INNATE AND ACQUIRED IMMUNITY IN HYPERTENSIVE TARGET ORGAN DAMAGE |
Dominik N. Müller, Berlin, Germany
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Chair: Giuseppe Bianchi, Milan, Italy |
Christos Chatziantoniou, Paris, France |
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Dr Dominik N. Müller
Max Delbrueck Center for Molecular Medicine Berlin, Germany |
Slide 1
Good morning Ladies and Gentlemen. I would like to raise the issue this morning whether the immune system is perhaps an important mediator of vascular and renal damage and I will start to explain what I mean by explaining immunity.
Slide 2
Normally, immunity is thought as a system which functions as a system to defend against pathogens. We have on the one hand innate immunity and on the other hand acquired immunity. Innate immunity is the unspecific response, the acquired or adaptive immunity is the specific response, both are divided in humoral and cellular and what you have here is the renin-angiotensin system. We know that the renin-angiotensin system is a system which activates inflammation while the formation of reactive oxygen species so promoting this event here. We know that the renin-angiotensin system affects T-cells it directs the T-cell switch to a Th1 response and we know that the renin-angiotensin system namely angiotensin II affects dendritic cells in the way that it promotes migration of dendritic cells and it does not promote maturation, this is done by other mechanisms. What we do not know or where we have little information is of the renin-angiotensin system and the complement system and also here. I will talk now in the next few minutes predominantly about the complement system.
Slide 3
We use another animal model, it’s also an angiotensin II dependent animal model, it’s a transgenic animal over-expressing the human renin and the human angiotensinogen gene, so these animals have high angiotensin II levels in the circulation, as well as in the tissues and what I want to stress here is that it is a non-immune model. So, if I talk now about cardiovascular damage, vascular damage and renal damage this is not a classical immune-dependent animal model, it’s a non-immune model, it’s just angiotensin which is changed, it’s just hypertensive, that’s what we think, it’s non-classical immune.
Slide 4
I want to address several questions. Is complement activated by angiotensin in this animal model? Does the complement activation occur perhaps earlier than the renal damage? Which components of the complement system are involved in this process?
Slide 5
So, we performed a chronic study in this animal model following untreated transgenic animals in a time course at week 5, 6 and 7, only up to week 7 because if you do not treat these animals, they die between week 7 and week 8. Then we compared these animals with classical inhibitors of the renin-angiotensin system like an AT1 receptor blocker, losartan and a new drug which is a renin inhibitor, a renin inhibitor which is at the moment in phase III studies in humans and compared it to non-transgenic animals.
Slide 6
So, here on this slide you see the blood pressure levels over time. In black the untreated double transgenic animals have already elevated blood pressure levels at week 5, blood pressure rises over levels of 200 at week 7. In white there’s the non-transgenic animal and here are the 2 inhibitors which normalise blood pressure. If you look at albuminuria as a marker of renal damage, we have slightly a different situation. If you look at this early phase at week 5, you see that the transgenic and the non-transgenic model are more or less the same.
Slide 7
Then between week 5 and week 6 you have an increase in albuminuria in the untreated animal which progresses over time and the inhibitors prevent the development of albuminuria. I do not want to say that there is no renal damage taking place here in this early phase but it’s not as pronounced as in the late phase and this is something that you should keep in mind during my next slides.
Slide 8
Then we measured the marker of inflammation, C-reactive protein and surprisingly or interestingly already at week 5 we have significantly elevated levels of inflammation which were stable over time indicating that we had already an inflammatory response prior to the end-renal failure.
Slide 9
The inhibitors prevented the production of C-reactive protein. TNF-α is a very important cytokine which promotes inflammation. It activates NF-kB which is an important transcription factor in the regulation of inflammation and here you see a staining in the upper panel of untreated double transgenic animals at week 5, 6 and 7. Here you see non-transgenics at week 5, 6 and 7 and here you see the inhibitor treated animals at week 7. So, what you can see here is that you have an increased expression of TNF- α in the vessel wall over the time but already as early as week 5, as well that you have it in the glomeruli, in damaged tubules which progress over time and which can be prevented by the 2 inhibitors and you have no staining in a healthy non-transgenic animal.
Slide 10
When we then analysed macrophage infiltration in the kidney we saw a similar picture like C-reactive protein levels. We had already increased inflammation in week 5 which progressed and the inhibitors were able to prevent inflammation.
Slide 11
The same picture occurs, if you look at CD4 or CD8 T-cells. Again already early you have this increased inflammation. So what I wanted to show with this set of slides is that before the maximal renal damage you have already signs of inflammation, cytokine expression which might trigger also this total process.
Slide 12
Here’s now the scheme of the complement cascade, you have different possibilities to activate the complement cascade. Importantly, these pathways merge at the level of complement C3 and this is what I will talk about in the next few minutes. Then different cleavage products, so these compounds are then always cleaved in a short form which is predominantly a pro-inflammatory form and a long form which is the classical form which promotes the function in the immune system. Then there are different products like the membrane attack complex, which is an end product of complement cascade.
Slide 13
There was indirect evidence and this was the only paper that I found when we did this study that angiotensin II might be involved in the complement activation. This was the remnant kidney model, a study by Remuzzi’s group and what you can see here is that if you follow up control animals and then the diseased animals over time that you get an increase of C3 expression, this is the quantification here, the white bars are the complement expression which increases from control towards the diseased model. They treated these animals with an ACE inhibitor, this you can see here. They started at day 1 or at day 7 and here are the non-treated animals for complement C3 and here are the significantly reduced levels by the ACE inhibitor.
Slide 14
So, this indirect evidence existed and we went on and looked at our animal model and here we stained for complement C1q. C1q was predominantly stained in the vessel wall and if you look at the quantification here, you see that there is an increased expression over time but already an early expression at week 5 and the blockade of the renin-angiotensin system completely prevents the expression of C1q.
Slide 15
The question was, is this just a phenomenon which is dependant on our transgenic animal model or is it a more generalised effect of angiotensin II models? Here is the model which you have heard in the last talk. The mouse renin transgenic rat, you see expression is in the study where we infused angiotensin II in Sprague-Dawley animals. You see expression in here again control rats.
Slide 16
Here is the situation for complement C3. Here upper panel again like before week 5, 6 and 7 untreated double transgenic animals here is the background staining of non-transgenic animals.
Slide 17
And what you can see here is that you have an expression in the vascular wall which is prevented by a blockade of the renin-angiotensin system but you have also expression in the glomerulus and in tubules. The expression increases over time with damage but it is already expressed in the early phase.
Now, the question was, is this complement immunoreactivity what we see? Deposition that it is the complement which is taken up from the blood stream or is it locally produced? Therefore, we isolated glomeruli from our animals and here our mRNA expression levels of complement C3 of glomeruli isolated from double transgenic animals compared to non-transgenic animals and you see a several fold increase of expression. This indicates that the complement immunoreactivity that you have seen in the glomerulus was due to synthesis, local synthesis rather than uptake.
Slide 18
Again here the 2 other animal models which give the same picture, the mouse renin transgenic animals show expression of complement C3 and the angiotensin II infused animals also show it and now we went back and looked at old samples which we had in the fridge to address a little bit the question that we had after the last talk, is angiotensin II doing something directly or is it more the blood pressure that is doing it?
Slide 19
So, what we have here is a staining of the untreated double transgenic animals. These animals have blood pressure over 200 and have albuminuria. Here we have a section of a double transgenic animal treated with dexamethasone and these animals have also a blood pressure over 200 but they do not have any albuminuria at this time point. We compared it to a treatment where we used a triple treatment of non-renin-angiotensin system blockers. So we used reserpine, hydralazine and hydrochlorothiazide to lower blood pressure. So, these animals were normotensive but since we used non-renin-angiotensin system blockers these animals still developed renal damage.
Slide 20
What you can see here is that these animals with low blood pressure but with renal damage express complement C3 while animals with high blood pressure without renal damage do not express it. So, it depends quite a lot on how you treat the animals and this was on the last slide of the previous speaker that the selection of the antihypertensive reagent might be quite important and signalling blockade might also be important.
Slide 21
Here’s just to finish up the analysis on the complement system, another slide for the membrane attack complex and you have a similar picture to what you have seen before. You have expression in the vessel wall which increases over time in the untreated double transgenic animals which can be prevented by the specific blockers. Now we went to in vitro studies. We isolated smooth muscle cells from untreated double transgenic animals and generated a smooth muscle cell line and from healthy Sprague-Dawley animals. What you can see here is that the cultured smooth muscle cells have a 4-fold higher expression of complement C3 compared to the non-transgenic ones and if you look at this side of the slide, you see that the proliferation of smooth muscle cells from the hypertensive angiotensin II dependent model has also a higher rate of proliferation compared to the non-transgenics. They also have different levels of molecules which are normally expressed in smooth muscle cells, like smooth muscle alpha actin and what we did here is we used a inhibitor, it is histone deacetylase inhibitor because we knew that this inhibitor reduces proliferation like you can see here and we asked if this inhibitor is able to reduce also complement C3 and this was the case.
Slide 22
So, here we had increased C3 levels with increased proliferation. Here we had decreased proliferation with decreased C3. We did this because we found this study published in 2004 which did an experiment where they compared smooth muscle cell lines from SHR animals and WKY controls and what they found in their analysis was that complement C3 was also increased in these animals in the hypertensive cell line and that the hypertensive SHR smooth muscle cells had also a higher rate of proliferation and when they used antisense against complement C3 then they could reduce the cell proliferation. So, what I wanted to show you with this set of experiments is that a classical immune component like complement C3 might have also completely different actions which could be interesting in respect of vascular remodelling and vascular function.
Slide 23
What we then did is we analysed the induction of complement C3, so we stimulated cells with either angiotensin II or TNF-α and to our surprise nothing happened neither in the Sprague-Dawley smooth muscle cells nor in the double transgenic smooth muscle cells when we used angiotensin II. So here you have the same levels and here you have the same levels. So angiotensin II did not directly promote complement expression while when we used TNF-α we got an increase, a six-fold increase in Sprague-Dawley cells but a huge increase in these double transgenic animals.
Slide 24
This was surprising and we were wondering why this happened and we looked at another marker that was known to be induced and this was IL-6, interleukin 6 and we found that TNF-α induced IL-6 in both cell lines in a similar manner. So we were surprised but we said ok, let’s see what CRP does to our cells and complement C3 and we found an identical picture like I’ve shown you before that in Sprague-Dawley smooth muscle cells we got a slight induction in these smooth muscle cells from the diseased model. We got a huge induction while we got a similar induction for IL-6.
Slide 25
So, this indicates that for complement C3 we have a special regulation of the molecule which is a sensitive regulation when we have smooth muscle cells with a certain phenotype which were not healthy and these cultures were taken from animals which had this damage before.
Slide 26
Now, in the end I want to show you quickly 2 studies, intervention studies where we interrupted signalling in vivo. So, on the one hand we used a TNF receptor blocker, etanercept which scavenges free TNF-α and prevents it from signalling. If you look at the data here, this receptor blocker had absolutely no effect on blood pressure, so they were equally hypertensive. It partially reduced albuminuria and it partially reduced inflammatory cells. Here macrophages were partially reduced, CD4, CD8 T cells, dendritic cells were almost normalised and here are molecules of the co-stimulatory pathway.
Slide 27
When we looked and analysed what happened to the complement components, we found that vascular complement C1q was dramatically reduced by TNF blockade. We found that vascular complement C3 expression was significantly reduced, as well as glomerular and tubular complement C3.
Slide 28
Then we did another study where we interrupted with an immunosuppressive reagent, mycophenolate mofetil, this is a drug which is used in transplantation for immunosuppression due to the suppression of T and B cell proliferation.
Slide 29
We found in these chronic treated animals a slight reduction of blood pressure but still significantly high blood pressure levels over 160 and a dramatically reduced albuminuria and a complete normalisation of all inflammatory cells.
Slide 30
There is more and more data that if you immunosuppress animals of different animal models also non-immune models like the Dahl salt-sensitive animals, you see an improved albuminuria, improved glomerular injury index, as well as if you use ischemia reperfusion injury models but there is also new data available about SHR animals and these mouse renin-transgenic animals always treated with MMF and all cardiovascular models were improved with this treatment.
Slide 31
So, in summary I could show you that the classical and alternative complement pathway are activated in our angiotensin II dependent model, that the complement system is activated most likely while an indirect mechanism that angiotensin II triggers molecules like TNF-α which then promote the direct expression of complement system. That the complement activation seems to be involved before the development of complete renal damage and that complement activation was not activated as a consequence of hypertension.
Slide 32
Here’s the lab which contributed to all these results and I want to thank you for your attention.
Slide 33
Chairman: Thank you very much for this clear presentation. Now, the paper is open for discussion. So it seems that there are no questions. May I ask? You certainly succeeded in convincing us that we have some activation of complement that is independent of blood pressure. TNF-α and other mediators can be involved. However, we can now approach this problem by precise molecular mechanisms, let’s say angiotensin II increases some transcription factors that then can produce transcription of genes. Have you looked at the problem from this point of view?
Dr Müller: We have done different experiments. We know that, for example, the transcription factor NF-kB is involved in the pathogenesis of angiotensin II mediated end-organ damage because we have interrupted inhibitors which inhibited NF-kB. We have done in vitro studies also looking at the regulation of complement C3 using NF-kB inhibitors and we could show that complement activation depends on NF-kB as well. So, there is a signalling cascade which starts with angiotensin II while the classical AT1 receptor which goes to the production of radicals and then divides into different other pathways activating different other transcription factors and one important transcription factor is the pro-inflammatory transcription factor NF-kB which also regulates complement and other pro-inflammatory molecules.
Chairman: Ok. There are other questions? Thank you very much.
Dr Müller: Thank you.