Monday, Hamza Ziad Bani-Younis Al-Nasser 25/7/2011 Complement Wednesday, Ziad Emad Subject AlAl-Gazo -4 35 16 Yousef Odeh System

6/7/2011 Nasser

Immuno Lec#16 Monday, 25/7/2011 Done By: Hamza Bani-Younis Yousef Odeh

Note: nearly, the first 5 pages of this lecture is review for the previous lecture, so it is up to you to read or leave them. & I just wanna point out that the vast majority of this lecture is Additional Notes!! Fa enjoy ;)

Y
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esterday , we talked about the section 3 which is about innate system. If u remember, the 2 arms of the immune system are: the innate and the

adaptive. We said also that u should know the differences between the

innate and the adaptive!! The innate is:

- Fast, its almost there. The problem with it that it doesnt have a memory, and the number of genes that involved is not that high, and it doesnt use the antigen recognition molecules. It uses Pattern-recognition molecules.

We talked about Toll-like receptor and how they can be activated by group antigen (e.g., lipopolysaccharide), and how those Pattern-

recognition molecules when they stimulate the macrophages and phagocytic cells, then macrophages and the phagocytic cells start producing cytokines; where they initiate signals in the adaptive immune response that we have in attack, so the initiation signal comes from the innate system. We will see how the innate and the adaptive systems cooperate together, and of course the ultimate call of these arms as I said is to get rid of pathogens or abnormal cells, and of course here the recognition is of self VS. non-self, where the adaptive immune response is specialized in this. Well talk about very few cases where the innate system can recognize self from non-self. In the adaptive we talked about antigen recognition molecules, the antigenic determinant, the epitopes, paratopes, and also the Tcell receptor and the MHC antigens, where lots of genes are involved. While in the innate it is not that many that are involved. Then we started talking about the the 1st, 2nd and 3rd.

lines of defensive;

It involves the physical barriers, mucus membranes and mucus, we said its very important, we talked about keratin, dead and living keratinocytes present in the skin, langerhans cells (which is APC) as well, and how those can produce even some interleukins and the tumor necrosis factor.

All of those present in the keratocytes. It is dry and the pH is low coz of fatty acids produced from the sebaceous glands which they produce the sebum (this is why we take a shower every day; to get rid of the sebum) which is acidic, so we have to take it out. This low pH favors the growth of certain MOs (Microorganisms) like the Coagulase-negative bacteria, staphylococcus epidermidis. And where we have moisture in our body outside it favors growth of so many others bacteria as well.

Mucu s

We talked about that in the respiratory tract and how the mucociliary function is so important as a brush border to wash thing out.

And we talked about smoking and how smoking can wipe out the cilia. We have a disease called

Primary

Dyskinesia. Dyskinesia means that the cilia do not move.

This is an inherited disease. Those patients who have this condition are considered as Immunocompromised.

Low pH

So bacteria and so on will adhere to mucus, and then we cough the mucus, then we swallow the mucus into the stomach, where we have

low pH

around 2 which is sufficient to kill all the MOs. Then they pass into the small intestine, where the pH is

high which can get up to 11, in pancreatic secretions. And what really manages to pass to the Colon

Normal Flora

makes what we call the

normal flora, which

around 10^9 MO/1gm of X2, and the normal flora in the colon provide us with non-specific immune defensive, they primed (activate) the immune

system. (I know it's not obvious, but that wt the Dr. said) And that answers the question of many students that how the B and T cells get primed if they did not get the usual prime signals?? So the normal flora that develops in our Gastrointestinal in the colon has the ability to prime the immune system. And so thats why the cells go to secondary lymphoid organs and develop memory cells against these normal flora!! Also the normal flora blocks the attachment of pathogens as well. So we need to keep it in, we need to keep the normal balance, we dont have to abuse antibiotic to wipe it out, we want to keep it. Btw they did lot of experiments about what happen if we sterilize our gastrointestinal tract or let a baby or an animal live in a barrel that completely sterile, they noticed that those become immunocompromised, those of sterile GIT become so thin, and they bleed, and sometimes could die. We call these animals in experiment: Gnotobiotic, Gnotobiotic means sterile. So its not healthy to live in complete sterile environment, we need to challenge our immune system continuously. So itll provide u with these primed lymphocytes that we have been talking about.

Urine Flow

Genitourinary tract, flow of urine is very healthy, we need to drink lot of

water especially in RAMADAN, and nowadays where its very hot.

So the wash of urine is extremely important to wash all MOs as well and any blockage to the flow of urine can lead to infection. So if u have kidney stones or bladder stones or

urethral stones or ureater stones; then u r going to have an infection. So number 1 advice, u r going to give people that they have UTI (urinary tract infection), simply, drink lot of fluids . How many as such as u can. 10 glasses per day..20things will washed up and MOs are going to be cleared out. The same thing of the pH in vagina provides us also with nonspecific immune defense. Glycogen that develops because of estrogen (the secondary sexual character) makes the pH of the vagina acidic, so favor certain MOs there.

Then we started talking about the blood and its constituents other than lymphocytes, we said we have plasma and serum and in plasma we have what we call, acute phase proteins. Also we have collectins, complement, and type 1 interferons. Acute phase proteins like the C-reactive protein; are proteins that will cover the MOs and help in the phagocytosis of these microbes. We talked about ESR (erythrocyte sedimentation rate); we use those as marker of inflammation. In general, when somebody has an inflammatory process thats going on, whether it is infectious or autoimmune, we follow up the patient with these specific markers (C-reactive proteins & ESR).

You cannot tell whats the problem thats going on, but at least u can tell that there is an inflammatory process thats going on in ur body, and u follow up these inflammations; whether the patient is improving or not!! By the ESR and the C-Reactive proteins.

Then we started talking about the complement system, and as we said complement comes from what we call complementary. Complementary complements the function of immunoglobulins, its not specific, so it is innate. The complement simply is a set of proteins, 9 groups, from C1 to C9, and they are activated in sequence. So the 1st one gets activated will act as an enzyme activates the one next, so the one next will be activated and act as an enzyme activate the ones that will follow , till C9 is activated. When C9 is activated it makes holes on the surface of the cell where those complement is going to be activated. So water & electrolytes imbalance and damage to that particular cell is going to take place. Whatever that cell is, tumor cell, bacteria, RBC and so on..its going to be destroyed. So this is the number 1 function of the complement and I will tell you how these things are going to happen.

Complement Function:

Destruction of cells.

Many inflammatory mediators are going to be

produced during the activation of the complement. Complement components can activate mast cells and basophils at the same time, so we r going to have inflammation. Also it might cause hypersensitivity types of reaction.

: complement components act as chemotactic

factor. So theyll call upon cells like neutrophils, eosinophils..etc.

: Complement components can clear out immune

complexes from our bodies, and we do that in the surface of RBCs. So if u have a deficiency with any of the complement components ull become immunocompromised.

It is extremely important to remember these functions; destruction of cells, inflammation, chemotactic factors, clearance of immune complexes.
Second important point that its important to remember that these complement reactions (cascade) needs o be controlled. So its need to be activated in infection or inflammation, and it needs to be inhibited by inhibitory molecules when the infection is gone. If these inhibitory molecules are deficient, the reaction will keep going on, so for the minor infection well have a severe type of reaction, its exactly the same, like the hypersensitivity reactions and it could lead to death.

The Complement system complements antibody function; we have three pathways of complement:
The first 1 is the classical pathway: classical pathway is activated by antigen-

antibody reaction, and the antibody has to be an IgM which is a pentamer or two IgG.
Those are the only 2 Ab's that activate complement.

In the IgGs, igG4 dont activate complement. You require 2 IgG or 1 IgM pentamer that they have to bind to antigen, then complement is going to be fits.

Note: Ill use some figures beside the Dr. Explanation, and these are from the youtube.com. Btw, u can follow them up in full animated view in this link: (http://www.youtube.com/watch?v=vbWYz9XDtLw)

Classical Pathway Step:

Endless Thanxez goes to Hussein Qasim for providing this video

We start here with antigen-antibody reaction, on the surface of the cell, and u have here on top 2 immunoglobulins in the surface. Now the first component thatll bind to CH2 area of the immunoglobulin is the C1 component, and the C1 component if formed of three components: C1q, C1r, C1s. All of them form the C1 complex. Here is the C1q, it has 6 arms. The first component thats going to bind is the C1q, when it binds to antigen then its going to bind C1r, I mean its going to be activated and acts as an enzyme to activate C1r. C1r has like 2 heads, and its going to stimulate C1s, which looks the same C1r. So started with C1q, then C1r then C1s.

Now C1 is activated, itll act as an enzyme. The one thats activated we put a bar on top of it. So the C1 means activated C1. Now the C1 will act on the one next in the cascade, and the one next is NOT C2. Instead, it is C4. So C1 acts first on C4 and then on C2. It will split C4 into 2 Components, C4a and C4b, we call them complement components. C4a has very weak chemotactic and C4b has weak opsonins activity. So they will bind here into the surface. C2 here it will be acted upon and it will be splitted into 2 components as well, C2a and C2b. Usually, when we talk about the complement components, b component is the bound one, usually that bind to the surface, and a is the active, or the one that usually flies away. Although in some books they considered C2a as the larger component thats bind but I want u to remember thats C2b here is the largest component, u see in many books that they considered C2a as the larger component and C2b as the smaller. [The Dr. mix up things here

regarding the size, so it-daggego$ in the next line :P].

Now the larger component C2a binds with C4b together, so u have now in the surface C4b2a (also called C3 convertase). [See the Dr.s figure in

the right].
Now this combination will act as an enzyme and it will act on the one next. We started C1 C4 C2 . Why C4 like this in order?? Bcz when they discover it , it was in sequence of 4, but when they realize that the cascade starts with 4 they dont want to change it, so they kept it as it is. This combination (C4b2a) will act on C3, so we call this combination C3 convertase, and its going to act on C3.

[Please check these figures sequence to follow up with the Dr.]

So upon acting on C3, it (C4b2a) will split the C3 into 2 components: C3b (the bind part, the larger component) & C3a (the smaller component). C3b now can bind to the surface of the cell and it acts as opsonins. As we said before, opsonization can be done with either immunoglobulins, like the IgG antibody, or with complement.

So the major complement part that involves in opsonization is C3b.

And the complement receptor that we call Complement Receptor 1 is where C3b is going to bind. Now, we have C3b thats bound & acts as opsonins and we have C3a that has left. C3a acts as a 1] Chemotactic factor, it calls upon neutrophils to come to the area, & it acts also as 2]Anaphalatoxins, and that means it can acts on mast cells and basophils to be activated and to produce vasactive amines & inflammation. Its stronger than C4a but weaker than C5a as well see now. By now the combination thats left in the surface is C4b2a3b. C4b2a3b now on the surface and it is going to be activated and act as an enzyme and activates the one next which is C5. C4b2a3b will split C5 into C5a and C5b. C5a is 100-times more powerful than C3a in acting as chemotactic factor as well

as anphalatoxins. They can stimulate so strongly the mast cell and basophils to produce the vasoactive amines and call upon neutrophils to come into the area, this is so interesting. C5b on the surface start process called membrane attack complex. i.e., the complex that will start attacking the membrane start with C5b. C5b will never be created unless C1 is activated from the beginning. Now C5b will bind to the surface, the minute that C5b is bound, C6 will join, and C6 will be activated, and C6 will activate C7. So now we have C5b67. And this is going to activate C8, so we have now C5b678. When C8 is bound, then the process will start creating imbalance between water and electrolytes at the level of cytoplasmic membrane of the cell. But it is not enough; it requires the activation of C9. So C9 will be activated by C5b678. Now, This combination of C5b6789 we call it the

membrane

attack complex.
C5b6789 leads to the activation of C9 at the end, and C9 creates the imbalance of the water and electrolytes in the cytoplasmic membrane, & the cell is going to be destroyed.

Review for the Whole Steps in classicalNew pathway: info.s are

You can see how the system has been created as such, started with C1, C1 qrs, qrs are kept together with calcium, acting on C4 and C2 (this step is clearly seen in the up mentioned video), and makes the C3 convertase (C4b2a) that will act on C3 and splitting C3 into C3a and C3b, this C3b will bind strongly to the cytoplasmic membrane thorough magnesium as well, and make a strong binding.

And then C5 convertase (C4b2a3b) is going to develop when C3 binds, and C5 will be splitted into C5a & C5b, where the C5b start the process of the membrane attack complex. C6, C7, C8 and C9 create the damage. This process needs to be controlled; the most important step in the control is at the level of serum. So when our body feels that we dont need this reaction and we need to stop it, C proteins is going to be developed, we call it C1-inhibitor. C1 inhibitor will act on C1 and prevents its activation, so it cannot act on C4 or C2 or C3, and the reaction will stop. Whats going to happen if we dont have the C1 inhibitor is a disease we call it angioneurotic edema; lots of C3a, lots of C4a, lots of C5a are going to be produced which is not needed, and those will going to stimulate lots of mast cells and basophiles to produce there vasoactive amines and we will have massive inflammation and edema all over our body. Whats really trigger this type of the reaction is any minor infection or sometimes trauma or even psychological factor which could stimulate this type of reaction, and because you dont have the C1 inhibitor massive inflammation could take place that could lead to death. We have such a case maybe you heard about it 3 weeks ago when patient came to the emergency room was 50 years old and that patient has chest pain, and they want to do catheterization to see whether he has CAD (coronary artery disease) or not. And during the assessment, they discover that he has hypertension, so they gave him just a tablet of a drug - enalapril- thats usually doesnt cause any type of hypersensitivity reaction, they give him just 1 tablet of drug, and then the patient went into angioneurotic edema. He was edematous all over his face, his lips. And then they started to give him hydrocortisone and anti-histamine, and then they send him to the ICU trying to incubate and put a tube in the trachea to maintain airway (this is the most important in life saving measures). But they couldnt because of the spasm and the contraction of

the smooth muscles, so they took him to the operating theaters to make tracheostomy, but unfortunately the patient die on the table :( This could happen occasionally, maybe once every 2-3 million, you could replace the C1 inhibitor, you could give hydrocortisone, they said that hydrocortisone can induce the production of C1 inhibitor, but if the gene for that is missing then C1 inhibitor cant be produced, you can give fresh frozen plasma that contain C1 inhibitor. Maybe in this case you couldnt think of angioneurotic edema at that time maybe they take it as hypersensitivity to a drug, that the only thing to do is to give hydrocortisone and antihistamine and so on. So I want you to remember angioneurotic edema and C1 inhibitor deficiency. This level (maybe he meant the C1 level :S Im not sure ) is the major control mechanism of the classical pathway. We could have another level like e.g., the level of C4b2a where we have a protein we call it C4b binding protein, so it binds to C4b preventing its binding to C2a to form the C3 convertase, and then the reaction will stop. And we have another one we called decay activating protein that binds between C4b and C2a. C1 receptor can play a role in binding and prevention the formation of the C3 convertase. Also maybe at the level of membrane attack complex when C5b67 develops we have a protein we call it CD59 - they was call it previously protein S which binds to C5b67 on S , preventing C8 and C9 from binding, then the reaction will stop. So you can see at many different levels, but the major one is the C1 inhibitor for the development of angioneurotic edema.

The second pathway we have what we call it the alternative pathway or the Properdin pathway, here the process start with C3 - and you will see that C3 is the key step of all the complement pathways - thats why C3 has the highest concentration in our serum. for the alternative pathway, well use this video (http://www.youtube.com/watch?v=qga3Wn76d9w) , Thx to Tariq Kewan In the alternative pathway you dont need an Ag-Ab reaction to activate it, the process here starts with the lipopolysaccharides of Gram-ve bacteria, endotoxin in particular. So if we are infected with Gram-ve bacteria, immediately the lipopolysaccharide will act on C3, and then C3 is going to be split into C3a and C3b. And the process will continue in a different format, the C3a has similar function of C3a of the classical pathway (anaphylatoxin, chemotactic factor), and the C3b is going to bind to the surface of the cell. Here, the interesting about the alternative pathway how it can recognize self from non-self?? But not to that degree of the adaptive immune system. If C3b binds to the self surface, then an inhibitory signal will form and the reaction will stop.

In case of foreign cells, the minute that C3b bind to the surface of foreign cells, another factor will come and bind to C3b, and this is called the B factor. Immediately when B factor bind to C3b it is going to be hit by another factor which is an enzyme we call it D factor, and it cant hit the B until it bind to C3b, and it will split the B into Ba and Bb. Now the C3bBb will be acting as an enzyme, and it will act again on C3 and it will split another C3 into C3a and C3b, and we call that the amplification loop. The C3bBb as an enzyme has a shorter half-life, but if its bind to another protein we call it the properdin (P) protein and thats why the pathway so named- it will become more stable and the half-life of it will be longer and it will act more on C3 and convert it into C3a and C3b. [The dr. said in the next few lines, The properdin (P) is optional]. Now you have C3 split continuously into C3a & C3b, and C3as will act as anaphylatoxins and chemotactic factors. The C3bs will bind to C3bBb. the minute that C3b binds to the rest of the complex (C3bBb (P)) then it will act as C5 convertase. C5 convertase is simply (C3b)2Bb, So C5 convertase is when we have more than one C3b binds to Bb, and this is going to act on C5 and split it into C5a and C5b. The P is optional dont forget its function is to make the complex stable. Then the C5b continues the membrane attack complex (MAC) and it will activate

C6 and then C7 to have C5b67 and then C8 will bind and then 9 and when 9 binds it will make imbalance between water and electrolytes and the cell is going to be destroyed. So the MAC in the classical pathway and in the properdin or the alternative pathway is the same, but the activation or the process to get the C5b is different.

How this mechanism is going to be controlled??

The process here will not control by C1 or C2 or C4 because we dont have any one of them. The regulation mechanism starts with the binding of the C3b to the surface of the cell; whether it is self or non-self. If it is foreign then the process will continue as we discussed, if it is self, immediately when C3b is bound, a factor we call it factor H is going to bind instead of factor D. And the minute that factor H binds, it will be hit immediately by factor I (Inhibitor), and thus C3bH will destroyed and then we will have what we call inactive C3b (or sometimes called C3bdg, I heard this word half a million times and I heard it like this, but couldnt find it online :\), and then the reaction will stop. So in the alternative pathway, the regulatory mechanism depends on whether we dealing with self or non-self.

The third reaction is the MBL when (mannan-binding mannan binds or lectin) to to pathway, this pathway starts lipopolysaccharide the cells.

carbohydrates on the surface of

This process is when mannan is going to act on C2 and C4. And it will form the same here C4b and C2a and they will act on C3 and split it into C3a and C3b, C3b will bind, and then C3b will make the C5 convertase and act on C5 and start MAC, So the only difference between MBL pathway and the classical pathway that in the MBL pathway you dont have C1, here the carbohydrates will act as C1.

Here, the Dr. turned to his slides....

We talked about the collectins and how the collectins can help in the phagocytosis when bacteria bind. They have receptors in the surface of macrophages that helps in phagocytosis, and they also activate complement in the case of MBL, the collectin-microorganism complex can bind to trigger complement system C4 C2 and it will continue as such. So we have 3 pathways: 1The classical pathway: Ag-Ab

reaction & complement. 23The alternative pathway. The MBL pathway.

In the three of them, the most important point is the cleavage of C3, which amplify the initial signal by activating so many downstream components, and these will lead to activation of mast cells and basophiles, chemotactic factors as well, which cause damage to cells by creating holes in the cytoplasmic membrane, as well the complement can binds to immune complexes and can clear them from the circulation.

Now the Dr. read slide #9

 So we have 9 components C1-C9, with different functions by receptor on cells. So when Im talking about C3, the receptor on that here is C3b receptor where opsonization is going to take place. Complement the function of antibodies. Key component is C3, it has the highest concentration. Many times they ask you this question: If you have do serum analysis on patient and you found that C3 is low (or any one of the complement component) what does that mean?? You have 2 options here: The first one this component is missed genetically so you have genetic disease with deficiency of one of the complement components, which is rare!! The second explanation is you have utilized it, you have excessive reaction. This makes sense more than the first one; you have severe reaction that this component has been utilized. So C3 is the most important one to measure because all these 3 pathways depend on it.

Note: You can find the classical & the alternative pathways cascade on the last page of this lecture. .

The End