I. INTRODUCTION

Inflammation is defined as the succession of changes that occurs in a living, vascularized tissue when it receives a sublethal injury. "Vascularized" means having a blood supply; as we will see, the reactions of inflammation intimately involve the small blood vessels and leukocytes (white blood cells).

The process of inflammation is designed to dilute, destroy, or otherwise inactive the agent that caused the injury in the first place. Ultimately, the goal of inflammation is to restore damaged or infected tissue to its original state, insofar as possible.

Anything that damages tissues can initiate the reactions of the inflammatory response. These include infection, physical trauma, chemical toxins, thermal injury (either heat or cold), and radiation of various types (including UV irradiation, as in a sunburn).

There are two types of inflammation, acute and chronic. Acute inflammation is of fairly short duration (lasting from a few minutes to a couple of days) and is characterized by accumulation of leukocytes known as neutrophils. If the acute inflammatory response is not sufficient to deal with the problem at hand, then chronic inflammation may ensue. Chronic inflammatory responses may last from a few days up to a lifetime, in the case of certain chronic inflammatory diseases (e.g., rheumatoid arthritis). In areas of chronic inflammation, macrophages and lymphocytes (rather than neutrophils) accumulate. Longer-lasting changes in the architecture of the affected tissues may also be seen, such as proliferation of fibroblasts and blood vessels. These changes are part of the normal would healing process, as we will see at the end of this lecture.

It is important that you know the functions of the principal cell types that are involved in inflammation:

Due to time constraints, this lecture will focus almost exclusively on acute inflammation. Chronic inflammation will be mentioned only briefly, but remember that chronic inflammation plays a central part in many diseases that you will encounter in this course.

An area of acute inflammation is characterized by five typical changes, which constitute the five cardinal signs of inflammation:

In areas of acute inflammation, three physiological changes typically occur that account for the five cardinal signs. These are hyperemia (increased blood flow), increased vascular permeability (meaning that the blood vessels get leaky), and migration of leukocytes from the blood to the tissues, a process termed diapedesis or extravasation. Each of these changes will now be considered in detail.
 
II. HYPEREMIA

In the circulatory system, a network of branching capillaries connects the smallest arteries (called arterioles) to the smallest veins (called venules). Some of these capillaries (called preferential channels) are always open to the flow of blood. However, blood flow to other capillaries can be controlled by donut-like rings of smooth muscle cells, called precapillary sphincters. Normally, many of these sphincters are contracted to squeeze the capillary shut and prevent the flow of blood. Likewise, arterioles are surrounded by sheaths of smooth muscle cells, which can contract or relax to regulate the diameter of the vessel.

During inflammation, inflammatory mediators are released that cause smooth muscle cells to relax. Consequently, arterioles dilate. Also, precapillary sphincters relax, allowing many previously unused capillaries to fill with blood. The net effect is a great increase in the flow of blood to the inflamed tissue.

There are many mediators that can cause this relaxation of smooth muscle cells. Two of the most important are histamine and prostaglandins. Histamine is released from granules of mast cells in the tissues, as well as platelets and basophils in the blood. Note that it is very easy to persuade a mast cell to release histamine – a sharp blow or exposure to cold temperatures is enough to do the job (food for thought: why does your nose run in cold weather??). In contrast, prostaglandins do not exist pre-formed within cells. Rather, this group of closely related chemical compounds must be manufactured from scratch when cells receive the right signal. Many kinds of cells are capable of producing prostaglandins when they find themselves in inflammatory settings. The first step in production is release of a fatty acid, called arachidonic acid, from phospholipids in the cell membrane. This arachidonic acid is then converted into prostaglandins by an enzyme called cyclooxygenase. A different enzyme, called lipoxygenase, can convert arachidonic acid into compounds called leukotrienes.

It is important that you know how prostaglandins are produced, because it will help you understand how one of our most common anti-inflammatory drugs, aspirin, works. Aspirin inactivates cyclooxygenase, thus preventing the generation of prostaglandins. Other so-called non-steroidal anti-inflammatory drugs (NSAIDs) work in the same way. These include ibuprofen (the active ingredient in Motrin) and indomethacin. Acetominophen, the active ingredient in Tylenol, is a good pain reliever, but it does not suppress formation of prostaglandins. In general, then, NSAIDs are better than Tylenol for treatment of inflammation.

It has been discovered that there is more than one kind of cyclooxygenase. COX-1 makes prostaglandins that are important for "housekeeping" jobs of the body, such as maintaining the protective mucosal lining of the stomach. COX-2 makes the prostaglandins that are important in inflammation. Recently, the Food and Drug Administration has approved a new anti-inflammatory drug that inhibits COX-2 but not COX-1 (other NSAIDs inhibit both). As a result, this new drug, called Celebrex, is much less apt to cause stomach irritation and gastric bleeding than other NSAIDs. You may have heard about this "COX-2 inhibitor" in news stories and advertisements.
 
III. INCREASED VASCULAR PERMEABILITY

In general, blood vessels get leaky in areas of inflammation. The degree of leakiness depends on the extent of damage. In a severe injury, the blood vessels will be ripped apart. They will leak for some time (hours to days), until clotting and wound healing repair the vessels. This type of leakage is called immediate sustained leakage. Another pattern of leakage is called delayed prolonged leakage, which does not begin until several hours after the injury and may last from hours to days. The basis for this type of leakage is not well understood. A classic example is severe sunburn, where fluid-filled blisters form only several hours after you’ve left the beach.

A third type of pattern is called immediate transient leakage, which begins immediately after the injury is sustained and lasts perhaps 15 minutes to an hour or so. This type of leakage is seen in mild injuries. It is caused by a number of mediators, including histamine and leukotrienes (which, like prostaglandins, are formed from arachidonic acid; see above). These mediators cause the endothelial cells that line the blood vessels to contract, so that they round up and pull away from one another. (Normally, the endothelial cells are tightly connected to form a semi-permeable barrier.) When the cells retract from one another, gaps are formed that permit fluid and plasma molecules to flow freely from the bloodstream out into the tissues. This process does not damage the endothelial cells. After a time, they spread back out and re-establish connections with their neighbors. The excess fluid in the tissues is drained by the lymphatic system, and all is returned to normal. This type of leakage is thus rapidly reversible.

Increased permeability leads to edema, which is defined as any excess fluid in the tissues. There are two types of edema:

IV. DIAPEDESIS OF LEUKOCYTES

As mentioned, the movement of white blood cells from the blood to the surrounding tissues is called diapedesis or extravasation. This is a step-wise process, in which circulating leukocytes first adhere to the endothelial lining of the blood vessel wall. Next, the leukocytes squeeze through the junctional spaces between endothelial cells and cross the basement membrane that underlies the endothelium. Lastly, the leukocytes travel through the surrounding connective tissue until they reach the damaged or infected area.

Although gaps form between endothelial cells during inflammation, the mere presence of these gaps is not sufficient to allow diapedesis. Injection of an animal with histamine will cause vascular leakage, but it will not cause diapedesis of leukocytes. Other signals are required to induce the leukocytes to leave the blood. First, the endothelium of vessels in the inflamed tissue becomes more adhesive for neutrophils. This occurs when inflammatory substances stimulate the endothelial cells to manufacture adhesion molecules. These adhesion molecules are displayed on the surfaces of the stimulated endothelial cells, and they literally reach out and grab onto passing leukocytes in the blood. Two important inflammatory substances that can activate endothelium in this way are interleukin-1 (IL-1) and lipopolysaccharide (LPS). IL-1 is made by macrophages at the site of inflammation. LPS is a component of the cell wall of many kinds of bacteria and so is likely to be present in infected tissues.

Once the leukocytes have stuck to the vessel wall, they need yet another signal to tell them to move across the endothelium and out into the tissues. This signal is provided by substances called chemoattractants. Chemoattractants induce cells to undergo chemotaxis, which is defined as directed movement of a cell that is induced by a substance in the cell’s environment. Chemoattractants are generated in inflamed tissue. They then diffuse through the tissue to blood vessels, where they serve to alert the leukocytes that there is a problem requiring their urgent presence. The leukocytes respond by moving from the area where the chemoattractant is present in the lowest concentration (i.e., at the vessel wall) to the area of highest concentration (i.e., at the site of injury, where the chemoattractant is being generated). MANY different chemoattractants have been identified. Two that you should know are 1) certain peptides generated from the breakdown of bacterial proteins; and 2) complement component C5a, which is produced when the complement system is activated. So infection with bacteria or the presence of antibodies bound to antigens (which you will recall activates complement) will generate chemoattractants to alert the white cells that there is a problem. A third very important group of chemoattractants is the chemokines. The chemokines are a family of over forty structurally related proteins. During inflammation, they are producedby many different types of cells, most notably endothelial cells and macrophages. A unique property of the chemokines is that individual members attract specific types of leukocytes (as opposed to bacterial peptides and C5a, which attract almost all kinds of leukocytes). This specificity of chemokines is thought to account, at least in part, for the proper trafficking of leukocytes throughout the body and for accumulation of specific populations of leukocytes in different inflammatory settings. Two of the most abundant and best-studied chemokines are interleukin-8, which attracts neutrophils, and monocyte chemoattractant protein-1, which attracts monocytes, lymphocytes, and basophils.

Just as there are diverse chemokines, there are many known cellular receptors that bind to chemokines. Recently, it has been recognized that chemokine receptors play an extremely important role in HIV infection.  The cellular receptor for HIV is CD4, but it turns out that HIV cannot enter host cells unless a “co-receptor” is also present.  A chemokine receptor named CCR5 has been identified as such a co-receptor.  It is needed for so-called M-tropic strains of HIV to infect macrophages.  It is these strains that are thought to initiate infection in previously uninfected people.  Interestingly, there are certain men who have lifestyles that place them at high risk for contracting HIV, but they remain uninfected.  Some of these men have a genetic lack of CCR5.  It has been estimated that individuals who are completely deficient in CCR5 are protected from infection with HIV by 97%.  These men appear perfectly healthy despite their lack of CCR5, implying that other chemokine receptors can compensate for its loss.  Another chemokine receptor, CXCR4, may be a co-receptor for T cell-tropic strains of HIV, which tend to evolve later in the disease process.  You will hear more about this topic in the upcoming lecture on infectious diseases.

Nature has performed an experiment to show the importance of these mechanisms for leukocyte recruitment. Some children have a genetic disease called leukocyte adhesion deficiency (LAD), which results in severe, recurrent bacterial infections from the time of birth. These children have plenty of neutrophils; in fact, they often have abnormally high numbers of circulating neutrophils (a condition called neutrophilia). However, children with LAD do not form pus at sites of infection – that is, the neutrophils do not leave the bloodstream properly. Laboratory studies have shown that the neutrophils of these children cannot bind to endothelial cells, because they have an inherited lack of the protein that would normally allow them to do so. Since the neutrophils cannot bind to endothelial cells, they cannot be recruited to areas of infection, where they would ordinarily ingest and kill the invading bacteria. Children with the severest form of LAD are very ill and, even with intensive antibiotic therapy, often die before reaching adulthood. Their only hope is a successful bone marrow transplant, which would supply them with genetically normal neutrophils. Fortunately, LAD is a very rare disease; it is highly unlikely that you will ever see a case.
 
V. SUMMARY OF THE LOCAL CHANGES IN INFLAMMATION

These three changes that occur at an area of inflammation (hyperemia, increased vascular permeability, and diapedesis) account for the classic signs of an inflammatory response. Redness is due to increased blood flow, as is heat. Swelling is due to accumulation of fluid and, to a lesser extent, leukocytes in the damaged tissue. Pain results from direct damage to nerves, from pressure of the swollen tissue on nerves, and from chemical mediators. Those of you with allergies know that histamine causes an itching type of pain. Another mediator released during inflammation, called bradykinin, causes a burning sensation. Since aspirin relieves pain, you might conclude, correctly, that prostaglandins are involved in producing pain. Although prostaglandins by themselves do not cause pain, they enhance one’s perception of pain – that is, prostaglandins intensify the pain-causing properties of agents such as histamine and bradykinin.
 
VI. THE NEUTROPHIL

Now that you know how a neutrophil gets where it is going, you need to learn what it does when it arrives. Neutrophils (as well as monocytes and macrophages) ingest microbial invaders by the process of phagocytosis. Once ingested, these leukocytes have a wide array of chemical weapons available to destroy the microorganisms. Usually, bacteria are not ingested very efficiently unless they are first opsonized (coated) with either antibody molecules or complement proteins. The term "opsonized" comes from the Greek meaning "to prepare for eating." If a bacterium is opsonized with antibody molecules, the Fc portions of these antibody molecules are available to bind to specific Fc receptors on the leukocyte, which then triggers phagocytosis. Alternatively, bacteria may activate the complement system. The bacteria then become coated with complement proteins, which again bind to specific receptors on the leukocytes to help facilitate ingestion.

As the bacterium is ingested, it is completely surrounded by the plasma membrane of the leukocyte to form a phagosome (also called a phagocytic vacuole). Membrane-bound granules (lysosomes) within the leukocyte then fuse with the phagocytic vacuole to form a phagolysosome. Upon fusion, the contents of these lysosomes, which include many antimicrobial substances, are injected into the phagosome and can therefore attack the ingested bacteria. (There is a diagram of the process of phagocytosis in the slide set on cell injury).

The antimicrobial weapons of the neutrophil can be divided into two categories: those that do not require the presence of oxygen (oxygen-independent) and those that do (oxygen-dependent). The oxygen-independent weapons include the following substances, all of which are contained within the lysosomes of the neutrophil:

Some of the most potent mechanisms by which neutrophils kill bacteria require the presence of oxygen, which is converted by the cell into a series of highly toxic compounds. First, an enzyme within the phagolysosome converts O2 into H2O2 (hydrogen peroxide). A second enzyme then combines the H2O2 with Cl- to form HOCl (hypochlorous acid, the active component of bleach). You probably are aware that both hydrogen peroxide and bleach are very powerful disinfectants. Children with a genetic lack of the enzyme that produces hydrogen peroxide suffer from chronic granulomatous disease, which is characterized by severe, recurrent bacterial infections. Unlike children with LAD, these children form pus at sites of infection. However, their neutrophils lack the capacity to kill many types of ingested microorganisms efficiently, since they cannot produce either hydrogen peroxide or hypochlorous acid.

Toxic products can leak from neutrophils and actually make the damage worse than it started out. The oxidants that neutrophils produce (i.e., hydrogen peroxide and hypochlorous acid) react very non-specifically to disrupt the structures of all sorts of molecules, especially lipids. If these oxidants leak out of the phagolysosome, they can attack host cell components just as they do bacterial components. Likewise, the lysosomal enzyme elastase can break down elastin, which gives tissues resiliency (for example, the lung is particularly rich in elastin). Another lysosomal enzyme called collagenase degrades collagen, which is the most abundant protein in the extracellular matrix of tissues.

How do these substances escape from the neutrophils? Usually, lysis of dead neutrophils is not much of a problem. Neutrophils die by apoptosis (see the cell injury lecture) and are then eaten up by macrophages. A bigger problem is what is called regurgitation during feeding, meaning that neutrophils are sloppy eaters. Lysosomes start fusing with the developing phagosome before it has fully formed, meaning that there is a direct pathway for lysosomal contents to reach the environment outside the cell (see the slide set if you need help visualizing this process.) This sloppy eating is probably necessary to allow the neutrophil to attack large clusters and long chains of bacteria that cannot be fully engulfed. Another mechanism of leakage is called frustrated phagocytosis, when a neutrophil tries to eat antigen-antibody complexes that are deposited in tissues of the body. The neutrophil cannot distinguish between antibody molecules that are bound to a bacterium and those that are abnormally deposited somewhere; it just tries to eat them all, even if the task is impossible.

A classic example of frustrated phagocytosis is the disease poststreptococcal glomerulonephritis. This is a disease that may affect the glomerulus of the kidney following infection of the throat with certain strains of streptococcus. If the strep infection is not treated, the body will mount an immune response and form antibodies to the strep bacteria. The antigen-antibody complexes that form travel through the blood and, with some types of strep, are just the right size and charge to become lodged in the walls of capillaries in the glomerulus of the kidney. (The glomerulus of the kidney is where filtration of wastes from the blood takes place. The capillaries in the glomerulus have pores [called fenestrations] that aid in the filtration process. It is in these pores that the antigen-antibody complexes become lodged.) The immune complexes that deposit in the glomerulus trigger activation of the complement system. C5a is released, which attracts neutrophils to the area. The neutrophils try to ingest the complexes, and, in so doing, release lysosomal molecules that damage the underlying basement membrane. (The pictures presented in lecture will really help in visualizing this whole process.) A child can survive this process once or twice without too much permanent damage, but if it happens repeatedly, he or she may end up on dialysis. This scenario is one reason pediatricians are so quick to treat suspected cases of strep throat with antibiotics: if the strep is prevented from growing, the child will not make antibodies, and the possibility of kidney complications will be avoided. Note that poststreptococcal glomerulonephritis does not involve infection of the kidney with strep. The bugs infect the throat; it is the antigen-antibody complexes and neutrophils that damage the kidney. Also, note that this is a very good example of a Type III hypersensitivity (see the immunology lecture).

Luckily, we are not totally at the mercy of leaky neutrophils. We have evolved some defenses that help limit the damage from their toxic substances. One very important defense consists of anti-proteinases that are contained in the blood plasma. These anti-proteinases bind to and neutralize many types of enzymes in the neutrophils. The importance of anti-proteinases is underscored by patients who have a genetic lack of alpha-1-proteinase inhibitor, which blocks the action of elastase. These individuals are at greatly increased risk for developing the lung disease emphysema. In emphysema, the elastic recoil of the lung is lost, so that air cannot be easily expelled. In normal people, infections of the lung are handled well by neutrophils, and alpha-1-proteinase inhibitor quickly neutralizes any elastase that leaks out. In people who lack this inhibitor, every little lung infection leads to release of elastase that is not properly neutralized and therefore chews away at the elastin in the lung. Once gone, this elastin can never be replaced. Over time, resiliency of the lung is lost, and emphysema results. Note that lack of alpha-1-antiproteinase is a rare cause of emphysema; smoking is the number one culprit.
 
VII. SYSTEMIC CHANGES IN INFLAMMATION

So far, we have considered only the local changes that constitute the inflammatory response – that is, those that occur right at the site of infection or damage. However, severe inflammation may also involve a number of systemic, or body-wide, changes. These systemic changes are collectively known as the acute phase response or acute phase reaction. Systemic changes in inflammation are mediated by cytokines, which can be defined simply as proteins produced by one cell that have effects on other cells. Macrophages are important sources of the major cytokines that produce the acute phase response. These include interleukin 1 (IL-1), which has already been discussed in regard to its effects on endothelial cells, and a closely related protein, tumor necrosis factor alpha (TNF). These two proteins have very similar activities. For the sake of simplicity, the discussions below will mention only IL-1, but you should note that TNF can serve much the same purpose.

During inflammation, macrophages in the tissues will be stimulated to produce IL-1. If the inflammation is severe enough, IL-1 and TNF will get into the bloodstream and travel to distant parts of the body to produce systemic symptoms. These symptoms include:

Another simple test to look for an acute phase response also depends on the production of acute phase proteins by the liver. Increased amounts of fibrinogen in the blood will cause red blood cells (erythrocytes) to clump together (only in a test tube, not in the body!) These clumps, called rouleaux, settle more quickly to the bottom of a tube than do normal, unclumped erythrocytes. Thus, the presence of increased fibrinogen in the blood, indicative of an acute phase reaction, leads to an increased erythrocyte sedimentation rate (sometimes just called a "sed rate"). The advantage to this test is that it is easy to perform in any doctor’s office; all that is needed is a calibrated tube and a timer. The disadvantage is that is not terribly specific – the sed rate may increase due to conditions other than an acute phase reaction.

 VIII. BENEFITS OF INFLAMMATION

Inflammation is often thought of as an undesirable condition, and, indeed, we have seen how toxic components from leukocytes can actually contribute to tissue damage at a site of inflammation. However, it is very important to bear in mind that the inflammatory response is critical to the body’s ability to defend itself against invaders and injurious agents. Inflammation is often referred to as a "two-edged sword" because it can be beneficial or detrimental, depending on the circumstances. Some of the very real benefits of inflammation include:

IX. CHRONIC INFLAMMATION

The importance of chronic inflammation in disease processes should not be overlooked, even though we will not have time to consider it thoroughly. Chronic inflammation occurs if the acute inflammatory response is not able to eliminate whatever caused the injury in the first place. Common causes of chronic inflammation include intracellular pathogens, toxic substances that cannot be degraded (e.g., silica particles), and autoimmune reactions. As acute inflammation turns into a chronic situation, short-lived neutrophils are replaced by longer-lived macrophages and lymphocytes. These cells may cause destruction of tissue. At the same time, the tissue is trying to heal, and processes associated with normal wound healing (described below) may occur. These include proliferation of blood vessels and fibroblasts, which lay down increased amounts of connective tissue. A subtype of chronic inflammation that you will encounter in this course is called granulomatous inflammation. Areas of granulomatous inflammation, called granulomas, are characterized by collections of modified macrophages. These are macrophages that contain indigestible foreign material. Because they cannot eliminate the material, the macrophages attempt to at least keep it from spreading through the body by becoming immobile. The immobilized macrophages take on the appearance of epithelial cells and thus are called epithelioid cells. Some of the epithelioid cells may fuse to form giant cells. Again, the purpose of this process is thought to be to "wall off" injurious substances that cannot be degraded or otherwise eliminated. Granulomas are typically seen in tuberculosis as the body tries to confine the spread of the intracellular bacillus.
 
X. WOUND HEALING

The purpose of inflammation is to pave the way for healing to take place. Damaged tissues can be mended in two different ways. If the normal architecture of the tissue has not been too greatly perturbed, then lost cells will be replaced by identical ones, a process called regeneration. In the face of greater damage, the wounded tissue may have to be replaced by a scar, a process called repair. The extent to which the healing process can restore an injured tissue depends not only on the severity of the damage, but also on the type of cell that was injured. It is relatively easy for the body to replace labile cells, which are cells that continually divide under normal circumstances. These include blood cells and epithelial cells. Stable cells can also be replaced. These are cells that normally do not divide much, but they can be stimulated to replicate when necessary. Examples of stable cells include endothelial cells, fibroblasts, and cells of the liver and kidney. You are really out of luck if you damage permanent cells, which cannot divide under any circumstances. These include nerve cells, cardiac muscle cells, and skeletal muscle cells.

The process of healing is an orderly one. The slides presented in lecture show the events that occur during the healing of a simple cut of the skin. Soon after injury, a clot forms, and the cut scabs over. The inflammatory response increases the permeability of vessels in the area, and neutrophils are recruited to fight off potential infection. Within the first couple of days, growth factors are produced that stimulate the replication of the epithelial cells surrounding the wound. Two to three days after the injury, neutrophils begin to disappear, and macrophages come in to phagocytose bacteria and debris from the damaged tissue. Fibroblasts migrate into the wound and proliferate, forming new connective tissue. Some of these fibroblasts take on certain characteristics of muscle cells and are thus called myofibroblasts. Myofibroblasts contract, pulling the edges of the wound together. This contraction is a very important part of healing, since it means the gap that will have to be filled by new tissue will be smaller. New blood vessels are also forming, a process called angiogenesis. These blood vessels are not completely formed, so they are somewhat leaky. If the scab is removed a this stage, the tissue underneath is pink and slightly wet due to the presence of all these leaky vessels. This pink, wet tissue is called granulation tissue.

By Day 5, the epidermis should be completely restored; the scab is gone. The wound is now filled with granulation tissue as new vessels continue to proliferate. After two weeks, the leukocytes and exudate are gone, and the blood vessels are beginning to degenerate. Connective tissue, composed largely of collagen, is still being made by fibroblasts. Collagen may continue to accumulate for many months, and the molecules are becoming cross-linked to one another to give the wound additional strength. The redness typical of a new scar fades as the blood vessels continue to regress.

In some people, particularly African-Americans, excessive collagen is formed in response to injury. These lesions are called keloids. They are a difficult problem to treat, since attempts to remove the keloid only stimulate formation of more scar tissue.
 
COMMENTS ON YOUR TEXT (Damjanov)
pp. 23: Swelling during inflammation is more due to leakage of fluid from the blood into tissues, rather than to increased blood flow as your text implies. Also, your text says that transmission of signals from nerves are important in dilation of vessels and opening of precapillary sphincters, but it is chemical mediators (such as histamine) that are most directly involved.

p. 24: Your book says that leakiness of vessels due to histamine allows blood cells to escape. In fact, other signals, such as chemotactic factors and increased adhesiveness of endothelium for the blood cells, are required to allow white blood cells to move out from the vessel. Leaky vessels alone are not enough!

p. 26: The definitions of transudation, exudate, and edema are inaccurate. Use the ones given in class.

p. 29: Although they have similar functions, basophils are not precursors of mast cells. Also, platelets do not release their granules upon contact with normal endothelium, only damaged endothelium.