28
Agents and Actions vol. 6.1/2/3 (1976) Birkh~iuserVedag, Basel
Lymphocyte Depletion in Patients with Rheumatoid Arthritis C.M. Pearson* Department of Medicine, School of Medicine, The Center for the Health Sciences, Los Angeles, California 90024, USA
Abstract Sixteen patients with severe rheumatoid arthritis, marked inflammation of the synonvial membrane and high rheumatoid titer were cannulated by the thoracic duct for a period ranging between 82 up to 100 days. The patients being not under any medication during that time. Quantitative and qualitative analysis of the lymphocytes were performed, as well as responses to mitogens, rheumatoid factor, circulating antibodies and delayed hypersensitivity. By the 14th day nearly all the patients had a partial or almost complete remission of their disease. No complications were observed. These results will be discussed.
Discussion
Svartz (Sweden) This piece of work on lymph drainage is very interesting, considering that so few examples of rheumatoid arthritis occur in lymphatic tissues. I f you consider for instance how much of the granulations are in the skin, and also large amounts in the neighbourhood of the joints, it must be interesting to study both the T cells and B cells. As far as I can understand there was also a decrease in the granulation tissue in the capsule of the joints. This is very remarkable. In our treatments of rheumatoid arthritic joints with injections and so on, scarification was usually found. I don't think you found the same thing here. It is also interesting to take tests from the skin while the drainage was in progress. In your lecture, you spoke about the change in cell populations, but you did not speak about dead cells. There is a great difference between your treatment and the treatment using various drugs.
Pearson (USA) T h a n k you for your comments Dr. Svartz. We have data and I didn't have time to discuss this. We have done serial needle biopsies of the synovial membrane, demonstrating that there is a great decrease in cellularity, of cell types of the synovium, and I will not go into the details of them. Great decrease in the amount of cells, in the thickness of cells but very little scarification of the lining cells that do occur during this situation. Therefore, I feel that we have removed m a n y of the cells that are inducing and continuing and propagating the inflammatory response. Actually, and amazingly, the n u m b e r of lining cells of the synovium - the synovial sites - decreases from seven or eightcell thicknesses, occurring as you know in active rheumatoid arthritis, down to one or two during this period of time, and then gradually builds up again after the drainage is discontinued. Turk (UK) In that connection, Carl, have you investigated the mononuclear phagocytic system, both in numbers, quantitatively, and in function after thoracic duct drainage in man? Pearson (USA) Yes, John, we have done some of that, and I am afraid I can't give you the finite details of those. Dr. Leonard C;oldberg in our unit has done some studies of the phagocytic monocytes,
*Unfortunatelythe completemanuscriptdid not arrivein time for publication,it ishoped to publishthisarticlein alater edition of Agents and Actions.
LymphocyteDepletion in Patients with Rheumatoid Arthritis and so forth, in the peripheral blood and occasionally in the synovial fluid. He finds them not to be changed to any significant extent during the period of time during drainage or afterwards. So it appears, that we are removing the T lymphocytes that do not convert to monocytes, if they do supposedly do so, at the site. They are not changed to any great extent. Ford (UK) In the studies of the immunological function of these patients undergoing chronic thoracic-duct drainage, I suppose the results tie in very well with animal experiments, in which the antibody responses are depressed rather little and delayed hypersensitivity tends to be depressed rather more. The exception in animal experiments, is with so-called strong histocompatibility differences, where grafts a cross an H 2 and AGB locus tend to be rejected in very little longer than the usual time, even in quite severely-drained rats and mice. Yet in your patients, husbands and wives presumably differing in HLA locus, you have survivals of a year, which is quite remarkable, and I wonder if we could go into that a little more? Were the patients receiving any other treatments, e.g. steroid treatments; do you know that there is a strong histocompatability difference? Are you sure that the tissues actually survive and weren't overgrown with the host tissue? Pearson USA) Yes we do have some evidence on this. Thank you for raising that point. We have H L A profiles on all of our patients and their husbands during this period of time. The grafts actually have survived during this period of time and it was a great surprise to us, as there was a great deal of differential in H L A types obviously, as you have implied, between husband and wife. Yet they have survived for reasons that are perhaps explainable, and perhaps need some more theory. The H L A types were very significantly different, and we had not started these patients after drainage on any therapy, except for a small number on aspirin, that they would require. We did not give them immunosuppressants or anything else, or cortico-steroids which might suppress the skin graft rejection. No Sir. Kacaki (Netherlands) Does the rebound of cell-mediated immu-
29 nity that you have seen in the majority of patients coincide with the recurrence of the clinical symptoms? The second question is do you think that the T cells that you have removed and the T cells reappearing in the patient after thoracic duct drainage have similar characteristics? Pearson (USA) T h a n k you for your questions. In regard to the second question I am not sure that I can answer you about the character of the T cells. Our impressions are that they would be the same, but the technique of identifying subpopulations of T cells is still in a great deal of debate, and we have not been able to clarify these - are these suppressor cells or whatever? I don't think I can say this for certain. On the first question on rebound, yes it does, Sir. The rebound of delayed hyper-sensitivity does occur, in essence, in parallel with the reappearance of the activity of the rheumatoid arthritis. So, at 30 days for instance, if the individual before drainage had been PPD positive, he or she is liable to be barely positive to PPD. By 90 days very strongly, if they were strongly before. So in essence it does parallel, quite considerably and quite precisely in the majority of our patients; delayed hypersensitivity skin test reactivity, PHA response, and so forth, to the lymphocytes and the reappearance of the rheumatoid arthritis activity. Poulter (UK) Can I just ask one or two questions of you if I may? Firstly, are you satisfied that the reduction in the delayed skin reaction is truly a decrease in the cellular immunity? Now what you are really showing here is that you have got a suppression of lymphocytes, in the periphery. Have you looked at the lymphocytes in the spleen and lymph nodes because what you are really showing is an expression of the cellular immunity from one particular parameter, and this in itself may not reflect immunity in general. Pearson (USA) Yes, I would most assuredly agree with you, that although I made a dogmatic statement about the suppression of immunity in regard to skin test positivity and so on. I think the only evidence I can mount to support my statement, is that by taking the cells we have removed, and
30
C.M. Pearson
attempting to stimulate them into blastogenesis by specific antigen to which they were previously responsive, or to mitogens, they are relatively, or absolutely, unresponsive. I cannot tell you about the reactivity of the lymphocytes of the spleen. In general we have done liver spleen scans, and we have seen that the spleen does not shrink to any great extent, and therefore, probably has a fairly significant cellular population of one type or another. We have done some lymph node biopsies, demonstrating that the lymph nodes appear to be depleted in the T cell areas of the lymph nodes in the paracortical areas primarily, but that is of course, a very gross measure. So those are the only measurements we have in regard to your question, but it is still an open question or an open answer if you will.
been four people who still want to ask questions.
Poulter (UK) Are you really saying that all you are really doing is depleting the cells which normally make up the inflammatory infiltrate in the active joints? Because if that is true, if you wait long enough, and I agree you have waited for up to 8 months as I saw in your slide there, if you wait long enough after that period, if the reactive lymphocytes wich might be affecting the arthritis are still left in the spleen and lymph nodes, won't the active joints eventually return?
Pearson (USA) T h a n k you very much Dr. Dick. I really can't answer that question and you knew I could not (Laughter).
Pearson (USA) I would most assuredly agree with you with that. I would feel that we are not depleting as you say the inflammatory cells per se, but perhaps the immunologic cells that are important in the immunologic inflammatory cycle. Perhaps there is some difference in that regard, but certainly we are not taking away the rheumatoid arthritis, nor the antigen or antigens that are responsible. But at least we are breaking the cycle temporarily, during this period of time, in order to observe some of the effects that have been demonstrated here, but by no means is this permanent. We hope to use this as a handle or handles to get a better understanding of some of the mechanisms involved in rheumatoid arthritis, but by no means are we claiming this to be a reasonable type of treatment, nor reasonable type of cure. No Dr. Poulter. Turk (UK) At some stage we will have to call this discussion to a close but there seem to have
Dick (UK) I think that with Professor Pearson I would have to say that the clinical interpretation of results like this are very difficult in the absence of a control, which is almost impossible in this situation. At the moment, for example, penicillamine is being claimed to make nodules disappear so for the non-clinician there is always the difficulty with this disease. But what I think is far more exciting, and what I would like to ask Professor Pearson is, why do the patients get better before any of the immunological tests alter? That is what I would like to hear him answer.
Turk (UK) T h a n k you Carl (Pearson), can we have the next question then. Can we have quick questions and quick answers. Zeitlin (UK) I would like to thank Professor Pearson for a very interesting presentation. I wonder if you could tell us the n u m b e r of patients who took part in the study? The percentage of patients who showed improvement during the treatment? The percentage of patients who showed rebound after the treatment was completed? Pearson (USA) In the study there are a total of 16 patients. 14 in w h o m adequate drainage was established, in others they had collaterals and we did not get enough lymph. In those 14 patients that I can talk about all 14 had a response, a very favourable response, and all 14 had a rebound after a reasonable period of time following discontinuance. Is that short enough John (Turk)? Brune (Switzerland) Dr. Pearson, it is well known that in patients treated for long periods on immunosuppressants there is a grave risk of them suffering from malignant disease. Have you any idea of the possible incidence, or would you like to
LymphocyteDepletion in Patients with Rheumatoid Arthritis speculate on the risk involved in your patients after this prolonged thoracic duct drainage?
31 our concept at the present time, with a small and moderate amount of antigen, but nevertheless we can't get rid of everything by this technique, or by this means. T h a n k you.
Pearson (USA) A very appropriate question, Sir, which ' has worried us all along. We have fortunately so Willoughby ( U K / F r a n c e ) far had no adverse experiences in regard to Carl (Pearson), I would just like you to malignancy, even although we have had our 4comment on the present vogue of using i m m u 5-year patients under continous surveillance. nostimulants for the treatment of rheumatoid disease. Even so this is not to say that in 1985 this is not going to occur. However we are just keeping our fingers tightly crossed. Pearson (USA) Well obviously, Derek (Willoughby), that Loewi (UK) is in direct contrast. Levamisole and the others I Apart from the very interesting and ob- assume you are discussing, that is in direct viously therapeutic implications, there is a theo- contrast with the observations and thoughts we retical question here which is that of the re- have here at the present time, using an i m m u circulation of lymphocytes. As you, I am sure, nosuppressant which has been much more satisknow well, Ziff showed, and we also showed this factory. We are in the process of attempting to once, that if you give an antigenic stimulus to study levamisole and other similar stimulants in patients, the response of the local synovial this regard, but I have no direct answer to you, m e m b r a n e lymphoid tissue was not like that of but would believe that if we remove enough of the rest of the body. This raises the whole these types of cells that are dedicated towards question of the re-circulation of lymphocytes the synovial m e m b r a n e adversely, that is probathrough this tissue, as opposed to other tissues bly going to be of considerable help. Levamiof the body. It would seem from your work sole could have effects on suppressor cells and here, from your results, that there is a circula- other things too, that I would not know about. tion which is necessary to keep this thing up, Turk (UK) which is an important theoretical concept. T h a n k you, Carl (Pearson), for your 'immuno-stimulating' discussion and I am sorry Pearson (USA) Yes, thank you for your comment. I you did not have time to tell us about the scleroderma story but you see there was enough believe we have come to that conclusion, that there is a continuous re-circulation going on at discussion on the rheumatoid without having that. Thank you very much. And now we pass all times, and that given a period of time one on to Professor Delphine Parrott. can catch most of those re-circulating cells and trap them outside of the body. At least that is
Agents and Actions vol. 6.1/2/3 (1976) Birkh~iuserVedag, Basel
Lymphocyte Depletion in Patients with Rheumatoid Arthritis C.M. Pearson* Department of Medicine, School of Medicine, The Center for the Health Sciences, Los Angeles, California 90024, USA
Abstract Sixteen patients with severe rheumatoid arthritis, marked inflammation of the synonvial membrane and high rheumatoid titer were cannulated by the thoracic duct for a period ranging between 82 up to 100 days. The patients being not under any medication during that time. Quantitative and qualitative analysis of the lymphocytes were performed, as well as responses to mitogens, rheumatoid factor, circulating antibodies and delayed hypersensitivity. By the 14th day nearly all the patients had a partial or almost complete remission of their disease. No complications were observed. These results will be discussed.
Discussion
Svartz (Sweden) This piece of work on lymph drainage is very interesting, considering that so few examples of rheumatoid arthritis occur in lymphatic tissues. I f you consider for instance how much of the granulations are in the skin, and also large amounts in the neighbourhood of the joints, it must be interesting to study both the T cells and B cells. As far as I can understand there was also a decrease in the granulation tissue in the capsule of the joints. This is very remarkable. In our treatments of rheumatoid arthritic joints with injections and so on, scarification was usually found. I don't think you found the same thing here. It is also interesting to take tests from the skin while the drainage was in progress. In your lecture, you spoke about the change in cell populations, but you did not speak about dead cells. There is a great difference between your treatment and the treatment using various drugs.
Pearson (USA) T h a n k you for your comments Dr. Svartz. We have data and I didn't have time to discuss this. We have done serial needle biopsies of the synovial membrane, demonstrating that there is a great decrease in cellularity, of cell types of the synovium, and I will not go into the details of them. Great decrease in the amount of cells, in the thickness of cells but very little scarification of the lining cells that do occur during this situation. Therefore, I feel that we have removed m a n y of the cells that are inducing and continuing and propagating the inflammatory response. Actually, and amazingly, the n u m b e r of lining cells of the synovium - the synovial sites - decreases from seven or eightcell thicknesses, occurring as you know in active rheumatoid arthritis, down to one or two during this period of time, and then gradually builds up again after the drainage is discontinued. Turk (UK) In that connection, Carl, have you investigated the mononuclear phagocytic system, both in numbers, quantitatively, and in function after thoracic duct drainage in man? Pearson (USA) Yes, John, we have done some of that, and I am afraid I can't give you the finite details of those. Dr. Leonard C;oldberg in our unit has done some studies of the phagocytic monocytes,
*Unfortunatelythe completemanuscriptdid not arrivein time for publication,it ishoped to publishthisarticlein alater edition of Agents and Actions.
LymphocyteDepletion in Patients with Rheumatoid Arthritis and so forth, in the peripheral blood and occasionally in the synovial fluid. He finds them not to be changed to any significant extent during the period of time during drainage or afterwards. So it appears, that we are removing the T lymphocytes that do not convert to monocytes, if they do supposedly do so, at the site. They are not changed to any great extent. Ford (UK) In the studies of the immunological function of these patients undergoing chronic thoracic-duct drainage, I suppose the results tie in very well with animal experiments, in which the antibody responses are depressed rather little and delayed hypersensitivity tends to be depressed rather more. The exception in animal experiments, is with so-called strong histocompatibility differences, where grafts a cross an H 2 and AGB locus tend to be rejected in very little longer than the usual time, even in quite severely-drained rats and mice. Yet in your patients, husbands and wives presumably differing in HLA locus, you have survivals of a year, which is quite remarkable, and I wonder if we could go into that a little more? Were the patients receiving any other treatments, e.g. steroid treatments; do you know that there is a strong histocompatability difference? Are you sure that the tissues actually survive and weren't overgrown with the host tissue? Pearson USA) Yes we do have some evidence on this. Thank you for raising that point. We have H L A profiles on all of our patients and their husbands during this period of time. The grafts actually have survived during this period of time and it was a great surprise to us, as there was a great deal of differential in H L A types obviously, as you have implied, between husband and wife. Yet they have survived for reasons that are perhaps explainable, and perhaps need some more theory. The H L A types were very significantly different, and we had not started these patients after drainage on any therapy, except for a small number on aspirin, that they would require. We did not give them immunosuppressants or anything else, or cortico-steroids which might suppress the skin graft rejection. No Sir. Kacaki (Netherlands) Does the rebound of cell-mediated immu-
29 nity that you have seen in the majority of patients coincide with the recurrence of the clinical symptoms? The second question is do you think that the T cells that you have removed and the T cells reappearing in the patient after thoracic duct drainage have similar characteristics? Pearson (USA) T h a n k you for your questions. In regard to the second question I am not sure that I can answer you about the character of the T cells. Our impressions are that they would be the same, but the technique of identifying subpopulations of T cells is still in a great deal of debate, and we have not been able to clarify these - are these suppressor cells or whatever? I don't think I can say this for certain. On the first question on rebound, yes it does, Sir. The rebound of delayed hyper-sensitivity does occur, in essence, in parallel with the reappearance of the activity of the rheumatoid arthritis. So, at 30 days for instance, if the individual before drainage had been PPD positive, he or she is liable to be barely positive to PPD. By 90 days very strongly, if they were strongly before. So in essence it does parallel, quite considerably and quite precisely in the majority of our patients; delayed hypersensitivity skin test reactivity, PHA response, and so forth, to the lymphocytes and the reappearance of the rheumatoid arthritis activity. Poulter (UK) Can I just ask one or two questions of you if I may? Firstly, are you satisfied that the reduction in the delayed skin reaction is truly a decrease in the cellular immunity? Now what you are really showing here is that you have got a suppression of lymphocytes, in the periphery. Have you looked at the lymphocytes in the spleen and lymph nodes because what you are really showing is an expression of the cellular immunity from one particular parameter, and this in itself may not reflect immunity in general. Pearson (USA) Yes, I would most assuredly agree with you, that although I made a dogmatic statement about the suppression of immunity in regard to skin test positivity and so on. I think the only evidence I can mount to support my statement, is that by taking the cells we have removed, and
30
C.M. Pearson
attempting to stimulate them into blastogenesis by specific antigen to which they were previously responsive, or to mitogens, they are relatively, or absolutely, unresponsive. I cannot tell you about the reactivity of the lymphocytes of the spleen. In general we have done liver spleen scans, and we have seen that the spleen does not shrink to any great extent, and therefore, probably has a fairly significant cellular population of one type or another. We have done some lymph node biopsies, demonstrating that the lymph nodes appear to be depleted in the T cell areas of the lymph nodes in the paracortical areas primarily, but that is of course, a very gross measure. So those are the only measurements we have in regard to your question, but it is still an open question or an open answer if you will.
been four people who still want to ask questions.
Poulter (UK) Are you really saying that all you are really doing is depleting the cells which normally make up the inflammatory infiltrate in the active joints? Because if that is true, if you wait long enough, and I agree you have waited for up to 8 months as I saw in your slide there, if you wait long enough after that period, if the reactive lymphocytes wich might be affecting the arthritis are still left in the spleen and lymph nodes, won't the active joints eventually return?
Pearson (USA) T h a n k you very much Dr. Dick. I really can't answer that question and you knew I could not (Laughter).
Pearson (USA) I would most assuredly agree with you with that. I would feel that we are not depleting as you say the inflammatory cells per se, but perhaps the immunologic cells that are important in the immunologic inflammatory cycle. Perhaps there is some difference in that regard, but certainly we are not taking away the rheumatoid arthritis, nor the antigen or antigens that are responsible. But at least we are breaking the cycle temporarily, during this period of time, in order to observe some of the effects that have been demonstrated here, but by no means is this permanent. We hope to use this as a handle or handles to get a better understanding of some of the mechanisms involved in rheumatoid arthritis, but by no means are we claiming this to be a reasonable type of treatment, nor reasonable type of cure. No Dr. Poulter. Turk (UK) At some stage we will have to call this discussion to a close but there seem to have
Dick (UK) I think that with Professor Pearson I would have to say that the clinical interpretation of results like this are very difficult in the absence of a control, which is almost impossible in this situation. At the moment, for example, penicillamine is being claimed to make nodules disappear so for the non-clinician there is always the difficulty with this disease. But what I think is far more exciting, and what I would like to ask Professor Pearson is, why do the patients get better before any of the immunological tests alter? That is what I would like to hear him answer.
Turk (UK) T h a n k you Carl (Pearson), can we have the next question then. Can we have quick questions and quick answers. Zeitlin (UK) I would like to thank Professor Pearson for a very interesting presentation. I wonder if you could tell us the n u m b e r of patients who took part in the study? The percentage of patients who showed improvement during the treatment? The percentage of patients who showed rebound after the treatment was completed? Pearson (USA) In the study there are a total of 16 patients. 14 in w h o m adequate drainage was established, in others they had collaterals and we did not get enough lymph. In those 14 patients that I can talk about all 14 had a response, a very favourable response, and all 14 had a rebound after a reasonable period of time following discontinuance. Is that short enough John (Turk)? Brune (Switzerland) Dr. Pearson, it is well known that in patients treated for long periods on immunosuppressants there is a grave risk of them suffering from malignant disease. Have you any idea of the possible incidence, or would you like to
LymphocyteDepletion in Patients with Rheumatoid Arthritis speculate on the risk involved in your patients after this prolonged thoracic duct drainage?
31 our concept at the present time, with a small and moderate amount of antigen, but nevertheless we can't get rid of everything by this technique, or by this means. T h a n k you.
Pearson (USA) A very appropriate question, Sir, which ' has worried us all along. We have fortunately so Willoughby ( U K / F r a n c e ) far had no adverse experiences in regard to Carl (Pearson), I would just like you to malignancy, even although we have had our 4comment on the present vogue of using i m m u 5-year patients under continous surveillance. nostimulants for the treatment of rheumatoid disease. Even so this is not to say that in 1985 this is not going to occur. However we are just keeping our fingers tightly crossed. Pearson (USA) Well obviously, Derek (Willoughby), that Loewi (UK) is in direct contrast. Levamisole and the others I Apart from the very interesting and ob- assume you are discussing, that is in direct viously therapeutic implications, there is a theo- contrast with the observations and thoughts we retical question here which is that of the re- have here at the present time, using an i m m u circulation of lymphocytes. As you, I am sure, nosuppressant which has been much more satisknow well, Ziff showed, and we also showed this factory. We are in the process of attempting to once, that if you give an antigenic stimulus to study levamisole and other similar stimulants in patients, the response of the local synovial this regard, but I have no direct answer to you, m e m b r a n e lymphoid tissue was not like that of but would believe that if we remove enough of the rest of the body. This raises the whole these types of cells that are dedicated towards question of the re-circulation of lymphocytes the synovial m e m b r a n e adversely, that is probathrough this tissue, as opposed to other tissues bly going to be of considerable help. Levamiof the body. It would seem from your work sole could have effects on suppressor cells and here, from your results, that there is a circula- other things too, that I would not know about. tion which is necessary to keep this thing up, Turk (UK) which is an important theoretical concept. T h a n k you, Carl (Pearson), for your 'immuno-stimulating' discussion and I am sorry Pearson (USA) Yes, thank you for your comment. I you did not have time to tell us about the scleroderma story but you see there was enough believe we have come to that conclusion, that there is a continuous re-circulation going on at discussion on the rheumatoid without having that. Thank you very much. And now we pass all times, and that given a period of time one on to Professor Delphine Parrott. can catch most of those re-circulating cells and trap them outside of the body. At least that is
