Slide 1

Well, thank you Jurgen. Ladies and Gentlemen it’s a pleasure to be here and to talk with you a little bit about complement, about immunity and then finally, about IgA nephropathy.

Slide 2

Rosanna Coppo has already indicated to you that functionally, let’s say conceptually it’s important to see that there are actually two compartments of immunity, namely innate immunity on which you’re very much dependent early in life and acquired immunity which you acquire after a number of months in life. Innate immunity is composed of a number of cells but next to these cells there are a large a number of soluble components and soluble mediators which actually together determine how you can defend yourself early in life and then there are conditions where your acquired immune response somewhere or other are suppressed. The combination of these cells together with these soluble mediators will determine how antigens are recognised and how they are presented via antigen presenting cells to B and T cells leading to the production of activated T cells resulting in the production of cytokines and chemokines and as very important the formation of antibodies. A lot of evidence suggests that it is the combination of activated T cells together with the production of antibodies which determines how and how far we are protected against all types of invading microrganisms.

Slide 3

Today I will talk mainly about complement and as you see, complement is only one of the many mediators which together with the other participants in innate immunity determine how acquired immunity finally is shaped.
The complement system cannot function alone and it has been shown by a large number of studies that regulation of complement can be determined by all these components which are there or which are generated following an active immune response in the acquired system.
So what is complement and what can it do? So, the first is that we know that there are many, many biological functions of complement. For you early on it was thought that complement was produced mainly by the liver which is still true but there are also local productions of complement and these are very important studies from the group of Steve Saxs which have shown that the local production, for instance, of complement in the kidney is of extreme importance to determine and to drive for instance, immune reactions in the kidney and ultimately, for instance, in tissue transplantation determine whether a kidney will be rejected or not.
So complement is involved in direct pathogen elimination so that’s probably one of the main functions of complement. It’s additionally involved in the induction of phagocytosis via opsonization. So by that mechanism actually it upregulates within the innate immune system the capacity of the innate immune system to clear pathogens, to clear viruses, bacteria etc. It’s a very strong inductor of inflammation and chemotaxis. By that mechanism it upregulates the potential of your innate immune system to increase your defence against pathogens. It’s a direct interface between the innate immune system and the adaptive immune system. In that sense that it recognises pathogens directly and serves as a kind of bridge to antigen presenting cells to get these antigens within antigen presenting cells and finally, lead to let’s say driving an initiation of adaptive immunity.
One of the very important things of complement is that it directs the clearance of immune complexes in such a way that immune complexes are not deposited at all tissue sites and cause inflammation. A very important function of complement is its role in tissue degeneration and regeneration and that is one of the fields where it’s going to play a major role. For instance, it’s known that if you take rats you remove a piece of the liver, that liver will just grow on but if you deplete that animal first of complement liver doesn’t grow on and it has been shown that that’s dependent on the activation of your host complement system.

Slide 4

So as you see many, many functions and these days you can read about complement almost in every journal. So what is complement? Complement is composed of a large number of molecules which are present in the circulation but also in all types of body fluids and in all types of tissues in so-called biologically non-activated form. So it has to be activated to acquire biological activity. There are about 35 complement components and a lot of receptors which are required to lack complement function. In order to lack complement function it has to be activated and there are 3 pathways of activation, the classical pathway, the lectin pathway and the alternative pathway and each of these pathways alone or in combination will lead to conversion or activation of the central component of complement C3 and to an activated form which can attach itself to let’s say the activator and a small fragment which generally has a vasoactive function by interaction with specific receptors for instance, on endothelial cells. Once sufficient C3 is activated it can lead to activation of the terminal components of complement C5 till C9. It can insert itself in tissue and cause injury and inflammation.

Slide 5

So what are the mechanisms which lead to complement activation? In the first place what you know is that immune complexes are composed mainly of antigen with antibody and these antibodies are mainly of the IgM and IgG class, they can activate the classical pathway via C4 and C2 leading to activation of C3. More recently, actually the most ancient pathway of complement, the lectin pathway has been discovered and here actually carbohydrates are the main recognition molecules and as Rosanna indicated, these are kind of patterns which are recognised on pathogens like bacterial viruses, injured tissue etc and these can drive the lectin pathway which finally feeds in and I’ll show some information later, within the classical pathway and then activates C3. The alternative pathway there we know that especially microbial products derived from lots of types of bacteria can activate the alternative pathway and here very recent information suggests that properine is the focusing and driving point of the alternative pathway.
So as you see there are 3 pathways of activation and today because we’re talking about IgA nephropathy I would like to focus on this lectin pathway of complement. So the lectin pathway of complement, since I come from a country, Holland where actually tulips are a major income source there everybody on their table has this type of tulips almost every day and it’s very cheap so you should come to Holland to buy it and enjoy it.

Slide 6

Actually the recognition unit of the lectin pathway of complement is mannose binding lectin and this molecule is composed of this recognition site which interacts with carbohydrates in a specific type of pattern and it requires calcium additionally for the recognition.
For you it’s very important to know that as you sit here about 70% of you have the so-called bunch of tulips, the wild type MBL which is able to recognise this very specific pattern and this will lead to binding of a number of serum proteases associated to this part of the molecule and lead to complement activation and generation of biological active material.
On the other hand, there are about 30% of you sitting here who has this type of single tulips these are I would say not the rich people who cannot buy this whole bunch but they can only buy one or two single units of tulips and these single variants or these types of single tulips occur because there’s a change or a variant in this part of the molecule, so that you cannot hold this structure together. If you have this variant type of mannose binding lectin, then you have in the first place that it will bind less to the activator, less to bacteria pathogens etc. it of course has an impaired interaction with these serum associated proteases, it activates the complement to a lesser extent and this phenotype is especially apparent in those people who are homozygous who only have this type or this type. In heterogeneous carriers which have some of this and some of that there is a kind of intermediate function.

Slide 7

So it is a direct relationship between this wild type and the non wild type.
So the question there actually is is there some way another relation with IgA nephropathy? But before I go there I would like to bring you into the scheme again. So the classical pathway is activated via antigen-antibody complexes via complement component 1 which activates C4 and C4 directly leading to formation of this complex which then activates C3.
In the lectin pathway mannose binding lectin and what I did not mention two ficolins, ficolin 1 and ficolin II, they can also recognise carbohydrate structures but they recognise other types of compositions. These molecules also feed in at the level of C4 and finally activate C3.

Slide 8

So what do we know of IgA nephropathy? In IgA nephropathy we know that there’s always deposition of IgA and that this IgA is mainly of the IgA 1 subclass and additionally lot of studies suggest that it’s mostly polymeric which means that it’s large in size. Additionally, most, up to 90% of the patients will show deposition of C3 which indicates that some way or another this IgA has activated the complement system up to C3.

Slide 9

So studies with Maria Pia Rastaldi in Milan actually showed that if you took biopsies from patients with IgA nephropathy and stained these four mannose binding lectin that there was a deposition pattern very compatible with the deposition of IgA.

Slide 10

So what do we know about IgA and complement? So very early studies actually from the group of Ratlebeck in Heidelberg from a long time ago have shown that the alternative pathway of complement is activated in IgA nephropathy and they found actually that there’s a strong association between deposition of the terminal complex of complement, the C5-C9 in IgA nephropathy patients.
So we asked the question since we find some MBL in these patients whether there’s an association also with the MBL pathway of complement in IgA nephropathy and whether it has to do anything with the severity of the disease?

Slide 11

So first of all we asked the question whether MBL can bind to IgA isolated from normals? What you see is that with increasing concentrations of MBL, there’s a very strong binding to IgA and while it doesn’t occur to other molecules which was not supposed to bind.

Slide 12

So the next question came up does it bind to all types of IgA or does it only bind to high molecular rate IgA? So therefore, we first isolated IgA from plasma by affinity chromatography and this is the profile of the isolated IgA of gel filtration column. Most of you know that large material will appear first from a gel filtration column and monomeric IgA which is smaller than polymeric IgA will appear later. When you take these fractions and then ask the question which ones bind MBL, then you see that MBL binding is mainly found in the large molecular rate IgA fraction. So this actually indicated that the binding of IgA which we saw in this one biopsy maybe dependent on the binding to IgA.
To get a better over view we stained biopsies for MBL for its serum protease associated to MBL and also whether it would activate complement and here you see that there’s a beautiful co-localization or in a similar pattern as the deposition of IgA.

Slide 13

Additionally which we have not investigated further you also see that L-ficolin is also found in biopsies of patients with IgA nephropathy.

Slide 14

So the question then comes is there an association between the deposition of MBL and the disease severity? Together with Maria Pia we took 60 IgA nephropathy patients and we stained that first for MBL and we found that from these 60 there were 15 which were positive for MBL like here and there were 45 which were negative.

Slide 15

So now the question is what do we see in these biopsies? So here there are these 15 patients who were positive for MBL and here are the 45 who were negative. If you stain these biopsies for the serum protease associated with the MBL, you see that all of them are positive for L-ficolin as well and they can activate complement and they showed clear deposition of C4d together with C4 binding protein which is a molecule which I did not discuss until now which also binds to C4. additionally what you see is that there is no classical pathway involvement because even q deposition was low, most of the biopsies were positive for C3 as they should be and in all cases there was IgA 1 and we were not able to demonstrate any IgA2 in these biopsies. On the other hand, if you take these negative cases for MBL in other biopsies none of them show them – no ficolins, no C4 activation and no C4d, while there can still be positivity for C3 activation and all of them are still IgA1 positive.

Slide 16

So the question is is there any association with disease activity? So here on the left you have the MBL negative cases and here on the right the MBL positive cases. As you see that these MBL positive cases had a higher proteinuria, significantly higher than the one which was MBL negative. I have to mention to you also that in this case where we found MBL positivity none of these patients had this single tulip type of MBL in their circulation.

Slide 17

Also when we look to the MBL positive cases we see that glomerular MBL positivity is associated with more mesangial proliferation and also with more extracapillary proliferation. So there seems to be a direct relationship between the deposition of MBL in the kidney together with IgA, together with complement activation and injury to the kidney.

Slide 18

These are two selected cases. MBL negative patients you see that there’s less interstitial damage and less glomerular damage as compared to the MBL positive cases.

Slide 19

So overall what we can conclude now is that in the MBL positive cases there is lectin pathway activity and this lectin pathway activity or activation is associated with more severe IgA nephropathy. While the MBL negative cases are associated with less severe disease and presumably since they all have or the majority has C3 deposition as well, we feel that this group of patients may be dependent on an alternative pathway activation while these are mainly driven by classical or lectin pathway activation.

Slide 20

So there is a distinction within the IgA nephropathy patients concerning progressiveness for disease. So taken together in IgA nephropathy patients about 25% in our hands are MBL positive, C4d positive, clearly indicating lectin pathway activation and this is associated with more severe disease. So in this case you will say that MBL will potentially be one of the biomarkers for following progression of disease. In the MBL negative cases we find that C4 activation negative but C3 positive so here we feel that probably the alternative pathway is involved. So one of the points will be to go and try to find parameters with which we can clearly distinguish lectin pathway activation and alternative pathway activation because this may determine or give us an impression to which patients will follow for more severe follow up than those who have a less severe disease.

Slide 21

So overall what we think is that in IgA nephropathy somewhere or another IgA, polymeric IgA deposits on the mesangium and here factors like deglycosylation, upregulation of mesangial receptors can lead to activation of the mesangial cells leading to proliferation and in this case when the lectin pathway’s activated that is associated with more inflammation and there’s more chance for renal failure. While an alternative pathway in some way the same mechanisms are operational but the driving force for inflammation is reduced. So if we will be able to really distinguish these two types of cases or groups of cases then I think that we will have some way to go in clinical medicine.

Slide 22

With that I come to the end and I would like to thank a lot of people but especially I think Anja Roos and Cees van Kooten. Most people think that I’m driving this research but I have to assure you that cases driving me these days to do all the nice work which we think we are generating in Leiden. So with that I would like to thank you and hope that you will have some questions so that we can clarify those points that have not been clear yet. Thank you very much Mr Chairman.