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Vasculitis and Inflammation

There are clearly complex immunological events and genetic events that initiate and drive lupus. This disease may be amplified in certain genetic impairments of processing of immune complexes or compliment receptors, as you are well aware. Abnormalities of the Fc receptor polyarteritis nodosa alllergic angiitis.

What I am going to talk about as a person interested in inflammation are the consequences of these events. That is, how we injure the blood vessels in particular, as a result of the autoantibodies in the immune complexes, and particularly as a result of complement activation. So beginning at the centerpiece in lupus where tissue gets injured in many of the end organs, complement is where I would like to begin the discussion. Itís a cascade that Iím sure is familiar to all of you in the audience. What I would like to really just focus on, is that as you activate C3 by either the classical or the alternative pathway and you begin to generate C3b and C5b in the membrane attack complex, which will settle on cells and activate cells.

Finally, itís been recognized over the past five years that in human search situations, the assembly of the membrane attack complex on a cell doesnít necessarily cause lysis. But that can cause calcium translocation and activate a variety of cells, including endothelial cells. So C5b-9 is also a biologically active produce of complement activation. The molecule that is of most interest to us in inflammation is the chemoattractant C5a, which is shown to the left. And this just simply says that a target of C5a ligation is the neutrophil and the neutrophil will engage C5a, as it will immune complexes, to release inflammatory mediators such as enzymes, oxygen derived free radicals such as superoxide anion, and products of arachidonic acid, that is LTB4. So this is what a neutrophil will do in response to C5a generation. Less appreciated is that neutrophils also respond to chemoattractant by clumping the way that platelets clump or aggregate. So hereís scanning electron micrograph in a control, unstimulated neutrophil as shown to the left, and on the right are a clump of neutrophils that are at a different scale, activated by a chemoattractant.

In studies we looked in the early 80ís at complement activation and as you would imagine, as you get sicker with lupus, when you go from stable to severe disease, you activate C5a and C3a and these can be measured in the plasma. They actually are a more sensitive indicator of disease activity than are drops in C3, which reflect both synthesis and degradation. So these molecules can be measured. What is shown on this cartoon is what happens when neutrophils are activated by C5a.

So in the course of this story that I am describing, we knew the complement was activated. We asked whether circulating neutrophils in patients with lupus were overexpressing CR3 and in fact they were. So when we compared them to normal, again arbitrarily in those years, simply having definitions for stable, mild, and severe disease.

About through the 1980ís it was really felt that this was a neutrophil-driven process, but as we all know, the endothelium plays an active role in inflammation and there are a number of inflammatory responses which the endothelial cell mediates in its addition to its barrier function so it can express per coagulant molecules tissue factor, it can participate via adhesion molecules to recruit leukocytes and cause cellular adhesion and even produce cytokines. So we became interested in what role the endothelium was playing in lupus, particularly as the story was evolving with respect to the activation of the endothelium as shown here.

Now this may be the mechanism whereby this interaction between neutrophils and endothelial cells is also thought to play a role in re-perfusion injury and there are clinical trials with antibodies to ICAM-1 to prevent re-perfusion injury, in essence to block this molecular interaction. So after 50 or 60 years, one understands on a molecular basis why that refractory period was necessary.

So what does this have to do with lupus? We have postulated over the years that you donít always need immune complex deposition. That you can get a Schwartzman-like lesion in SLE. When I was a fellow, this woman was brought to the Bellevue Hospital Emergency Room about 11:30 at night. A younger fellow, Bill Given, went down and actually I was a young attending and Bill was one of these fellows at 11:30 he started drawing blood. And he drew blood on this lady who was a patient with lupus who came with cerebritis. She was seizing. She was thrombocytopenic. She was a couple of months in the hospital.

So we think that there is a subset of lupus patients, some of these extremely ill people, who have ischemic events with a Schwartzman-like pathogenesis. So in the early 1990ís that Barringer Ingleheim article came out and so we asked, "Well, do lupus patients have activated endothelium?" and so in a study involving several individuals, including Joe Byon, at our institution we began to take a look at biopsies of normal appearing skin of the buttocks. So non-sun exposed, non-lesional skin in people who were sick and people who were well with SLE and we stained as they did in that BI article for E-selectin and for ICAM. This is a typical patient with lupus thatís active. This is a hospitalized patient with several organ systems involved.

When this was quantitated in a number of patients, circulating endothelial cells per ml of blood, one can see that in active SLE circulating endothelium are released. So the endothelium are activated, they are being damaged and they are peeling off from the basement membrane and ending up in the circulation. We asked whether these cells, these endothelium, were expressing ICAM (P means patients, lupus patients and this is healthy controls) and these are the circulating endothelial cells. One can see marked increase ICAM-1 expression in the cells, INOS expression in the patients and not in the healthy controls.

I wanted to lay out to you that itís not just inflammation that drives this, for us. We are interested in two kinds of blood clots; those on the arterial side that we believe have something to do with platelets, plaque rupture, atherothrombosis. What weíve found is that you can measure things like fibrinogen, factor 7, Von Willibrandís, RFR, fibrinolytic markers, TPA and Pi-1, platelet function, LPTLA and these intriguing inflammatory markers, the C-reactive proteins. You can actually predict future arterial events but have a big goose-egg for venous events. That to us is very important because it says that the hyper-coagulable stasis phenomenon of coagulation is different than the plaque rupture phenomenon. In contrast, you may be more familiar with our genetic work factor; prothrombin mutation, mutations in protein C and protein S predicting venous events, but on a population basis, having little to do with arterial events. Now you all probably get referral patients who are quite unique. Where you have these abnormalities and you are getting venous and arterial clots. But on a population basis this is the key issue.

Why inflammation? What has really happened over the last five years is that a fundamental wealth of basic laboratory work -which was very translational coming out of molecular labs that many of you were involved in, and then moving into the vascular biology community - have led to a number of hypotheses that we felt needed to be tested. I think those hypotheses are really two-fold. The first is that, from a cardiologists standpoint, what makes one plaque rupture as compared to another plaque remaining stable, we believe is fundamentally an underlying inflammatory process. Which is to say that histologically, unstable plaques have increased leukocyte infiltrates. You can find abundant T cells and macrophages at the sites of the plaque rupture itself. And a lot of evidence that cytokines and NPs are in that process of plaque degradation. Whatís very interesting to the cardiology community is the idea that lipid lowering, particularly with the statins, does a lot more than just lower LDL cholesterol. And in fact might well be - if I can use it in quotes for this audience - "antiinflammatory". Again there is some experimental evidence toward that effect. To make it very simple -which is what you have to do for cardiologists - is say, "Look, over here in this cartoon we have some inflammatory cells. This is an unstable lesion, this is a bad one to have." "Over here the inflammatory cells are gone and thatís a good lesion."