 3, at wvhich time it was similar to tChat in normal skin. Between days 3 and 1. it inccreased rap(idly, reaching a peak arounid (lay 4 in both1 autografts and homografts. 1i Llie autografts the peak blood flow was maiiitairmed until day 6 and this gradually decreased until it reached normal skin blood flow. In hlie lhomogra-ft, however, the peak blood flow wvas maintained for a 12-h period only, after which the blood flow fell dramatically until 11o -flow could be detected at day 5, hour 6. Histamine and prostaglandins When wve looked at the histamine and prostaglandin E, (PGE2) contents of the grafts we found that in the autografts the histamine content increased as the blood flow increased, wrhereas the prostaglandin content remaine(d low throughout. If this histamine is responsible for the blood flow changes in the autografts it would appear to be released in a form not accessible to histamine antagonists because the blood flow changes were not altered by histamine H, or H, receptor antagonists. In the homograft there was a different pattem. The histamine content formed two distinct peaks, the first during day 4 at which time there was also a smaller but significant rise in PGE,. Then after day 5-that is, after the onset of rejection-both histamine and PGE, increased significantly. When compared with the blood flow changes in the homograft it was dear that when the histamine content

I97

increased so did the blood flow, but at the hiighest concentration of histamine the blood flow in the homografts stopped completely. Again if the histamine is responsible for the increase oif blood flow during this healing-in process it is unaffected by the H, and H, antagonists as in the autografts. However, the lhigh,. hist amine content may be partally resl)onsible for the vasoconstriction that occurs at tlhe onset of rejection because the cessation of blood flow is delayed in an animal treated Wiith antihistamine. In rabbits treated with mcl))'ramine blood flow ceased on days 7-IO cornl)ared with day 5 in the untreated animals. Tlhe healing-in process involves growth of new tissuie and it has been suggested earlier tl.at bistamine formation is associated with new gt-ovthlan.(ldthat the actions of this histamine are resistant: to antihistamine, which fits weH %vith ouir present findings. Whether histamine is (lil ectly involved in the growth process is nothlerdt and interesting matter. Professor McMinni is going to round off this symposium wvith. a talk about tissue repair, although he Will concentrate on cellular aspects rather than

mnediators:r. Fro,m anlloher point of view we know that histcamine in hiigher concentrations causes vasocoustriction via an H, receptor (at least in the rabbit). The findings firstly that the histamine ont:enlt of the homograft (3 times that of aiftografts) occurred just before the cessation of blod flow and secondly that the stoppage of blood flow was delayed by antihistamine and mepyramine both suggest that histamine inight be involved in the vascular events at the onset of graft rejection. Prtostaglandins, however, appear to play a

miniior role in this reaction because in animals

lrieated with a dose of indomethacin which was sufficient to inhibit prostaglandin formation the only effect on the blood flow changes was a reduction of peak blood flow in homografts which corresponded to the small increase in PGE, content of the homografts. There was io delay in the cessation of blood flow. I think we can still regard histamine and br'adykinin, which we have not yet examined very closely in homograft tissue, as primary mediators of acute inflammation, whereas prostaglandins are to be considered more as modulators of the actions of other mediators.

I98

Inflammation

We can see a good example of this when we examine acute pain. Both histamine and bradykinin produce pain when low concentrations are injected intradennally, whereas prostaglandins in similar concentrations cause no pain. If, on the other hand, histamine or bradykinin is injected at the same site as the prostaglandin the pain produced is greatly potentiated in intensity and duration. Tim Williams, a member of my department, is particularly interested in this interaction of prostaglandin with histamine and bradykinin with regard to their effect on vascular permeability. In this respect too, the prostaglandins potentiate the action of the other two mediators. It might well be, and this will be one of the points he will discuss, that this potentiation is the result of the vasodilatation produced by the prostaglandins. Even Hunter some 200 years ago pointed out that 'the colour [vasodilatation] and the swelling [increased permeability] correspond very much since they both depend on the same principle'. There appears to be another kind of modulating effect exerted by prostaglandins which works in the opposite direction, resulting in an inhibitory effect. It has been shown, in the US as well as in this country, in isolated systems that some inflammatory cells have receptors which, when stimulated, cause an increase in intracellular cyclic adenosine monophosphate which in some way inhibits the release of mediators. It has also been shown that prostaglandins stimulate such a receptor and cause a reduction in the release of other mediators. Dr Priscilla Piper and her colleagues, also in the Department of Phar-

macology, have shown this to occur in an immunological response in sensitised guineapig lungs. She will show later that when the prostaglandin system is greatly reduced by giving indomethacin, this reaction produces a great release of histamine and slow-reacting substance of anaphylaxis (SRS-A). This finding is a good illustration of the inhibition of a negative feedback mechanism. SRS-A is one of the acute mediators about which not very much is known, and Dr Piper is kindly going to bring us up to date on that particular subject. In spite of all the research that has been carried out on the subject of inflammation since Hunter's Treatise of I794 it is still a subject which 'requires our greatest attention'.

References Dumonde, D C, and Glynn, L E (I962) British Journal of Experimental Pathology, 43, 373. 2 Lewis, G P (I967) Journal of Physiology, I9I, 59'. 3 Lewis, G P (I969) Journal of Physiology, 205, I

6I9.

4 Boyles, S, Lewis, G P, and Westcott, B (1970) British Journal of Pharmacology, 39, 798. 5 Bach, C, and Lewis, G P (I973) Journal of Physiology, 235, 477. 6 Bach, C, and Lewis, G P (I974) British Journal of Pharmacology, 52, 359. 7 Butler, K, and Lewis, G P (1976) Journal of Pathology, II9, I75. 8 Jasani, M K, and Lewis, G P (I97I) Journal of Physiology, 219, 525. 9 Bullock, G, Jasani, M K, and Lewis, G P (0977) Cell Biology International Reports, i, 123. I0 Dumonde, D C (1970) Annals of Immunology, 2, I 29. ii

Lewis, G P, and Mangham, B (1978) British Journal of Pharmacology, 62, 420.

THE ROLE OF PROSTAGLANDINS IN INFLAMMATION T J WilliaMS BSC Department of Pharmacology, Royal College of Surgeons of England

Introduction John Hunter was the first to recognise that the redness of inflammation was due to an increase in blood supply to the affected tissues and that tissue swelling was due to an extravasation of fluid from the blood'. A contemporary of Hunter, the Reverend Edmund Stone, discovered2 that willow bark contained an active ingredient, later to be called aspirin.

Two centuries passed before the evidence was obtained which linked these independent observations. Firstly, Wilis3 detected prostaglandins in inflammatory exudates, and secondly, Vane and his co-workers4 discovered that aspirin inhibited the synthesis of prostaglandins in vitro. However, the reports coinceming the exact mechanism of action of prostaglandins in inflammation have been as

Inflammation, with emphasis on its mediation.

Annals of the Royal College of Surgeons of England (I978) vol 6o HUNTERIAN FESTIVAL, 1978 Inflammation Papers* contributed to a symposium arranged b...
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